Anti-DLL3 antibodies, antibody-drug conjugates thereof and pharmaceutical uses thereof

Abstract

Anti-DLL3 antibody, its antibody-drug conjugate and medical use thereof. The present invention relates to an anti-DLL3 antibody-ecteinascidin drug conjugate represented by the general formula (Pc-LD), in which Pc is an anti-DLL3 antibody. JPEG2025500952000113.jpg95170

Description

[Technical field]

[0001] This application claims priority to a Chinese patent application (CN202111592539.7) filed on December 23, 2021.

[0002] The present disclosure relates to an anti-DLL3 antibody, its drug conjugate and its preparation method. At the same time, the anti-DLL3 antibody or its ADC described in the present disclosure is applicable to the preparation of a medicament for treating a disease or condition. [Background technology]

[0003] The statements herein are not necessarily intended to constitute prior art, but merely to provide background information relevant to the present disclosure.

[0004] Small cell lung cancer (SCLC) is a relatively malignant type of lung cancer that accounts for 10% to 15% of all lung cancer cases. Small cell lung cancer has a fast tumor growth rate, is prone to metastasis, and has a 5-year survival rate of less than 7%. Platinum-based / etoposide combination chemotherapy is often used to treat small cell lung cancer. Patients with small cell lung cancer respond well to chemotherapy in the early stages, but are highly susceptible to drug resistance and recurrence. Recent immunotherapies, such as PD-L1 antibodies and PD1 antibodies, have some effect on patients with small cell lung cancer, but the efficacy rate is about 15%. The current situation is that specific targeted therapeutic drugs have not yet been developed.

[0005] DLL3 is a ligand that inhibits Notch. Under normal conditions, DLL3 is located in the Golgi apparatus, and in cancer cells (e.g., small cell lung cancer cells), DLL3 reaches the cell surface and binds to Notch in cis, inhibiting cell-cell binding and endocytosis of Notch into target cells, thereby suppressing the Notch signaling pathway and promoting tumor cell growth. DLL3 is mainly expressed in neural or neuroendocrine tumors, including SCLC, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumor, small cell bladder cancer, glioblastoma multiforme, metastatic castrated prostate cancer, melanoma, etc. In particular, for SCLC, more than 80% of SCLCs have positive expression of DLL3, but normal lung cancer tissues and paracancer tissues do not express it. This difference in expression makes DLL3 a highly potential therapeutic target for treating SCLC.

[0006] Ecteinascidin derivatives, such as lurbinectedin, are inhibitors of RNA polymerase II that can selectively inhibit the transactivated RNA polymerase II-mediated transcription process by covalently binding to the minor groove in the DNA double helix structure, without affecting RNA polymerase I and mitochondrial RNA polymerase activity or the normal transcription process of mRNA. RNA polymerase II tends to be overactivated during the transcription of tumor cells, and lurbinectedin can cause tumor cells to become abnormal during mitosis and apoptosis, ultimately reducing cell proliferation. Tumor cells support their proliferation by a fast-operating transcription process, and these tumor cells are particularly sensitive to lurbinectedin, including SCLC, BRCA1 / 2-mutated breast cancer, platinum-resistant ovarian cancer, and sarcoma due to chromosomal translocation.

[0007] Antibody drug conjugates (ADCs) are made by linking a monoclonal antibody or antibody fragment to a biologically active drug via a linker, taking advantage of the specificity of the antibody to bind to normal and tumor cell surface antigens and the high efficiency of the drug (e.g., cytotoxic agent), while avoiding the drawbacks of the antibody's relatively low therapeutic effect and the drug's excessive toxicity and side effects. Compared with traditional chemotherapy drugs, antibody drug conjugates can kill tumor cells more precisely and reduce the impact on normal cells. Summary of the Invention

[0008] The present disclosure relates to anti-DLL3 antibodies, their ADCs, and their pharmaceutical uses. More specifically, the present disclosure provides novel anti-DLL3 antibodies of various sequences and their ADCs coupled with cytotoxic agents (e.g., ecteinascidin derivatives).

[0009] In some embodiments, the disclosure provides an anti-DLL3 antibody comprising a heavy chain variable region and a light chain variable region, i) the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 57; and The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, wherein SEQ ID NO: 57 is represented by PLYX1YGRSYNX2VAY, wherein X1 is Y or H, and X2 is A or G; or ii) the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; and The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:19, an LCDR2 comprising the amino acid sequence of SEQ ID NO:20, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:21.

[0010] In some embodiments, the anti-DLL3 antibody is the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, 30, or 31; and The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:25, an LCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

[0011] In some embodiments, the anti-DLL3 antibody is the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24; and The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:25, an LCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

[0012] In some embodiments, the anti-DLL3 antibody is the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:22, an HCDR2 comprising the amino acid sequence of SEQ ID NO:23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:30; and The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:25, an LCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

[0013] In some embodiments, the anti-DLL3 antibody is the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:22, an HCDR2 comprising the amino acid sequence of SEQ ID NO:23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:31; and The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:25, an LCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

[0014] In some embodiments, the anti-DLL3 antibody is i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:57, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively; Wherein, SEQ ID NO:57 is represented by PLYX1YGRSYNX2VAY, Wherein, X1 is Y or H, and X2 is A or G; or ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. In some embodiments, the anti-DLL3 antibody is a murine antibody, a chimeric antibody or a humanized antibody. In some embodiments, the anti-DLL3 antibody is preferably a humanized antibody.

[0015] In some embodiments, the anti-DLL3 antibody comprises a human immunoglobulin framework region (FR region).

[0016] In one embodiment, the anti-DLL3 antibody is The heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 57, where SEQ ID NO: 57 is represented by PLYX1YGRSYNX2VAY, where X1 is Y or H, and X2 is A or G, and the FR of the heavy chain variable region comprises one or more back mutations selected from 1E, 49A, and 94S; The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the FR of the light chain variable region comprises a 43I back mutation, and the back mutation site follows the Kabat numbering convention.

[0017] In one embodiment, the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 18, and the FR of the heavy chain variable region comprises one or more back mutations selected from 1E, 27Y, 30T, 38K, 43K, 48I, 67A, 68A, 69L, 71V, 73K, 75S, 76N, and 93A; The light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 19, LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the FR of the light chain variable region comprises one or more backmutations selected from 36L, 43S, 44F, 46G, 69A, 71Y, and 85D. The backmutation sites follow the Kabat numbering convention.

[0018] In one embodiment, the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 16, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 18, and the FR of the heavy chain variable region comprises back mutations of 1E, 68A, 69L, 71V, 73K, 75S, and 76N; The light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 19, LCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the FR of the light chain variable region comprises back mutations of 36L, 46G, 69A, 71Y, and 85D, and the back mutation sites follow the Kabat numbering convention.

[0019] In one specific embodiment, the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the FR of the heavy chain variable region comprises back mutations of 1E and 94S; and The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the backmutation sites follow the Kabat numbering convention.

[0020] In one embodiment, the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, 30, or 31, and the FR of the heavy chain variable region comprises one or more backmutations selected from 1E, 49A, and 94S; and The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the FR of the light chain variable region comprises a 43I backmutation. The backmutation site follows the Kabat numbering convention.

[0021] In one embodiment, the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the FR of the heavy chain variable region comprises one or more backmutations selected from 1E, 49A, and 94S; The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the FR of the light chain variable region comprises a 43I backmutation. The backmutation site follows the Kabat numbering convention.

[0022] In one embodiment, the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the FR of the heavy chain variable region comprises one or more backmutations selected from 1E, 49A, and 94S; The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the FR of the light chain variable region comprises a 43I backmutation. The backmutation site follows the Kabat numbering convention.

[0023] In one embodiment, the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 31, and the FR of the heavy chain variable region comprises one or more backmutations selected from 1E, 49A, and 94S; The light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the FR of the light chain variable region comprises a 43I backmutation. The backmutation site follows the Kabat numbering convention.

[0024] In some embodiments, the anti-DLL3 antibody is i) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14, 50, 51, 52, 53 or 54; and / or the light chain variable region comprises a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 15, 55 or 56; or ii) the heavy chain variable region comprises a sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44; and / or The light chain variable region comprises a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13, 45, 46, 47, 48 or 49.

[0025] In some embodiments, the anti-DLL3 antibody is i) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14; and / or the light chain variable region comprises a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 15; or ii) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 50, 51, 52, 53 or 54; and / or the light chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 55 or 56; or iii) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12; and / or the light chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13; or iv) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44; and / or The light chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:45, 46, 47, 48 or 49.

[0026] In some embodiments, the anti-DLL3 antibody is i) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 50; and / or the light chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:55; or ii) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 43; and / or The light chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:48.

[0027] In some embodiments, the anti-DLL3 antibody is i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15, or ii) the heavy chain variable region comprises any one of the amino acid sequences selected from SEQ ID NOs: 50, 51, 52, 53, and 54, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NOs: 55 or 56; or iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; or iv) the heavy chain variable region comprises any one of the amino acid sequences selected from SEQ ID NOs: 43, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 44, and / or the light chain variable region comprises any one of the amino acid sequences selected from SEQ ID NOs: 48, 45, 46, 47, and 49.

[0028] In some embodiments, the anti-DLL3 antibody is the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:50, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:55; The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:43, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:48.

[0029] In some embodiments, the anti-DLL3 antibody is an antibody fragment, wherein the antibody fragment is a Fab, Fab', F(ab')2, Fab'-SH, Fd, Fv, scFv, dsFv, bibody, or domain antibody.

[0030] In some embodiments, the anti-DLL3 antibody comprises a light chain constant region and a heavy chain constant region.

[0031] In some embodiments, the anti-DLL3 antibody comprises an IgG1, IgG2, IgG3, or IgG4 constant region.

[0032] In some embodiments, the anti-DLL3 antibody comprises a lambda or kappa chain constant region.

[0033] In some embodiments, the anti-DLL3 antibody comprises an IgG1 heavy chain constant region and a kappa light chain constant region.

[0034] In some embodiments, the anti-DLL3 antibody, wherein the heavy chain constant region comprises the sequence of SEQ ID NO:28 and / or the light chain constant region comprises the sequence of SEQ ID NO:29.

[0035] In some embodiments, the anti-DLL3 antibody comprises a heavy chain and a light chain, the heavy chain comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 60, and / or the light chain comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 61, or The heavy chain comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:58 and / or the light chain comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:59.

[0036] In some embodiments, the anti-DLL3 antibody, wherein the heavy chain comprises the sequence set forth in SEQ ID NO:60 and / or the light chain comprises the sequence set forth in SEQ ID NO:61; or The heavy chain comprises the sequence given in SEQ ID NO:58 and / or the light chain comprises the sequence given in SEQ ID NO:59.

[0037] In another aspect, the disclosure provides an isolated anti-DLL3 antibody that binds to DLL3 or an epitope thereof in competition with any one of the antibodies described above.

[0038] In some embodiments, the anti-DLL3 antibody, wherein the antibody has at least one of the following properties: a) the anti-DLL3 antibody has a KD value for binding to human DLL3 or an epitope thereof of ≦3 nM, ≦2 nM, ≦1 nM, ≦0.9 nM, ≦0.8 nM, ≦0.7 nM, ≦0.6 nM, ≦0.5 nM, or ≦0.4 nM, and the KD value is measured by Biacore; b) the anti-DLL3 antibody has an EC50≦3 nM, EC50≦2 nM, EC50≦1 nM, EC50≦0.5 nM, EC50≦0.2 nM, EC50≦0.1 nM, EC50≦0.09 nM, EC50≦0.08 nM, EC50≦0.07 nM, EC50≦0.06 nM for binding to H1184 cells expressing DLL3, wherein the EC50 is detected by FACS; c) the anti-DLL3 antibody is capable of being endocytosed by cells expressing DLL3; d) the anti-DLL3 antibody has an EC50 of ≦0.1 nM (≦0.09 nM, ≦0.08 nM, ≦0.07 nM, ≦0.06 nM, ≦0.05 nM, ≦0.04 nM) for binding to DLL3 or an epitope thereof, wherein the EC50 is measured by ELISA; and e) The above anti-DLL3 antibody and the positive antibody (e.g., BI-764532) recognize different DLL3 epitopes.

[0039] In another aspect, the disclosure provides an isolated nucleic acid encoding any one of the above anti-DLL3 antibodies.

[0040] In another aspect, the disclosure provides a vector comprising the isolated nucleic acid described above.

[0041] In a further aspect, the present disclosure provides a host cell comprising the above vector or any one of the above anti-DLL3 antibodies.

[0042] In one aspect, the disclosure provides a method of preparing an antibody that binds to DLL3, the method comprising culturing the host cell under conditions suitable for expression of the antibody.

[0043] In a further aspect, the present disclosure provides an antibody-drug conjugate comprising any one of the above described anti-DLL3 antibodies, or a pharma- ceutically acceptable salt thereof.

[0044] In a further aspect, the present disclosure provides an antibody-drug conjugate, or a pharma- ceutically acceptable salt thereof, comprising any one of the above-described anti-DLL3 antibodies and a drug coupled to the anti-DLL3 antibody.

[0045] In some embodiments, the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof, wherein the drug is selected from one or more of a cytotoxic agent, a radioactive marker, a fluorophore, a chromophore, a developing agent, an immunomodulatory agent, an angiogenesis inhibitor, a cell growth inhibitor, an apoptosis promoter, and a cytolytic enzyme.

[0046] In some embodiments, the drug is an ecteinascidin Et-743 (Trabectedin) or a derivative thereof. In some embodiments, the drug is an ecteinascidin derivative, such as lurbinectedin.

[0047] In some embodiments, the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof has a structure represented by the general formula (Pc-L-Da): [ka] In the above, Pc is any one of the anti-DLL3 antibodies, L is a linker, and n is 1 to 10 (including integers and decimals), and preferably 3 to 5.

[0048] In some embodiments, the antibody-drug conjugate or a pharma- ceutically acceptable salt thereof is represented by the general formula (Pc-LD) or a pharma- ceutically acceptable salt thereof: [ka] In the above, Pc is any one of the anti-DLL3 antibodies, L is a linker, and n is 1 to 10, preferably 3 to 5.

[0049] In some embodiments, the antibody-drug conjugate or pharma- ceutically acceptable salt thereof is represented by the above general formula (Pc-L-Da) or (Pc-LD), in which n is the average number of drug modules of each antibody, and may be an integer or a decimal. In some embodiments, n is an average value of 1 to 10, or 1 to 9, or 2 to 10, or 2 to 9, or 1 to 8, or 2 to 8, or 2 to 7, or 2 to 4, or 1 to 4, or 1 to 3, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 4 to 7, or 4 to 6, or 4 to 5, and in some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0050] In some embodiments, the antibody-drug conjugate represented by any one of the general formulas (Pc-L-Da) or (Pc-LD) described above or a pharma- ceutically acceptable salt thereof, wherein the linker-L- is L 1 -L 2 -L 3 -L 4 -Of which, L 1 -(succinimid-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, where W is C 1-6 Alkylene group, C 1-6 Alkylene-C 3-6 cycloalkyl groups, among which the above C 1-6 Alkylene group, C 1-6Alkylene-C 3-6 each cycloalkyl group is independently optionally further substituted with one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl; L 2 -NR 4 (CH2CH2O) p CH2CH2C(O)-, -NR 4 (CH2CH2O) p CH2C(O)-, -S(CH2) p C(O)- and a chemical bond, wherein p is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acid residues, among which the amino acids are selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) and aspartic acid (D), and are optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group and cycloalkyl group; L 4 -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH2) t - and a chemical bond, wherein t is an integer from 1 to 6; R 3 , R 4 and R 5 are the same or different and each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 6 and R 7 are the same or different and each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0051] In some embodiments, the antibody-drug conjugate represented by any one of the general formulas (Pc-L-Da) or (Pc-LD) described above or a pharma- ceutically acceptable salt thereof, wherein the linker-L- is L 1 -L 2 -L 3 -L 4 -Of which, L 1 teeth [ka] and s 1 is an integer from 2 to 8, including but not limited to 2, 3, 4, 5, 6, 7, or 8; L 2 is a chemical bond, L 3 is a tetrapeptide residue, preferably L 3 is a tetrapeptide residue containing glycine-glycine-phenylalanine-glycine, L 4 is -NH(CH2)t-, where t is 1 or 2; Among them, the above L 1 The termini are ligated to Pc.

[0052] In some embodiments, the antibody-drug conjugate is represented by any one of the general formulas (Pc-L-Da) or (Pc-LD) described above, or a pharma- ceutically acceptable salt thereof, wherein -L- is [ka] It is.

[0053] In some embodiments, the antibody-drug conjugate represented by any one of the general formulas (Pc-L-Da) or (Pc-LD) or a pharma- ceutically acceptable salt thereof described above, wherein the antibody-drug conjugate has a structure selected from the following: [ka] wherein Pc is any one of the anti-DLL3 antibodies described above, and n is 1-10.

[0054] In some embodiments, the antibody-drug conjugate has the general formula (Pc-L-Da) or (Pc-LD) described in any one of the above, or a pharma- ceutically acceptable salt thereof, and the antibody-drug conjugate has the following structure: [ka] Among them, n is 1 to 10; Pc is an anti-DLL3 antibody according to any one of the above, preferably Pc is an anti-DLL3 antibody, which is a heavy chain as set forth in SEQ ID NO: 60 and a light chain as set forth in SEQ ID NO: 61; or It comprises a heavy chain as shown in SEQ ID NO:58 and a light chain as shown in SEQ ID NO:59.

[0055] In some embodiments, the antibody-drug conjugate has the general formula (Pc-L-Da) or (Pc-LD) described in any one of the above, or a pharma- ceutically acceptable salt thereof, and the antibody-drug conjugate has the following structure: [ka] Among them, Pc is an anti-DLL3 antibody according to any one of the above, preferably Pc is an anti-DLL3 antibody, which is a heavy chain as set forth in SEQ ID NO: 60 and a light chain as set forth in SEQ ID NO: 61; or comprising a heavy chain as set forth in SEQ ID NO:58 and a light chain as set forth in SEQ ID NO:59; n is 1 to 8.

[0056] In some embodiments, the antibody-drug conjugate has the general formula (Pc-L-Da) or (Pc-LD) described in any one of the above, or a pharma- ceutically acceptable salt thereof, and the antibody-drug conjugate has the following structure: [ka] Among them, Pc is an anti-DLL3 antibody according to any one of the above, preferably Pc is an anti-DLL3 antibody, which is a heavy chain as set forth in SEQ ID NO: 60 and a light chain as set forth in SEQ ID NO: 61; or comprising a heavy chain as set forth in SEQ ID NO:58 and a light chain as set forth in SEQ ID NO:59; n is 3 to 5.

[0057] In some embodiments, the antibody-drug conjugate has the general formula (Pc-L-Da) or (Pc-LD) described in any one of the above, or a pharma- ceutically acceptable salt thereof, and the antibody-drug conjugate has the following structure: [ka] Among them, Pc is an anti-DLL3 antibody according to any one of the above, preferably Pc is an anti-DLL3 antibody, which is a heavy chain as set forth in SEQ ID NO: 60 and a light chain as set forth in SEQ ID NO: 61; or comprising a heavy chain as set forth in SEQ ID NO:58 and a light chain as set forth in SEQ ID NO:59; n is 3.73 to 4.27.

[0058] In some embodiments, the antibody-drug conjugate has the general formula (Pc-L-Da) or (Pc-LD) described in any one of the above, or a pharma- ceutically acceptable salt thereof, and the antibody-drug conjugate has the following structure: [ka] Among them, Pc is an anti-DLL3 antibody according to any one of the above, preferably Pc is an anti-DLL3 antibody, which is a heavy chain as set forth in SEQ ID NO: 60 and a light chain as set forth in SEQ ID NO: 61; or comprising a heavy chain as set forth in SEQ ID NO:58 and a light chain as set forth in SEQ ID NO:59; n is 3.72 to 4.88.

[0059] The present disclosure further provides a method for preparing an antibody-drug conjugate, the method comprising reducing an anti-DLL3 antibody described above and coupling it with a compound represented by (LD) or (L-Da) to obtain an antibody-drug conjugate described in the present disclosure.

[0060] In another aspect, the present disclosure provides a pharmaceutical composition comprising any one of the above-described anti-DLL3 antibodies, or any one of the above-described antibody-drug conjugates or pharma- ceutically acceptable salts thereof, and one or more pharma- ceutically acceptable excipients, diluents, or vectors. In some embodiments, the unit dose of the pharmaceutical composition comprises 0.1-3000 mg or 1-1000 mg of the anti-DLL3 antibody or the antibody-drug conjugate.

[0061] In another aspect, the present disclosure provides the use of any one of the anti-DLL3 antibodies described above, or any one of the antibody-drug conjugates or pharma- ceutically acceptable salts thereof, or a pharmaceutical composition comprising same, as a medicament.

[0062] In another aspect, the present disclosure provides a use of any one of the anti-DLL3 antibodies described above, or any one of the antibody-drug conjugates or pharma- ceutically acceptable salts thereof, or the pharmaceutical composition described above, in the preparation of a medicament for treating a DLL3-mediated disease or condition. In some embodiments, the DLL3-mediated disease or condition is a tumor or cancer. In some embodiments, the DLL3-mediated disease or condition is a disease or condition expressing DLL3.

[0063] In another aspect, the present disclosure provides a use of any one of the above-described anti-DLL3 antibodies, or any one of the above-described antibody-drug conjugates or pharma- ceutically acceptable salts thereof, or any one of the above-described pharmaceutical compositions, in the preparation of a medicament for treating or preventing a tumor or cancer, preferably, wherein the tumor or cancer is The tumor or cancer is selected from lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, large cell lung cancer), head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer (e.g., medullary thyroid cancer), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), liver cancer, hepatic and gallbladder cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer (e.g., colon cancer and rectal cancer), renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer, glioblastoma, skin cancer and melanoma, more preferably, the tumor or cancer is small cell lung cancer.

[0064] More preferably, the tumor or cancer is a tumor or cancer that expresses DLL3.

[0065] In another aspect, the present disclosure further relates to a method for treating and / or preventing a tumor or cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of any one of the anti-DLL3 antibodies described above, or any one of the antibody-drug conjugates or pharma- ceutically acceptable salts thereof described above, or the pharmaceutical composition described above, preferably wherein the tumor or cancer is a tumor or cancer that expresses DLL3.

[0066] In another aspect, the present disclosure further relates to a method for treating or preventing a tumor or cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of any one of the anti-DLL3 antibodies described above, or any one of the antibody-drug conjugates or pharma- ceutical salts thereof described above, or the pharmaceutical composition described above, wherein the tumor or cancer is The tumor or cancer is selected from lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, large cell lung cancer), head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer (e.g., medullary thyroid cancer), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), liver cancer, hepatic and gallbladder cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer (e.g., colon cancer and rectal cancer), kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer, glioblastoma, skin cancer, and melanoma, preferably, the tumor or cancer is small cell lung cancer.

[0067] In another aspect, the present disclosure further provides the above-mentioned anti-DLL3 antibody, or the above-mentioned antibody-drug conjugate, as a medicament, preferably as a medicament for treating cancer or tumor, more preferably as a medicament for treating cancer or tumor expressing DLL3.

[0068] The anti-DLL3 antibodies and antibody-drug conjugates provided in the present disclosure have good affinity for cell surface antigens, can be effectively endocytosed by cells expressing DLL3, and have strong tumor growth inhibitory effects while also having good safety. [Brief description of the drawings]

[0069] [Figure 1A] ~ [Figure 1C] Figure 1 shows the binding of Hu6 and Hu100 antibodies to DLL3 from different species, of which Figure 1A shows the FACS results of the binding of Hu6 and Hu100 antibodies to H1184 cells, Figure 1B shows the FACS results of the binding of Hu6 and Hu100 antibodies to cynoDLL3 / CHO-s cells, and Figure 1C shows the FACS results of the binding of Hu6 and Hu100 antibodies to ratDLL3 / CHO-s cells. [Diagram 2]1 shows the results of a competitive binding experiment of different anti-DLL3 antibodies, which indicates that Hu6 and Hu100 do not compete with BI-764532, and therefore that the antibodies Hu6 and Hu100 bind to different epitopes than BI-764532. [Diagram 3] The results show that Hu6 and Hu100 antibodies are endocytosed by cells, indicating that both Hu6 and Hu100 can be endocytosed by cells. [Figure 4A] ~ [Figure 4E] The inhibitory effects of ADC-1 and ADC-2 on the growth of different cells are shown in Figure 4A, which shows the inhibitory effect of ADC-1 and ADC-2 on the growth of DMS53 cells with low DLL3 expression, Figure 4B shows the inhibitory effect of ADC-1 and ADC-2 on the growth of H1184 cells with high DLL3 expression, Figure 4C shows the inhibitory effect of ADC-1 and ADC-2 on the growth of HT-29 cells without DLL3 expression, Figure 4D shows the inhibitory effect of ADC-1 and ADC-2 on the growth of U-2 OS cells without DLL3 expression, and Figure 4E shows the inhibitory effect of ADC-1 and ADC-2 on the growth of CHO-K1 cells without DLL3 expression. [Diagram 5] The results of the bystander cytotoxicity effect of ADC-1 and ADC-2 are shown. [Figure 6] 1 shows the results of ADC-1 and ADC-2 inhibiting the growth of subcutaneously implanted DMS53 cell tumors in mice. [Figure 7] 1 shows the results of ADC-1 and ADC-2 inhibiting the growth of subcutaneously implanted H1184 cell tumors in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] 1. Terminology In order to make the present disclosure more readily understandable, the following provides an explanation of some technical and scientific terms. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0071] Unless specifically stated to the contrary, terms used in the specification and claims have the following meanings.

[0072] As used in the specification and claims, the singular forms "a," "an," and "the above" include plural referents unless the context clearly dictates otherwise.

[0073] Unless the context clearly indicates otherwise, in the patent specification and claims, the words "containing," "having," "including," and the like, are to be understood in the sense of "including, but not limited to," rather than in the exclusive or exhaustive sense.

[0074] The term "and / or" is intended to include the dual meaning of "and" and "or." For example, the phrase "A, B and / or C" is intended to cover each of A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone) and C (alone). When trade names are used in this disclosure, they are intended to include formulations of products under that trade name, drugs and active drug portions of products under that trade name.

[0075] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0076] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in the same manner as naturally occurring amino acids. Naturally occurring amino acids are those amino acids that are coded for by codons, and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α carbon attached to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that act in the same manner as a naturally occurring amino acid.

[0077] The term "amino acid mutation" includes amino acid substitution (also called amino acid replacement), deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, provided that the final construct has the desired properties, such as reduced binding to Fc receptors. Deletion and insertion of amino acid sequences include deletion and insertion at the amino and / or carboxy termini of the polypeptide chain. A specific amino acid mutation may be an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods of modifying amino acid side groups other than genetic engineering, such as chemical modification, may also be available. As used herein, the same amino acid mutation may be designated by various names. As used herein, the amino acid residue at a particular site may be designated in the form of position + amino acid residue, for example, 366W indicates that the amino acid residue at site 366 is W. T366W indicates that the amino acid residue at site 366 is replaced by W for the original T.

[0078] The term "antibody" is used in the broadest sense and covers various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), provided that they exhibit the desired antigen-binding activity. A complete antibody generally comprises two light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has one variable region (VH), also called variable heavy chain domain, heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has one variable region (VL), also called variable light chain domain or light chain variable domain, followed by one constant light chain domain (light chain constant region, CL).

[0079] The terms "full length antibody," "complete antibody," and "whole antibody" may be used interchangeably herein and refer to an antibody having a structure generally similar to that of a native antibody or having an Fc region in the heavy chain. The light chain of a native complete antibody comprises a light chain variable region VL and a constant region CL, where VL is at the amino terminus of the light chain, the light chain constant region comprises a kappa chain and a lambda chain, the heavy chain comprises a variable region VH and constant regions (CH1, CH2, and CH3), where VH is at the amino terminus of the heavy chain and the constant region is at the carboxy terminus, of which CH3 is closest to the carboxy terminus, and the heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0080] The term "variable region" or "variable domain" of an antibody refers to the domain in the heavy or light chain of the antibody that is involved in binding the antibody to an antigen. As used herein, the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody each contain four conserved framework regions (FR) and three complementarity determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region in the variable domain that mainly promotes binding to an antigen, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, and VL contains three CDR regions, LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxy terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0081] The boundaries of the amino acid sequences of the CDRs can be determined by various known methods, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77(2003); Front Immunol. 2018 Oct 16, 9:2278), and the like, and the correspondence between various numbering systems is well known to those skilled in the art and is illustratively shown in Table 1 below.

[0082] [Table 1]

[0083] Unless otherwise stated, all variable region and CDR sequences in this disclosure follow the Kabat numbering convention.

[0084] The term "antibody fragment" refers to a molecule distinct from a complete antibody and includes a portion of the complete antibody that binds to the antigen that the complete antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), and multispecific antibodies composed of antibody fragments.

[0085] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two subunits, which may be the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable Fc regions for use in the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. In some embodiments, the boundaries of the Fc region may be altered, for example, by deletion of the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region, or deletion of the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system) of the Fc region. Unless otherwise stated, the numbering convention for the Fc region is the EU numbering system, also referred to as the EU index.

[0086] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from another, different source or species.

[0087] The term "humanized antibody" is an antibody that maintains the reactivity of a non-human antibody while having low immunogenicity in humans, which may be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterparts (i.e., the constant and variable region framework region portions).

[0088] The terms "human antibody", "humanized antibody", "fully human antibody", and "fully human antibody" may be used interchangeably and refer to antibodies in which the variable and constant regions are human sequences. The terms cover antibodies that are derived from human genes but have sequences that, for example, reduce potential immunogenicity, increase affinity, remove cysteines or glycosylation sites that may cause undesirable folding, etc. The terms cover such antibodies that are recombinantly produced in non-human cells (which may confer glycosylation not characteristic of human cells). The terms also cover antibodies that have been bred in transgenic mice that contain human immunoglobulin heavy and light chain loci. The definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0089] The term "affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity and reflects the interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be expressed as a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including the methods described herein.

[0090] As used herein, the term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody can be measured by methods known in the art. For example, the affinity in solution is measured by a biosensing system such as a surface plasmon resonance system, or by solution equilibrium titration (SET).

[0091] The term "effector function" refers to a biological activity that can be attributed to an antibody Fc region (either a native sequence Fc region or an amino acid sequence mutated Fc region) and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and activation of B cells.

[0092] The term "monoclonal antibody" refers to a population of essentially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules within the population are the same except for minor natural mutations that may exist. In contrast, a polyclonal antibody preparation typically contains a number of different antibodies that have different amino acid sequences in their variable domains, which are typically specific for different epitopes. "Monoclonal" should not be construed as requiring production of the antibodies by any particular method.

[0093] In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0094] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent, including, for example, an antigen binding protein (e.g., an antibody). An antigen may have one or more epitopes that are capable of interacting with different antigen binding proteins (e.g., antibodies).

[0095] The term "epitope" refers to an area or region on an antigen capable of specific binding to an antibody or antigen-binding fragment thereof. An epitope may be formed by a contiguous string of amino acids (linear epitope) or may comprise non-contiguous amino acids (conformational epitope) that are spatially proximate, for example, due to folding (i.e., tertiary folding) of the antigen. Conformational and linear epitopes differ in that the binding of an antibody to a conformational epitope is not detectable in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed by methods routine in the art, including, but not limited to, alanine scanning, peptide blots, peptide truncation analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496) and cross-blocking.

[0096] The terms "anti-DLL3 antibody" and "antibody that binds to DLL3" refer to an antibody that can bind to DLL3 or an epitope thereof with sufficient affinity. In one embodiment, the extent of binding of an anti-DLL3 antibody to an unrelated protein is at least about 10% less than the binding of the antibody to DLL3, as can be measured by BIACORE® surface plasmon resonance assays.

[0097] The terms "capable of specifically binding," "specifically binds," or "binding" refer to the ability of an antibody to bind to an antigen or epitope thereof with greater affinity than to other antigens or epitopes. Generally, antibodies bind to an antigen or epitope with greater affinity than an antibody ... -7 M or less (for example, about 1×10 -8An antibody binds to an antigen or an epitope thereof with an equilibrium dissociation constant (KD) of 10 M or less. In some embodiments, the KD of an antibody for binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody for binding to a non-specific antigen (e.g., BSA, casein). KD can be measured by known methods, for example, by BIACORE® surface plasmon resonance assay. However, an antibody that specifically binds to an antigen or an epitope thereof may be cross-reactive with other related antigens, for example, cross-reactive with corresponding antigens from other species (homologs) (e.g., humans or monkeys such as Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp)) or Callithrix jacchus (common marmoset, marmoset).

[0098] The term "nucleic acid" may be used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term covers backbone residues or linked nucleic acids containing known nucleotide analogs or modifications. The nucleic acids are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties as the reference nucleic acid and are metabolized in the same manner as the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphate, chiral-methyl phosphate, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell, as described below, which generally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a location different from its natural chromosomal location. An isolated nucleic acid encoding a polypeptide or a fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or a fusion protein, including one or more nucleic acid molecules in a single vector or separate vectors, and one or more nucleic acid molecules present in one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence also implicitly covers its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences and the sequence specified. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues.

[0099] The terms "polypeptide" and "protein" may be used interchangeably herein to refer to a polymer of amino acid residues in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, and apply to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly covers conservatively modified variants thereof.

[0100] The term sequence "identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are homologous at equivalent positions, and gaps are introduced as necessary to obtain the maximum percentage of identity when the two sequences are optimally aligned, and any conservative substitutions are not considered as part of the sequence identity. To measure the percentage of sequence identity, alignment can be achieved by techniques known in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to measure alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0101] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA circle that can be ligated to an additional DNA segment. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which another DNA segment can be ligated to the viral genome. Some vectors can autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell after being introduced into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming a host cell and containing nucleic acid sequences that direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and RNA splicing of the coding region operably linked thereto, if there is an intron.

[0102] The terms "host cell," "host cell line," and "host cell culture" may be used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the original transformed cell and its derived progeny, without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, and may contain mutations. Included herein are mutant progeny that have the same function or biological activity as the cells screened or selected from the original transformed cell. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Exemplary host cells are Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells, and HEK-293 cells, Pichia pastoris, Pichia finlandica, Candida albicans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei.

[0103] An "antibody drug conjugate" (ADC) is a conjugate in which an antibody (or its antigen-binding fragment) is linked to a drug directly or via a linker.

[0104] "Drug" (abbreviation: D) is any substance having biological or detectable activity (e.g., therapeutic agents, detectable labels, binding agents, etc.) and any prodrug that is metabolized to an active agent in vivo. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cell growth inhibitors, and immunomodulatory agents. Chemotherapeutic agents are chemical compounds that can be used to treat cancer. Exemplary therapeutic agents include cytotoxins, cytotoxic agents, and cell growth inhibitors.

[0105] A cytotoxic effect is the deletion, elimination and / or death of the target cell. A cytotoxic agent refers to an agent that has a cytotoxic and / or cell growth inhibitory effect on a cell. A cell growth inhibitory effect is the inhibition of cell proliferation. A cell growth inhibitory agent refers to an agent that has a cell growth inhibitory effect on a cell, thereby inhibiting the growth and / or proliferation of a specific subgroup of cells.

[0106] Additional representative therapeutic agents include radioisotopes, chemotherapeutic agents, immunomodulatory agents, antiangiogenic agents, antiproliferative agents, proapoptotic agents, and cytolytic enzymes (e.g., RNAses). These agent descriptive terms are not mutually exclusive, and thus the therapeutic agent may be described by one or more of the above terms. For example, the radioisotope selected is also a cytotoxin. The therapeutic agent may be prepared as a pharma- ceutically acceptable salt, acid, or derivative of any one of the above. In general, conjugates having a radioisotope as the drug are called radioimmunoconjugates, and conjugates having a chemotherapeutic agent as the drug are called chemoimmunoconjugates.

[0107] Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, CC-1065, dolastatin, duocarmycins, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinblastine, SN-38, tubulysin, hemiasterlin, eribulin, trabectedin, lurbinectedin, and stereoisomers, isosteres, analogs, or derivatives thereof. Chemotherapeutic agents, plant toxins, other biologically active proteins, enzymes (i.e., ADEPT), radioisotopes, photosensitizers (i.e., for photodynamic therapy) may also be used.

[0108] The term "label" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, the label may catalyze a detectable chemical change of a substrate compound or composition. Radioisotope labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. The label may also be an undetectable entity, such as a toxin.

[0109] The term "linker unit", "linker" refers to a chemical fragment or bond that is linked at one end to an antibody and at the other end to a drug, and may be linked to an antibody or drug via another linker. Linkers may be attached to antibodies in several ways, for example, via surface lysines, reductive coupling to oxidized carbohydrates, cysteine ​​residues released by reduction of interchain disulfide bonds, reactive cysteine ​​residues engineered at specific sites, and endogenous glutamines that become reactive upon modification of the polypeptide in the presence of transglutaminase and amines, or via tags that contain acyl donor glutamines. Several ADC linkage systems are known in the art, including hydrazone-, disulfide-, and peptide-based linkages.

[0110] The linker may include one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB").

[0111] The linker may be selected from the following elements: extenders, spacers, and amino acid units, or a combination thereof. The linker may be synthesized by methods known in the art, for example, as described in US20050238649A1. The linker may be a "cleavable linker" that facilitates the release of the drug in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0112] Linker elements include, but are not limited to, the following: MC=6-maleimidocaproyl, with the following structure: [ka] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease cleavable linker); Citrulline = 2-amino-5-ureidopentanoic acid, PAB = p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker element); Me-Val-Cit = N-methyl-valine-citrulline (in which the linker peptide bond is modified so that it is not cleaved by cathepsin B); MC(PEG)6-OH = maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines); SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate; SPDP = N-succinimidyl 3-(2-pyridyldithio)propionate, SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate; IT=iminothiolane.

[0113] "LD" is the linker-drug moiety produced by attaching a drug (D) to a linker (L).

[0114] The "drug loading amount" is also called the drug-to-antibody ratio (DAR), that is, the average amount of drugs coupled to each antibody in the ADC. It may be, for example, within the range of about 1 to about 10 drugs coupled to each antibody, and in some embodiments, within the range of about 1 to about 8 drugs coupled to each antibody, preferably selected from the ranges of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 1 to 3, 3 to 4, 3 to 5, 5 to 6, 5 to 7, 5 to 8, and 6 to 8. The general formula of the ADC according to the present disclosure includes a collection of antibody-drug conjugates within the above certain range. In the embodiment of the present disclosure, the drug loading amount may be represented by n, which is a decimal or an integer. The drug loading amount can be measured by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA test, HIC, and RP-HPLC.

[0115] In one embodiment of the present disclosure, the drug is coupled to a reactive group (eg, a mercapto group) of the antibody via a linker.

[0116] The drug loading of the ADC is (1) controlling the molar ratio of the linking reagent to the monoclonal antibody; (2) controlling reaction time and temperature; (3) selecting different reaction reagents; The amount of oxygen can be controlled by a number of methods, including but not limited to:

[0117] The term “alkyl group” refers to a saturated, straight- or branched-chain aliphatic hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C 1-20 The alkyl group is an alkyl group having 1 to 12 carbon atoms (i.e., C1-12 alkyl group) is preferred, and alkyl groups having 1 to 6 carbon atoms (i.e., C 1-6 Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 6-methylhexyl, 7-methylhexyl, 8-methylhexyl, 9-methylhexyl, 10-methylhexyl, 11-methylhexyl, 12-methylhexyl, 13-methylhexyl, 14-methylhexyl, 15-methylhexyl, 16-methylhexyl, 17-methylhexyl, 18-methylhexyl, 19-methylhexyl, 22-methylhexyl, 23-methylhexyl, 24-methylhexyl, 25-methylhexyl, 26-methylhexyl, 27-methylhexyl, 28-methylhexyl, 29-methylhexyl, 30-methylhexyl, 31-methylhexyl, 32-methylhexyl, 33-methylhexyl, 34-methylhexyl, 35-methylhexyl, 36-methylhexyl, 37-methylhexyl, 38-methylhexyl, 39-methylhexyl, 40-methylhexyl, 41-methylhexyl, 42-methylhexyl, 43-methylhexyl, 44-methylhexyl, 45-methylhex Examples of the alkyl groups include 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl and various branched chain isomers thereof. The alkyl group may be substituted or unsubstituted and, when substituted, it may be substituted at any available attachment point, and the substituents are preferably one or more selected from D atoms, halogens, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0118] The term “alkylene group” refers to a divalent alkyl group, wherein the alkyl group is as defined above, having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C 1-20 The alkylene group is an alkylene group having 1 to 12 carbon atoms (i.e., C 1-12 Alkylene groups having 1 to 6 carbon atoms (i.e., C alkylene groups) are preferred, and alkylene groups having 1 to 6 carbon atoms (i.e., C alkylene groups) are more preferred. Non-limiting examples include -CH-, -CH(CH)-, -C(CH)-, -CHCH-, -CH(CHCH)-, -CHCH(CH)-, -CHC(CH)-, -CHCHCH-, -CHCHCHCH-, and the like. The alkylene group may be substituted or unsubstituted, and if substituted, it may be substituted at any available attachment point, and the substituent is preferably one or more selected from D atoms, halogens, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups.

[0119] The term "alkenyl group" refers to an alkyl group containing at least one carbon-carbon double bond in the molecule, wherein the alkyl group is as defined above and has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) carbon atoms (i.e., C 2-12 The alkenyl group is an alkenyl group having 2 to 6 carbon atoms (i.e., C 2-6Alkenyl groups are preferred. Non-limiting examples include vinyl, propenyl, isopropenyl, butenyl, etc. The alkenyl group may be substituted or unsubstituted, and if substituted, it may be substituted at any available point of attachment, and the substituents are preferably one or more selected from D atoms, alkoxy groups, halogens, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups.

[0120] The term "alkynyl group" refers to an alkyl group, wherein the alkyl group is as defined above and contains at least one carbon-carbon triple bond in the molecule, and has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) carbon atoms (i.e., C 2-12 The alkynyl group is an alkynyl group having 2 to 6 carbon atoms (i.e., C 2-6 Alkynyl groups are preferred. Non-limiting examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. The alkynyl group may be substituted or unsubstituted, and if substituted, it may be substituted at any available point of attachment, and the substituents are preferably one or more selected from D atoms, alkoxy groups, halogens, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups.

[0121] The term "alkoxy group" refers to -O-(alkyl group), where alkyl group is as defined above. Non-limiting examples include methoxy, ethoxy, propoxy and butoxy groups. The alkoxy group may be substituted or unsubstituted, and if substituted, it may be substituted at any available attachment point, with the substituents being preferably one or more selected from D atoms, halogens, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0122] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic all-carbocyclic ring (i.e., a monocyclic cycloalkyl group) or polycyclic ring system (i.e., a polycyclic cycloalkyl group) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 3- to 20-membered cycloalkyl group). The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 ring atoms (i.e., a 3- to 12-membered cycloalkyl group), more preferably a cycloalkyl group having 3 to 8 ring atoms (i.e., a 3- to 8-membered cycloalkyl group), and most preferably a cycloalkyl group having 3 to 6 ring atoms (i.e., a 3- to 6-membered cycloalkyl group).

[0123] The monocyclic cycloalkyl groups include, by way of non-limiting example, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups.

[0124] The polycyclic cycloalkyl groups include spirocycloalkyl groups, fused cycloalkyl groups and bridged cycloalkyl groups.

[0125] The term "spirocycloalkyl group" refers to a polycyclic ring system in which the rings share one carbon atom (referred to as a spiro atom), which may contain one or more double bonds within the ring, or which may contain one or more heteroatoms within the ring selected from nitrogen, oxygen and sulfur (which may optionally be oxidized, i.e., to form nitrogen oxides, and which may optionally be substituted with an oxo group, i.e., to form sulfoxides or sulfones, but which do not include -OO-, -OS- or -SS-), provided that at least one all-carbocyclic ring is included and the point of attachment is at the all-carbocyclic ring, which has from 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5-20 membered spirocycloalkyl group). The spirocycloalkyl group is preferably a spirocycloalkyl group having 6 to 14 ring atoms (i.e., a 6- to 14-membered spirocycloalkyl group), more preferably a spirocycloalkyl group having 7 to 10 ring atoms (i.e., a 7- to 10-membered spirocycloalkyl group). The spirocycloalkyl group includes a monospirocycloalkyl group or a polyspirocycloalkyl group (e.g., a bisspirocycloalkyl group, etc.), preferably a monospirocycloalkyl group or a bisspirocycloalkyl group, more preferably a 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered, or 7-membered / 6-membered monospirocycloalkyl group. Non-limiting examples are: The connection point may be located anywhere. [ka] etc.

[0126] The term "fused cycloalkyl group" refers to a polycyclic system in which the rings share two adjacent carbon atoms, such as a monocyclic cycloalkyl group fused to one or more monocyclic cycloalkyl groups, or a monocyclic cycloalkyl group fused to one or more heterocyclyl, aryl, or heteroaryl groups, in which the point of attachment is on the monocyclic cycloalkyl group, which may contain one or more double bonds in the ring, and which has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5-20 membered fused cycloalkyl group). The fused cycloalkyl group is preferably a fused cycloalkyl group having 6 to 14 ring atoms (i.e., a 6-14 membered fused cycloalkyl group), and more preferably a fused cycloalkyl group having 7 to 10 ring atoms (i.e., a 7-10 membered fused cycloalkyl group). The fused cycloalkyl group includes bicyclic fused cycloalkyl groups and polycyclic fused cycloalkyl groups (e.g., tricyclic fused cycloalkyl groups, tetracyclic fused cycloalkyl groups, etc.), preferably bicyclic fused cycloalkyl groups or tricyclic fused cycloalkyl groups, more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered, or 7-membered / 6-membered bicyclic fused cycloalkyl groups. Non-limiting examples are: The connection point may be located anywhere. [ka] etc.

[0127] The term "bridged cycloalkyl group" refers to an all-carbon polycyclic ring system in which the rings share two carbon atoms that are not directly connected to each other, and may contain one or more double bonds in the ring, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., a 5-20 membered bridged cycloalkyl group). The bridged cycloalkyl group is preferably a bridged cycloalkyl group having 6 to 14 carbon atoms (i.e., a 6-14 membered bridged cycloalkyl group), and more preferably a bridged cycloalkyl group having 7 to 10 carbon atoms (i.e., a 7-10 membered bridged cycloalkyl group). The bridged cycloalkyl group includes a bicyclic bridged cycloalkyl group and a polycyclic bridged cycloalkyl group (e.g., a tricyclic bridged cycloalkyl group, a tetracyclic bridged cycloalkyl group, etc.), and is preferably a bicyclic bridged cycloalkyl group or a tricyclic bridged cycloalkyl group. Non-limiting examples are: [ka] Including, The connection point may be located in any position.

[0128] The cycloalkyl group may be substituted or unsubstituted and, if substituted, it may be substituted at any available attachment point, and the substituents are preferably one or more selected from D atoms, halogens, alkyl groups, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, oxo groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0129] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., a monocyclic heterocyclyl group) or polycyclic heterocyclic ring system (i.e., a polycyclic heterocyclyl group) containing at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur within the ring (wherein the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide, and the sulfur may be optionally substituted with an oxo group, i.e., to form a sulfoxide or sulfone, but does not include -OO-, -OS- or -SS-) and having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 3- to 20-membered heterocyclyl group). The heterocyclyl group is preferably a heterocyclyl group having 3 to 12 ring atoms (i.e., a 3- to 12-membered heterocyclyl group), more preferably a heterocyclyl group having 3 to 8 ring atoms (i.e., a 3- to 8-membered heterocyclyl group), even more preferably a heterocyclyl group having 3 to 6 ring atoms (i.e., a 3- to 6-membered heterocyclyl group), and most preferably a heterocyclyl group having 5 or 6 ring atoms (i.e., a 5- or 6-membered heterocyclyl group).

[0130] The monocyclic heterocyclyl groups include, by way of non-limiting example, pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl groups.

[0131] The polycyclic heterocyclyl groups include spiroheterocyclyl groups, fused heterocyclyl groups and bridged heterocyclyl groups.

[0132] The term "spiroheterocyclyl group" refers to a polycyclic heterocyclic ring system in which the rings share one atom (referred to as a spiro atom), optionally containing one or more double bonds within the ring, and containing at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur within the ring (wherein the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide, and the sulfur may be optionally substituted with an oxo group, i.e., to form a sulfoxide or sulfone, but does not include -OO-, -OS- or -SS-), provided that at least one monocyclic heterocyclyl group is included and the point of attachment is at the monocyclic heterocyclyl group having 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5-20 membered spiroheterocyclyl group). The above spiroheterocyclyl group is preferably a spiroheterocyclyl group having 6 to 14 ring atoms (ie, a 6- to 14-membered spiroheterocyclyl group), and more preferably a spiroheterocyclyl group having 7 to 10 ring atoms (ie, a 7- to 10-membered spiroheterocyclyl group). The spiroheterocyclyl group includes monospiroheterocyclyl groups and polyspiroheterocyclyl groups (such as bisspiroheterocyclyl groups), preferably monospiroheterocyclyl groups or bisspiroheterocyclyl groups, more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered or 7-membered / 6-membered monospiroheterocyclyl groups. Non-limiting examples are: [ka] etc.

[0133] The term "fused heterocyclyl group" refers to a polycyclic heterocyclic ring system in which the rings share two adjacent atoms, optionally containing one or more double bonds within the ring, and containing at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur within the ring, wherein the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide, and the sulfur may be optionally substituted with an oxo group, i.e., to form a sulfoxide or sulfone, but not limited to -OO-, -OS-, or -SS-. -), which is a monocyclic heterocyclyl group fused with one or more monocyclic heterocyclyl groups, or a monocyclic heterocyclyl group fused with one or more cycloalkyl groups, aryl groups, or heteroaryl groups, where the linking point is on the monocyclic heterocyclyl group and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5-20 membered fused heterocyclyl group). The above fused heterocyclyl group is preferably a fused heterocyclyl group having 6 to 14 ring atoms (i.e., a 6-14 membered fused heterocyclyl group), and more preferably a fused heterocyclyl group having 7 to 10 ring atoms (i.e., a 7-10 membered fused heterocyclyl group). The fused heterocyclyl groups include bicyclic and polycyclic fused heterocyclyl groups (e.g., tricyclic fused heterocyclyl groups, tetracyclic fused heterocyclyl groups, etc.), preferably bicyclic fused heterocyclyl groups or tricyclic fused heterocyclyl groups, more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered or 7-membered / 6-membered bicyclic fused heterocyclyl groups. Non-limiting examples are: [ka] etc.

[0134] The term "bridged heterocyclyl group" refers to a polycyclic heterocyclic ring system in which the rings share two atoms that are not directly linked, which may contain one or more double bonds within the ring, and which contains at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur within the ring (which nitrogen may optionally be oxidized, i.e., to form a nitrogen oxide, and which sulfur may optionally be substituted with an oxo group, i.e., to form a sulfoxide or sulfone, but does not include -OO-, -OS- or -SS-), which has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5- to 20-membered bridged heterocyclyl group). The bridged heterocyclyl group is preferably a bridged heterocyclyl group having 6 to 14 ring atoms (i.e., a 6- to 14-membered bridged heterocyclyl group), and more preferably a bridged heterocyclyl group having 7 to 10 ring atoms (i.e., a 7- to 10-membered bridged heterocyclyl group). Depending on the number of rings constituting the heterocyclyl group, it can be divided into a bicyclic bridged heterocyclyl group and a polycyclic bridged heterocyclyl group (e.g., a tricyclic bridged heterocyclyl group, a tetracyclic bridged heterocyclyl group, etc.), and is preferably a bicyclic bridged heterocyclyl group or a tricyclic bridged heterocyclyl group. Non-limiting examples are: [ka] etc.

[0135] The heterocyclyl group may be substituted or unsubstituted and, when substituted, it may be substituted at any available attachment point, and the substituents are preferably one or more selected from D atoms, halogens, alkyl groups, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, oxo groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0136] The term "aryl group" refers to a monocyclic all-carbon aromatic ring (i.e., a monocyclic aryl group) or a polycyclic aromatic ring system (i.e., a polycyclic aryl group) having a conjugated π-electron system and having 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 6-14-membered aryl group). The aryl group is preferably an aryl group having 6 to 10 ring atoms (i.e., a 6-10-membered aryl group). The monocyclic aryl group is, for example, a phenyl group. The polycyclic aryl group includes, as non-limiting examples, a naphthyl group, an anthryl group, a phenanthryl group, and the like. The polycyclic aryl groups further include those in which a phenyl group is fused to one or more of a heterocyclyl group or a cycloalkyl group, or a naphthyl group is fused to one or more of a heterocyclyl group or a cycloalkyl group, where the point of attachment is at the phenyl or naphthyl group, and in this case the number of ring atoms still refers to the number of ring atoms in the polycyclic aromatic ring system, non-limiting examples include: [ka] etc.

[0137] The aryl group may be substituted or unsubstituted and, when substituted, it may be substituted at any available attachment point, and the substituents are preferably one or more selected from D atoms, halogens, alkyl groups, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, oxo groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0138] The term "heteroaryl group" refers to a monocyclic heteroaromatic ring (i.e., a monocyclic heteroaryl group) or a polycyclic heteroaromatic ring system (i.e., a polycyclic heteroaryl group) having a conjugated π-electron system and containing at least one (e.g., 1, 2, 3, or 4) heteroatom selected from nitrogen, oxygen, and sulfur within the ring (which nitrogen may optionally be oxidized, i.e., to form a nitrogen oxide, and which sulfur may optionally be substituted with an oxo group, i.e., to form a sulfoxide or sulfone, but does not include -OO-, -OS-, or -SS-) and which has 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 5-14 membered heteroaryl group). The heteroaryl group is preferably a heteroaryl group having 5 to 10 ring atoms (ie, a 5- to 10-membered heteroaryl group), and more preferably a heteroaryl group having 5 or 6 ring atoms (ie, a 5- or 6-membered heteroaryl group).

[0139] Non-limiting examples of the monocyclic heteroaryl group include a furanyl group, a thienyl group, a thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, an oxadiazolyl group, a thiadiazolyl group, an imidazolyl group, a pyrazolyl group, a triazolyl group, a tetrazolyl group, a furazanyl group, a pyrrolyl group, an N-alkylpyrrolyl group, a pyridyl group, a pyrimidinyl group, a pyridonyl group, an N-alkylpyridone group (e.g., [ka] etc.), pyrazinyl group, pyridazinyl group, etc.

[0140] The polycyclic heteroaryl groups include, as non-limiting examples, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalyl, phthalazinyl, benzimidazolyl, benzothienyl, quinazolinyl, benzothiazolyl, carbazolyl, etc. The polycyclic heteroaryl groups further include monocyclic heteroaryl groups fused to one or more aryl groups, where the connection point is on the aromatic ring, and in this case the number of ring atoms still indicates the number of ring atoms in the polycyclic heteroaromatic ring system. The polycyclic heteroaryl groups further include monocyclic heteroaryl groups fused to one or more cycloalkyl or heterocyclyl groups, where the connection point is on the monocyclic heteroaromatic ring, and in this case the number of ring atoms still indicates the number of ring atoms in the polycyclic heteroaromatic ring system. Non-limiting examples are: [ka] etc.

[0141] The heteroaryl group may be substituted or unsubstituted and, if substituted, it may be substituted at any available point of attachment and the substituents are preferably one or more selected from D atoms, halogens, alkyl groups, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxy groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0142] The term "amino-protecting group" refers to a group that is easily removed and introduced to an amino group so that the amino group is not altered when other sites on the molecule are reacted. Non-limiting examples include (trimethylsilyl)ethoxymethyl, tetrahydropyranyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl, ethoxycarbonyl, phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), trityl (Trt), 2,4-dimethoxybenzyl (DMB), acetyl, benzyl, allyl, p-methoxybenzyl, and the like.

[0143] The term "hydroxy protecting group" refers to a labile group that is introduced into a hydroxy group to react with another functional group of a compound in order to block or protect the hydroxy group. Non-limiting examples include trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, p-nitrobenzoyl, and the like.

[0144] The term "cycloalkyloxy" refers to a cycloalkyl-O- group, where the cycloalkyl group is as defined above.

[0145] The term "heterocyclyloxy group" refers to a heterocyclyl-O- group, where the heterocyclyl group is as defined above.

[0146] The term "aryloxy group" refers to an aryl-O- group, where the aryl group is as defined above.

[0147] The term "heteroaryloxy group" refers to a heteroaryl-O- group, where the heteroaryl group is as defined above.

[0148] The term "alkylthio group" refers to an alkyl-S- group, in which the alkyl group is as defined above.

[0149] The term "haloalkyl group" refers to an alkyl group substituted with one or more halogens, where the alkyl group is as defined above.

[0150] The term "haloalkoxy" refers to an alkoxy group substituted with one or more halogens, where alkoxy is as defined above.

[0151] The term "deuterated alkyl group" refers to an alkyl group substituted with one or more deuterium atoms, where the alkyl group is as defined above.

[0152] The term "hydroxyalkyl group" refers to an alkyl group substituted with one or more hydroxy groups, where alkyl is as defined above.

[0153] The term "methylene group" refers to =CH2.

[0154] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0155] The term "hydroxy group" refers to --OH.

[0156] The term "mercapto" refers to -SH.

[0157] The term "amino group" refers to -NH2.

[0158] The term "cyano" refers to -CN.

[0159] The term "nitro group" refers to --NO.sub.2.

[0160] The term "oxo" or "oxo group" refers to "=O".

[0161] The term "carbonyl group" refers to C=O.

[0162] The term "carboxy" refers to -C(O)OH.

[0163] The term "carboxylic acid ester group" refers to a -C(O)O(alkyl), -C(O)O(cycloalkyl), (alkyl)C(O)O-, or (cycloalkyl)C(O)O-, where the alkyl and cycloalkyl groups are defined above.

[0164] The abbreviation "Me" in the chemical formula is the methyl group.

[0165] The abbreviation "Ph" in the chemical formula is the phenyl group.

[0166] The term "THF" refers to tetrahydrofuran.

[0167] The term "EtOAc" refers to ethyl acetate.

[0168] The term "MeOH" refers to methanol.

[0169] The term "DMF" refers to N,N-dimethylformamide.

[0170] The term "DIPEA" refers to diisopropylethylamine.

[0171] The term "TFA" refers to trifluoroacetic acid.

[0172] The term "MeCN" refers to acetonitrile.

[0173] The term "DMA" refers to N,N-dimethylacetamide.

[0174] The term "Et2O" refers to diethyl ether.

[0175] The term "DCE" refers to 1,2-dichloroethane.

[0176] The term "DIPEA" refers to N,N-diisopropylethylamine.

[0177] The term "NBS" refers to N-bromosuccinimide.

[0178] The term "NIS" means N-iodosuccinimide.

[0179] The term "Cbz-Cl" refers to benzyl chloroformate.

[0180] The term "Pd2(dba)3" refers to tris(dibenzylideneacetone)dipalladium.

[0181] The term "Dppf" refers to 1,1'-bisdiphenylphosphinoferrocene.

[0182] The term "HATU" refers to 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0183] The term "KHMDS" refers to potassium hexamethyldisilazide.

[0184] The term "LiHMDS" refers to lithium bis(trimethylsilyl)amide.

[0185] The term "MeLi" refers to lithium methide.

[0186] The term "n-BuLi" refers to n-butyllithium.

[0187] The term "NaBH(OAc)3" refers to sodium triacetoxyborohydride.

[0188] The term "DCM" refers to dichloromethane.

[0189] The term "DMAP" refers to 4-dimethylaminopyridine.

[0190] The term "DMBOH" refers to 2,4-dimethoxybenzyl alcohol.

[0191] The term "EDCI" refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0192] The term "MTBE" is methyl tert-butyl ether.

[0193] The term "DMF" refers to N,N-dimethylformamide.

[0194] The term "DMTMM" refers to 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride.

[0195] The term "EtOAc" refers to ethyl acetate.

[0196] The compounds of the present disclosure may have specific stereoisomeric forms. The term "stereoisomer" refers to isomers that have the same structure but differ in the spatial arrangement of atoms. It includes cis and trans (or Z and E) isomers, (-)- and (+)-isomers, (R)- and (S)-enantiomers, diastereomers, (D)- and (L)-isomers, tautomers, atropisomers, conformers, and mixtures thereof (e.g., racemates, mixtures of diastereomers). Substituents in the compounds of the present disclosure may have other asymmetric atoms. All such stereoisomers and mixtures thereof are within the scope of the present disclosure. Optically active (-)- and (+)-isomers, (R)- and (S)-enantiomers, and (D)- and (L)-isomers can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. Single isomers of certain compounds of the present disclosure can be prepared by asymmetric synthesis or chiral auxiliaries, or, if the molecule contains a basic functional group (e.g., an amino group) or an acidic functional group (e.g., a carboxy group), by forming a diastereomeric salt with an appropriate optically active acid or base, and then performing diastereomeric resolution by conventional methods known in the art to obtain the pure isomers. Furthermore, separation of enantiomers and diastereomers is typically accomplished by chromatography.

[0197] In the chemical structures of the compounds described in this disclosure, [ka] indicates that the configuration is not specified, i.e., if chiral isomers are present in the chemical structure, [ka] The bond [ka] or [ka] or [ka] and [ka] The above two types of arrangements may be included at the same time.

[0198] The compounds of the present disclosure may exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to a structural isomer that exists in equilibrium and in which the isomeric form is easily converted from one to the other. It includes all possible tautomers, i.e., exists in the form of a single isomer or in the form of a mixture of the above tautomers in any ratio. Non-limiting examples include keto-enol, imine-enamine, lactam-lactim, etc. The lactam-lactim equilibrium is as follows: [ka]

[0199] For example, reference to a pyrazolyl group should be understood to include any one or a mixture of the two tautomers of the following two structures: [ka]

[0200] All tautomeric forms are within the scope of the disclosure, and the naming of a compound does not exclude any tautomeric form.

[0201] The compounds of the present disclosure include all suitable isotopic derivatives of the compounds. The term "isotopic derivative" refers to a compound in which at least one atom is replaced with an atom having the same atomic number but a different atomic mass. Examples of isotopes that can be introduced into the compounds of the present disclosure include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, for example, 2 H (deuterium, D), 3 H (tritium, T),11 C. 13 C. 14 C. 15 N, 17 O. 18 O. 32 p, 33 p, 33 S, 34 S, 35 S, 36 S, 18 F, 36 Cl, 82 Br, 123 I, 124 I, 125 I, 129 I and 131 I, with deuterium being preferred.

[0202] Compared with non-deuterated drugs, deuterated drugs have the advantages of reducing toxicity and side effects, increasing drug stability, improving therapeutic efficacy, and extending the biological half-life of drugs. All isotopic variations of the compounds according to the present disclosure, whether radioactive or not, are included within the scope of the present disclosure. Each available hydrogen atom connected to a carbon atom can be independently replaced with a deuterium atom, where the replacement of deuterium can be partial or complete, and partial deuterium replacement means that at least one hydrogen is replaced with at least one deuterium.

[0203] When a position is specifically designated as "deuterium" or "D," it should be understood that the position has an abundance of deuterium at least 1000 times greater than the natural abundance of deuterium (i.e., at least 15% deuterium has been incorporated), which is 0.015%. In some embodiments, each designated deuterium atom has an abundance of deuterium at least 1000 times greater than the natural abundance of deuterium (i.e., at least 15% deuterium has been incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 2000 times greater than the natural abundance of deuterium (i.e., at least 30% deuterium has been incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 3000 times greater than the natural abundance of deuterium (i.e., at least 45% deuterium has been incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 3340 times greater than the natural abundance of deuterium (i.e., at least 50.1% deuterium incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 3500 times greater than the natural abundance of deuterium (i.e., at least 52.5% deuterium incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 4000 times greater than the natural abundance of deuterium (i.e., at least 60% deuterium incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 4500 times greater than the natural abundance of deuterium (i.e., at least 67.5% deuterium incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 5000 times greater than the natural abundance of deuterium (i.e., at least 75% deuterium incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 5500 times greater than the natural abundance of deuterium (i.e., at least 82.5% deuterium is incorporated), hi some embodiments, each designated deuterium atom has an abundance of deuterium at least 6000 times greater than the natural abundance of deuterium (i.e., at least 90% deuterium is incorporated).In some embodiments, each designated deuterium atom has an abundance of deuterium at least 6333.3 times greater than the natural abundance of deuterium (i.e., at least 95% deuterium is incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 6466.7 times greater than the natural abundance of deuterium (i.e., at least 97% deuterium is incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 6600 times greater than the natural abundance of deuterium (i.e., at least 99% deuterium is incorporated). In some embodiments, each designated deuterium atom has an abundance of deuterium at least 6633.3 times greater than the natural abundance of deuterium (i.e., at least 99.5% deuterium is incorporated).

[0204] "Substituted" or "substituted" refers to the replacement of one or more hydrogen atoms, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms in a group, with a corresponding number of substituents, independently of one another. A person skilled in the art can determine (experimentally or theoretically) possible or impossible substitutions without much effort. For example, an amino group or a hydroxy group having free hydrogen may be unstable when bonded to a carbon atom having an unsaturated bond (e.g., an olefin).

[0205] The present disclosure further includes various deuterated forms of the antibody-drug conjugate of formula (Pc-L-Da) or (Pc-LD). Each available hydrogen atom connected to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize the deuterated forms of the antibody-drug conjugate of formula (Pc-L-Da) or (Pc-LD) by referring to relevant literature. When preparing the deuterated forms of the antibody-drug conjugate of formula (Pc-L-Da) or (Pc-LD), commercially available deuterated starting materials may be used, or the antibody-drug conjugate of formula (Pc-L-Da) or (Pc-LD) may be synthesized by conventional techniques with deuterated reagents, including but not limited to deuterated borane, tritium borane tetrahydrofuran solution, lithium aluminum deuteride, deuterated iodoethane, deuterated iodomethane, etc.

[0206] "Optionally" or "optionally" means that the event or circumstance described next may or may not occur, and includes both cases where the event or circumstance occurs and where it does not occur. For example, "an alkyl group optionally (arbitrarily) substituted with a halogen or a cyano group" includes cases where the alkyl group is substituted with a halogen or a cyano group and cases where the alkyl group is not substituted with a halogen or a cyano group.

[0207] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or their physiologically / pharmaceutical acceptable salts or prodrugs with other chemical components, and other components such as physiologically / pharmaceutical acceptable vectors and excipients. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredients to further exert biological activity.

[0208] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable solvents and vehicles may include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin and processed to form a microemulsion. The injectable solution or microemulsion may be injected into the subject's bloodstream by localized bolus injection. Alternatively, the solution and microemulsion may be administered by a method capable of maintaining a constant cyclic concentration of the compounds of the present disclosure. A continuous intravenous administration device may be used to maintain such a constant concentration. An example of such a device is the Deltec CADD-PLUS.TM. 5400 type intravenous pump.

[0209] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oily suspension for use in intramuscular and subcutaneous administration. Such suspensions may be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents as described above. The sterile injectable preparation may be a sterile injectable solution or suspension prepared in a non-toxic, gastrointestinal-acceptable diluent or solvent, for example, a solution prepared in 1,3-butanediol. Sterile fixed oils are also conveniently used as a solvent or suspending medium. For this purpose, any suitable fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acids, may also be used to prepare injectables.

[0210] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of an antibody or antibody-drug conjugate according to the present disclosure, which has safety and efficacy when used in a subject, and has the desired biological activity. As an example, an antibody or antibody-drug conjugate according to the present disclosure contains at least one amino group, and therefore can form a salt with an acid, and non-limiting examples of medicinal salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0211] The term "pharmaceutically acceptable vector" refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to a subject. Pharmaceutically acceptable vectors include, but are not limited to, buffers, stabilizers, or preservatives.

[0212] The term "excipient" refers to an additive other than an active ingredient in a pharmaceutical formulation, and may be called an additive. For example, any of the following may be called an excipient: binders, fillers, disintegrants, and lubricants in tablets, base parts in ointments and creams that are semisolid preparations, preservatives, antioxidants, flavorings, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and coloring agents in liquid preparations.

[0213] The term "diluent" is also called filler, and its main use is to increase the weight and volume of tablets. The addition of diluent not only ensures a certain volume size, but also reduces the dose deviation of the main ingredient, improves the compression moldability of the drug, etc. When the tablet drug contains oily ingredients, it is necessary to add an absorbent to absorb the oily substance in order to maintain a "dry" state and contribute to the preparation into tablets. For example, starch, lactose, inorganic salts of calcium, microcrystalline cellulose, etc.

[0214] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, birds, amphibians, and reptiles. Unless otherwise indicated, the terms "patient" and "subject" may be used interchangeably herein. As used herein, the term "cyno" or "cynomolgus" refers to cynomolgus monkeys (Macaca fascicularis). In some embodiments, the individual or subject is a human.

[0215] "Administration" or "giving," as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid.

[0216] The term "sample" refers to a collection of fluids, cells or tissues isolated from a subject, as well as fluids, cells or tissues within a subject. Exemplary samples are biological fluids, such as blood, serum and serous fluid, plasma, lymphatic fluid, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavities fluids, fluids collected from bronchial washings, synovial fluid, liquid solutions that have come into contact with a subject or a biological source, such as cell and organ media (including cell or organ conditioned media), lavage fluids, tissue biopsies, fine needle aspirates, surgically excised tissues, organ cultures or cell cultures.

[0217] "Treatment" and "treatment" (and grammatical variations thereof) refer to a clinical intervention that seeks to alter the pathological process of the individual being treated, and can be performed prophylactically or during the clinical pathological process. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction / reduction of any direct or indirect pathological consequence of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of the disease state, and regression or improvement of prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the formation of disease or to slow the progression of disease.

[0218] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or underlying causes, prevent the appearance of symptoms and / or their underlying causes, and / or improve or ameliorate damage caused by or associated with a disease state. In some embodiments, the effective amount is a therapeutically or prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a condition or symptom associated with the disease state, or to prevent, inhibit, delay or reverse the progression of the disease state or any other undesirable symptoms associated with the disease in other ways. A "prophylactically effective amount" is an amount that, when administered to a subject, provides a desired prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete therapeutic or prophylactic effect does not necessarily occur when one dose is administered, but may occur after a series of doses are administered. Thus, a therapeutically or prophylactically effective amount can be administered in a single or multiple dose regime. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on a variety of factors, such as, for example, the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indications of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

[0219] II. Description of Specific Embodiments A. Exemplary Anti-DLL3 Antibodies In one embodiment, the disclosure provides an antibody that binds to DLL3. In one embodiment, the disclosure provides an isolated antibody that binds to DLL3. In one embodiment, the disclosure provides an antibody that specifically binds to DLL3.

[0220] In certain embodiments, the anti-DLL3 antibody has at least one of the following characteristics: a) the anti-DLL3 antibody has a KD value for binding to human DLL3 or an epitope thereof of ≦3 nM, ≦2 nM, ≦1 nM, ≦0.9 nM, ≦0.8 nM, ≦0.7 nM, ≦0.6 nM, ≦0.5 nM, or ≦0.4 nM, and the KD value is measured by Biacore; b) the anti-DLL3 antibody has an EC50≦3 nM, EC50≦2 nM, EC50≦1 nM, EC50≦0.5 nM, EC50≦0.2 nM, EC50≦0.1 nM, EC50≦0.09 nM, EC50≦0.08 nM, EC50≦0.07 nM, EC50≦0.06 nM for binding to H1184 cells expressing DLL3, wherein the EC50 is detected by FACS; c) the anti-DLL3 antibody is capable of being endocytosed by cells expressing DLL3; d) the anti-DLL3 antibody has an EC50 of ≦0.1 nM (≦0.09 nM, ≦0.08 nM, ≦0.07 nM, ≦0.06 nM, ≦0.05 nM, ≦0.04 nM) for binding to DLL3 or an epitope thereof, wherein the EC50 is measured by ELISA; and e) The above anti-DLL3 antibody and the positive antibody (e.g., BI-764532) recognize different DLL3 epitopes.

[0221] In one embodiment, the present disclosure provides an anti-DLL3 antibody, including those selected from the group consisting of: (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 57; (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25; (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26; and (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, wherein SEQ ID NO: 57 is represented by PLYX1YGRSYNX2VAY, wherein X1 is Y or H and X2 is A or G; or (i) HCDR1 having the amino acid sequence of SEQ ID NO: 16, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 17, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 18, (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 19, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 20, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 21.

[0222] In one embodiment, the present disclosure provides an anti-DLL3 antibody, including those selected from the group consisting of: (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, or (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 30, (iv) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, (v) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and (vi) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27, or (i) HCDR1 having the amino acid sequence of SEQ ID NO: 22, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 23, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 31, (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 25, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 26, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 27.

[0223] In one aspect, the disclosure provides an anti-DLL3 antibody, which comprises HCDRs in the heavy chain variable region of SEQ ID NO: 14 and LCDRs in the light chain variable region of SEQ ID NO: 15; or It comprises the HCDRs of the heavy chain variable region of SEQ ID NO: 12 and the LCDRs of the light chain variable region of SEQ ID NO: 13; or It comprises the HCDRs of the heavy chain variable region of SEQ ID NO: 52 and the LCDRs of the light chain variable region of SEQ ID NO: 15; or It comprises the HCDRs of the heavy chain variable region of SEQ ID NO:53 and the LCDRs of the light chain variable region of SEQ ID NO:15.

[0224] In some embodiments, the CDRs sequences are obtained according to numbering methods known in the art, including but not limited to Kabat, Chothia, IMGT, AbM and Contact.

[0225] In one aspect, the disclosure provides an anti-DLL3 antibody, HCDR1, the sequence of which is shown in SEQ ID NO: 22 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 22; HCDR2, the sequence of which is shown in SEQ ID NO: 23 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 23; an HCDR3 whose sequence is shown in SEQ ID NO: 24 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 24; LCDR1, the sequence of which is shown in SEQ ID NO: 25 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 25; LCDR2, the sequence of which is shown in SEQ ID NO: 26 or which contains 3, 2 or 1 amino acid mutations compared to the given SEQ ID NO: 26; LCDR3, the sequence of which is shown in SEQ ID NO: 27 or which contains 3, 2 or 1 amino acid mutations compared to the given SEQ ID NO: 27; Includes.

[0226] In one aspect, the disclosure provides an anti-DLL3 antibody, HCDR1, the sequence of which is shown in SEQ ID NO: 22 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 22; HCDR2, the sequence of which is shown in SEQ ID NO: 23 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 23; an HCDR3 whose sequence is shown in SEQ ID NO: 30 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 30; LCDR1, the sequence of which is shown in SEQ ID NO: 25 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 25; LCDR2, the sequence of which is shown in SEQ ID NO: 26 or which contains 3, 2 or 1 amino acid mutations compared to the given SEQ ID NO: 26; LCDR3, the sequence of which is shown in SEQ ID NO: 27 or which contains 3, 2 or 1 amino acid mutations compared to the given SEQ ID NO: 27; Includes.

[0227] In one aspect, the disclosure provides an anti-DLL3 antibody, HCDR1, the sequence of which is shown in SEQ ID NO: 22 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 22; HCDR2, the sequence of which is shown in SEQ ID NO: 23 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 23; an HCDR3 whose sequence is shown in SEQ ID NO: 31 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 31; LCDR1, the sequence of which is shown in SEQ ID NO: 25 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 25; LCDR2, the sequence of which is shown in SEQ ID NO: 26 or which contains 3, 2 or 1 amino acid mutations compared to the given SEQ ID NO: 26; LCDR3, the sequence of which is shown in SEQ ID NO: 27 or which contains 3, 2 or 1 amino acid mutations compared to the given SEQ ID NO: 27; Includes.

[0228] In one aspect, the disclosure provides an anti-DLL3 antibody, HCDR1, the sequence of which is shown in SEQ ID NO: 16 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 16; HCDR2, the sequence of which is shown in SEQ ID NO: 17 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 17; an HCDR3 whose sequence is shown in SEQ ID NO: 18 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 18; LCDR1, the sequence of which is shown in SEQ ID NO: 19 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 19; LCDR2, the sequence of which is shown in SEQ ID NO: 20 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 20; LCDR3, the sequence of which is shown in SEQ ID NO: 21 or which contains 3, 2 or 1 amino acid mutations compared to SEQ ID NO: 21; Includes.

[0229] In one aspect, the disclosure provides an anti-DLL3 antibody, which comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively; and The light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively.

[0230] In one aspect, the disclosure provides an anti-DLL3 antibody, which comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:30, respectively; and The light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively.

[0231] In one aspect, the disclosure provides an anti-DLL3 antibody, which comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:31, respectively; and The light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively.

[0232] In one aspect, the disclosure provides an anti-DLL3 antibody, which comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; and The light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively.

[0233] In another aspect, the heavy chain variable region of the anti-DLL3 antibody comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences selected from SEQ ID NOs: 14, 50, 51, 52, 53, or 54, respectively. In some embodiments, there are substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-DLL3 antibody comprising the sequence retains the ability to bind to DLL3. In some embodiments, the substitutions, insertions, or deletions are made in a region other than the CDRs (i.e., in the FRs).

[0234] In another aspect, the light chain variable region of the anti-DLL3 antibody comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences selected from SEQ ID NOs: 15, 55, or 56, respectively. In some embodiments, there are substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-DLL3 antibody comprising the sequence retains the ability to bind to DLL3. In some embodiments, the substitutions, insertions, or deletions are made in a region other than the CDRs (i.e., in the FRs).

[0235] In another aspect, the heavy chain variable region of the anti-DLL3 antibody comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences selected from SEQ ID NOs: 12, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44, respectively. In some embodiments, there are substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-DLL3 antibody comprising the sequence retains the ability to bind to DLL3. In some embodiments, the substitutions, insertions, or deletions are made in a region other than the CDRs (i.e., in the FRs).

[0236] In another aspect, the light chain variable region of the anti-DLL3 antibody comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences selected from SEQ ID NOs: 13, 45, 46, 47, 48, or 49, respectively. In some embodiments, there are substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-DLL3 antibody comprising the sequence retains the ability to bind to DLL3. In some embodiments, the substitutions, insertions, or deletions are made in a region other than the CDRs (i.e., in the FRs).

[0237] In some embodiments, the anti-DLL3 antibody comprises a heavy chain variable region set forth in any one of SEQ ID NOs: 50, 14, 51, 52, 53, or 54, and / or a light chain variable region set forth in any one of SEQ ID NOs: 55, 15, or 56.

[0238] In some embodiments, the anti-DLL3 antibody comprises a heavy chain variable region set forth in any one of SEQ ID NOs: 43, 12, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 44, and / or a light chain variable region set forth in any one of SEQ ID NOs: 48, 13, 45, 46, 47, or 49.

[0239] In some embodiments, the anti-DLL3 antibody comprises a heavy chain variable region set forth in SEQ ID NO:14 and / or a light chain variable region set forth in any one of SEQ ID NOs:15.

[0240] In some embodiments, the anti-DLL3 antibody comprises a heavy chain variable region selected from any one of SEQ ID NOs: 50, 51, 52, 53, and 54, and / or a light chain variable region selected from any one of SEQ ID NOs: 55 or 56.

[0241] In some embodiments, the anti-DLL3 antibody comprises a heavy chain variable region set forth in SEQ ID NO:12 and / or a light chain variable region set forth in SEQ ID NO:13.

[0242] In some embodiments, the anti-DLL3 antibody comprises a heavy chain variable region selected from any one of SEQ ID NOs: 43, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 44, and / or a light chain variable region selected from any one of SEQ ID NOs: 48, 45, 46, 47, and 49.

[0243] In some embodiments, the anti-DLL3 antibody comprises a heavy chain variable region set forth in SEQ ID NO:50 and a light chain variable region set forth in SEQ ID NO:55.

[0244] In some embodiments, the anti-DLL3 antibody comprises a heavy chain variable region set forth in SEQ ID NO:43 and a light chain variable region set forth in SEQ ID NO:48.

[0245] In some embodiments, the anti-DLL3 antibody comprises a heavy chain constant region and a light chain constant region.

[0246] In some embodiments, the anti-DLL3 antibody has a heavy chain constant region sequence set forth in SEQ ID NO:28.

[0247] In some embodiments, the anti-DLL3 antibody has a light chain constant region sequence set forth in SEQ ID NO:29.

[0248] In some embodiments, the anti-DLL3 antibody comprises a heavy chain and a light chain, the heavy chain comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 60, and / or the light chain comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 61; or The heavy chain comprises an amino acid sequence having at least 85% identity to SEQ ID NO:58 and / or the light chain comprises an amino acid sequence having at least 85% identity to SEQ ID NO:59.

[0249] In some embodiments, the anti-DLL3 antibody comprises a heavy chain set forth in SEQ ID NO:60 and a light chain set forth in SEQ ID NO:61.

[0250] In some embodiments, the anti-DLL3 antibody comprises a heavy chain set forth in SEQ ID NO:58 and a light chain set forth in SEQ ID NO:59.

[0251] B. Antibody Structure In certain embodiments, the antibodies provided herein are full-length antibodies.

[0252] In certain embodiments, the antibodies provided herein are antibody fragments.

[0253] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH or F(ab')2 fragment, particularly a Fab fragment. "Fab" is a monovalent fragment consisting of VL, VH, CL and CH1 domains. "Fab fragment" may be produced by dissolving an antibody with papain protease. "Fab'" includes VL, CL, VH and CH1, and further includes a region between the CH1 and CH2 domains, which allows the formation of an interchain disulfide bond between both heavy chains of two Fab' fragments to form a F(ab')2 molecule. "Fab'-SH" is a Fab' fragment in which a cysteine ​​residue in the constant region has a free sulfhydryl group. "F(ab')2" is a bivalent fragment including two Fab fragments linked by a disulfide bond at the hinge region.

[0254] In another embodiment, the antibody fragment is a diabody, a triabody or a tetrabody. A diabody is an antibody fragment with two antigen binding sites, the fragment comprising a VH and a VL linked on the same polypeptide chain (VH-VL). Using a linker that is too short prevents pairing of the two domains on the same chain, and these domains are forced to pair with complementary domains on another chain, producing two antigen binding sites, where the two antigens can be the same or different.

[0255] In another embodiment, the antibody fragment is a single-chain Fab fragment. A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of VH, CH1, VL, CL and a linker, in which the domains and the linker are arranged in one of the following orders from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In one embodiment, the linker is a polypeptide having at least 30 amino acids. In another embodiment, the linker is a polypeptide having 32 to 50 amino acids. The single-chain Fab fragment is stabilized by a natural disulfide bond between CL and CH1. Additionally, these single-chain Fab molecules can be further stabilized by the insertion of cysteine ​​residues (e.g., position 44 in the heavy chain variable region and position 100 in the light chain variable region, Kabat numbering) to generate interchain disulfide bonds.

[0256] In another embodiment, the antibody fragment is a single chain variable fragment (scFv). An "scFv" is a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, in which the light chain variable region and the heavy chain variable region are linked contiguously by a short flexible peptide linker and can be expressed as a single chain polypeptide, and in which the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise indicated herein, an scFv may have the VL and VH variable regions in any one order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, the scFv may comprise VL-linker-VH or VH-linker-VL.

[0257] In another embodiment, the antibody fragment is an Fd fragment consisting of the VH and CH1 domains.

[0258] In another embodiment, the antibody fragment is an Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0259] In another embodiment, the antibody fragment is a dsFv, which is obtained by linking polypeptides in which one amino acid residue in each of VH and VL is replaced with a cysteine ​​residue via a disulfide bond between the cysteine ​​residues. The amino acid residue to be replaced with a cysteine ​​residue can be selected based on the three-dimensional structure prediction of an antibody by a known method (Protein Engineering. 7:697 (1994)).

[0260] In another embodiment, the antibody fragment is a single domain antibody, which is an antibody fragment that comprises all or part of the heavy chain variable domain, or all or part of the light chain variable domain of an antibody.

[0261] In another embodiment, the antibody fragment is a domain antibody (dAb), see, e.g., U.S. Patent No. 6,248,516. A domain antibody (dAb) is a functional binding domain of an antibody that corresponds to the variable region of the heavy (VH) or light (VL) chain of a human antibody. dABs have a molecular weight of approximately 13 kDa, or less than one-tenth the size of a complete antibody. dABs are well expressed in a variety of hosts, including bacteria, yeast, and mammalian cell systems. Furthermore, dAbs are highly stable and active, even under harsh conditions such as, for example, lyophilization and heat denaturation. See, for example, U.S. Patents 6,291,158, 6,582,915, 6,593,081, 6,172,197, U.S. Series No. 2004 / 0110941, European Patent 0368684, U.S. Patent 6,696,245, WO04 / 058821, WO04 / 003019 and WO03 / 002609.

[0262] In some embodiments, the antibodies provided herein are chimeric antibodies. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody.

[0263] In certain embodiments, the antibody is a humanized antibody. Humanization of a non-human antibody typically reduces its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable regions in which the CDRs or portions thereof are derived from a non-human antibody and the FRs or portions thereof are derived from a human antibody. Optionally, the humanized antibody may also comprise a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be replaced with corresponding residues from a non-human antibody (e.g., the antibody providing the CDR sequences).

[0264] Humanized antibodies and methods for their production are reviewed, for example, by Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (describing grafting of specificity determining regions (SDRs)), Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfuacing"), Dall' Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the "guided selection" method of FR shuffling).

[0265] Human framework regions that can be used for humanization include framework regions selected by the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)), framework regions of consensus sequences of human antibodies derived from specific subclasses of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al., J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0266] C. Antibody Modifications In some embodiments, amino acid sequence variants of the antibodies provided herein are provided. For example, it is desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution can be made to obtain the final construct, provided that the final construct has the desired properties, such as antigen binding properties.

[0267] a) Substitution, insertion and deletion mutations In one embodiment, antigen variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". More substantial changes are provided in Table 2 under the heading of "exemplary substitutions" and as further described below with reference to amino acid side chain classifications. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0268] [Table 2] Amino acids can be grouped according to common side chain properties as follows: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0269] Non-conservative substitutions involve replacing a member in one of these classes with a member in another class.

[0270] In certain embodiments, substitutions, insertions or deletions may be made in one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) can be made to the CDRs that do not substantially reduce binding affinity. Such changes may, for example, be outside of the antigen contact residues in the CDR. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two or three amino acid substitutions.

[0271] A method that can be used to identify residues or regions that can be target sites for mutagenesis in an antibody is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to see if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Also, contact points between the antibody and the antigen can be identified by examining crystal structures of antigen-antibody complexes. These contact and adjacent residues can be targeted or removed as substitution candidates. Mutants can be screened to see if they contain the desired properties.

[0272] Amino acid sequence insertions include fusions at the amino- and / or carboxyl-terminus of polypeptides ranging in length from one residue to 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody with an enzyme or a polypeptide which extends the serum half-life of the antibody.

[0273] b) Modification of the Fc region In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein.

[0274] In some embodiments, the one or more amino acid modifications can reduce binding of the Fc to an Fc receptor, e.g., its binding to an Fcγ receptor, and reduce or eliminate effector function. In some embodiments, the altered Fc region has a 50%, 80%, 90%, or 95% or greater reduction in binding affinity to an Fc receptor compared to a native Fc region. In some embodiments, the Fc receptor is a human Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIB, FcγRIIIa. In some embodiments, the altered Fc region also has a reduced binding affinity to complement, such as C1q, compared to a native Fc region. In some embodiments, the altered Fc region has a stronger binding affinity to the neonatal Fc receptor (FcRn) compared to a native Fc region, e.g., M252Y / S254T / T256E mutations are introduced into the Fc region. In some embodiments, the modified Fc region has reduced effector functions, which may include, but are not limited to, one or more of: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen-presenting cell antigen uptake, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced cell apoptosis, and reduced dendritic cell maturation or reduced T cell triggering. For an IgG1 Fc region, effector functions can be reduced by amino acid residue substitutions at positions such as 238, 265, 269, 270, 297, 327, and 329. In some embodiments, the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. For the IgG4 Fc region, substitution of amino acid residues at positions such as 228 can reduce effector function.

[0275] In certain embodiments, the antibody comprises one or more amino acid substitutions that improve ADCC, for example substitutions at positions 298, 333 and / or 334 (using the EU numbering system) of the Fc region.

[0276] In certain embodiments, the Fc domain of the antibody herein comprises a "knob-in-hole" mutation. "Knob-in-hole" is a design strategy for engineering antibody heavy chain homodimers to heterodimerize (e.g., to effectively generate bispecific, multispecific, or single-arm antibodies). In general, such techniques involve introducing a protrusion ("knob") at the interface of a first polypeptide (e.g., the first CH3 domain in the first antibody heavy chain) and a cavity ("hole") at a corresponding location in the interface of a second polypeptide (e.g., the second CH3 domain in the second antibody heavy chain) such that the protrusion can be accommodated in the cavity, thereby promoting heterodimer formation and inhibiting homodimer formation. The protrusion is constructed by replacing a relatively small amino acid side chain from the interface of the first polypeptide (e.g., the first CH3 domain in the heavy chain of the first antibody) with a relatively large side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan). A complementary cavity having a similar or similar size to the protrusion is created at the interface of a second polypeptide (e.g., a second CH3 domain in a second antibody heavy chain) by replacing a relatively large amino acid side chain with a relatively small side chain (e.g., alanine, serine, valine, or threonine). The protrusion and cavity may be generated by altering a nucleic acid encoding the polypeptide (e.g., site-directed mutagenesis) or by peptide synthesis. In some embodiments, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, while the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In some embodiments, the subunit of the Fc domain that comprises the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc domain that comprises the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues allows the formation of disulfide bridges between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J. Immunol. Methods 248:7-15(2001)).Exemplary combinations of knob-in-hole mutations include, but are not limited to, those listed in Table 3.

[0277] [Table 3]

[0278] Details of knob-in-hole technology are described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, WO 2009 / 089004, US 2009 / 0182127, Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658, Kontermann, Acta Pharmacologica Sincia (2005) 26:1-9, Ridgway et al., Prot Eng 9:617-621 (1996), and Carter, J Immunol Meth 248:7-15 (2001).

[0279] The C-terminus of the Fc region may be a complete C-terminus terminating in amino acid residue PGK, or may be a truncated C-terminus, e.g., where one or two C-terminal amino acid residues have already been removed. In a preferred embodiment, the C-terminus of the heavy chain is a truncated C-terminus terminating in PG. Thus, in some embodiments, a composition of complete antibodies may comprise antibodies in which any K447 and / or G446+K447 residues have been removed. In some embodiments, a composition of complete antibodies may comprise antibodies in which any K447 and / or G446+K447 residues have not been removed. In some embodiments, a composition of complete antibodies comprises a mixture of antibodies with and without K447 and / or G446+K447 residues.

[0280] D. Recombinant Methods Anti-DLL3 antibodies can be produced by recombinant methods. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0281] In one embodiment, the disclosure provides an isolated nucleic acid encoding such an antibody. Each such nucleic acid can independently encode any one of the polypeptide chains described above. In another aspect, the disclosure provides one or more vectors (e.g., expression vectors) comprising such a nucleic acid. In another aspect, the disclosure provides a host cell comprising such a nucleic acid. In one embodiment, a method of preparing an anti-DLL3 antibody is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression as provided above, and optionally recovering the antibody from the host cell (or host cell medium).

[0282] To produce an antibody recombinantly, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using routine procedures, or produced by recombinant methods, or obtained by chemical synthesis.

[0283] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, they can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. After expression, they can be isolated from bacterial cell paste in a soluble fraction and further purified.

[0284] E. Measurement The polypeptides or fusion proteins provided herein can be identified, screened, or characterized for their physical / chemical characteristics and / or biological activity by various assays known in the art. In one embodiment, the activity of the polypeptides or complexes of the present disclosure is measured by known methods, such as, for example, ELISA, Western blotting, etc.

[0285] F. Treatment Methods and Routes of Administration Any of the above anti-DLL3 antibodies provided herein, or an antibody-drug conjugate comprising the same, or a pharma- ceutically acceptable salt thereof, can be used in the treatment of a disease.

[0286] In one aspect, the present disclosure provides the use of an anti-DLL3 antibody, an antibody-drug conjugate comprising the same, or a pharma- ceutically acceptable salt thereof, in the preparation of a medicament for treating a tumor or cancer, in some embodiments, the present disclosure provides the use of an anti-DLL3 antibody, an antibody-drug conjugate comprising the same, or a pharma- ceutically acceptable salt thereof, in the preparation of a medicament for treating a tumor or cancer. In some embodiments, the tumor or cancer is selected from lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, large cell lung cancer), head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer (e.g., medullary thyroid cancer), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), liver cancer, hepatic and gallbladder cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer (e.g., colon cancer and rectal cancer), renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer, glioblastoma, skin cancer, and melanoma, preferably, the tumor or cancer is small cell lung cancer.

[0287] In some embodiments, the present disclosure provides use of an anti-DLL3 antibody, an antibody-drug conjugate comprising the same, or a pharma- ceutically acceptable salt thereof in the preparation of a medicament for treating a DLL3-associated disease.

[0288] In one such embodiment, the use further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents).

[0289] In a further aspect, a pharmaceutical composition is provided comprising the anti-DLL3 antibody or its drug conjugate, e.g., it is used in any of the pharmaceutical uses or treatment methods described above. In one embodiment, the pharmaceutical composition comprises any of the polypeptides or drug conjugates provided herein and a pharma- ceutically acceptable vector. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0290] The anti-DLL3 antibodies or drug conjugates thereof according to the present disclosure may be used for therapy alone or in combination with other agents, for example, the antibodies according to the present disclosure may be administered with at least one additional therapeutic agent.

[0291] The anti-DLL3 antibody or drug conjugate thereof of the present disclosure can be administered by any suitable means, including parenteral, pulmonary, and intranasal administration, and can be administered intralesionally if localized treatment is required. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses at multiple time points, bolus administration, and pulse infusion.

[0292] The anti-DLL3 antibody or drug conjugate thereof of the present disclosure is formulated, dosed and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific condition being treated, the specific mammal being treated, the clinical situation of the individual patient, the cause of the condition, the site of delivery of the reagent, the method of administration, the administration schedule and other factors known to medical practitioners. The anti-DLL3 antibody or drug conjugate thereof may be formulated with one or more other reagents. The effective amount of such other reagents will depend on the amount present in the pharmaceutical composition, the type of condition or treatment and other factors. They are generally used in the same dosages and routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at another dosage and by any route determined empirically / clinically appropriate.

[0293] The appropriate dosage of an anti-DLL3 antibody or drug conjugate thereof of the present disclosure (whether used alone or in combination with one or more additional therapeutic agents) for preventing or treating a disease will depend on the type of disease being treated, the type of therapeutic molecule, the severity and course of the disease, whether the administration is prophylactic or therapeutic, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is administered to the patient at one time or over a series of treatments, as appropriate.

[0294] G.Product In another aspect of the disclosure, an article of manufacture (e.g., a kit) is provided that contains materials that can be used to treat, prevent, and / or diagnose the above-mentioned conditions. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as, for example, glass or plastic.

[0295] The container holds the anti-DLL3 antibody or drug conjugate thereof of the present disclosure alone or in combination with another composition. The container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper). At least one active agent in the composition is the anti-DLL3 antibody or drug conjugate thereof of the present disclosure. The label or package insert indicates that the composition is used for treating the selected condition.

[0296] Additionally, the article of manufacture may include (a) a first container containing an anti-DLL3 antibody or its drug conjugate, and (b) a second container containing a composition comprising another cytotoxic agent or other therapeutic agent.

[0297] Optionally, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer. From a business and user standpoint, it may further comprise other materials necessary, including other buffers, diluents, filters, needles and syringes.

[0298] Working Example The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present invention.

[0299] Experimental methods for which specific conditions are not specified in the Examples or Test Examples of this disclosure are generally in accordance with common conditions or conditions recommended by the raw material or product manufacturers. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Methods in Molecular Biology, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which specific sources are not specified are conventional commercially available reagents.

[0300] 1. Preparation of antibodies Example 1: Preparation of DLL3 antigen and detection protein and stable transformed cell line The DLL3 genes of different species and human DLL1 and DLL4 genes were transfected into Chinese hamster ovary CHO-s cells (Invitrogen, R80007) to construct CHO-s cell lines expressing DLL3 proteins of different species, which were used for subsequent antibody screening and identification. The amino acid sequences of the relevant proteins are as follows: Human DLL3 full length protein (Uniprot, Q9NYJ7): MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAA GGPwards EVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGAR CEFPVHPDGASALPAAPPGLRPGDPQRYLLPPALGLLVAAGVAGAALLLVHVRRRGHSQDAGSRLLAGTPEPSVHALPDALNNLRTQEGSGDGPSSSVDWNRPEDVDPQGIYVISAPSIYAREVATPLFPPLHTGRAGQRQHLLFPYPSSILSVK SEQ ID NO:1 Cynomolgus DLL3 full length protein (Uniprot, A0A2K5WSR4): MVSPRMSRLLSQTVILALIFIPQARPAGVFELQIHSFGPGPGPGAPRSPCSARGPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPEAPAPDLPLPNGLLQVPFRDAWPGTFSLIIETWREELGDQIGGPAWSLLARVTRRRRLAA GGPwards EVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRNCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGAR CEFPVHPDGVSALPAAPPGLRPGDPQRYLLPPALGLLVAAGVAGAALLLVHVRRRGHAQDAGSRLLAGTPEPSVHALPDALNNLRTQEGPGDVPSSSVDWNRPEDVDSRGIYVISAPSIYAREVAMPLFPPLHTGRAGQRQNLLFFPSSILSVK SEQ ID NO:2 Rat DLL3 full length protein (Uniprot, O88671): MVSLQVSSLPQTLILAFLLPQALPAGVFELQIHSFGPGPGPGTPRSPCNARGPCRLFFRVCLKPGVSQEAAESLCALGAALSTSGPVYTEQPGVPAAALSLPDGLVRVPFLDAWPGTFSLIIETWREQLGERAAGPAWNLLARVAGR RRLAAGAPWARDVQRTGAWELHFSYRARCEPAVGAACARLCRSRSAPSRCGPGLRPCTPFPDECEAPRESLTVCRAGCSPEHGYCEEPDECHCLEGWTGPLCTVPVSTSSCLNSRVSGPAGTGCLLPGPGPCDGNPCANGGSCSET PGSFECACPRGFYGPRCEVSGVTCADGPCFNGGLCVGGEDPDSAYVCHCPPAFQGSNCERRVDRCSLQPCQNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGARRCSCALGFGGRDCRERADPCASRPCA HGGRCYAHFSGLVCACAPGYMGVRCEFAVRPDGADAVPAAPRGLRQADSQRFLLPPAGLLAAAALAGAALLLIHVRRRGPGRDTGTRLLSGTREPSVHTLPDALNNLRLQDGAGDGPTSSADWNHPEDGDSRSIYVIPAPSIYAREA SEQ ID NO:3 Mouse DLL3 full length protein (Uniprot, O88516): MVSLQVSPLSQTLILAFLLPQALPAGVFELQIHSFGPGPGLGTPRSPCNARGPCRLFFRVCLKPGVSQEATESLCALGAALSTSVPVYTEHPGESAAALPLPDGLVRVPFRDAWPGTFSLVIETWREQLGEHAGGPAWNLLARVVGRR RLAAGGPWARDVQRTGTWELHFSYRARCEPPAVGAACARLCRSRSAPSRCGPGLRPCTPFPDECEAPSVCRPGCSPEHGYCEEPDECRCLEGWTGPLCTVPVSTSSCLNSRVPGPASTGCLLPGPGPCDGNPCANGGSCSETSGSFEC ACPRGFYGLRCEVSGVTCADGPCFNGGLCVGGEDPDSAYVCHCPPGFQGSNCEKRVDRCSLQPCQNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGSRRCSCALGFGGRDCRERADPCASRPCAHGGRCYA HFSGLVCACAPGYMGVRCEFAVRPDGADAVPAAPRGLRQADPQRFLLPPALGLLVAAGLAGAALLVIHVRRRGPGQDTGTRLLSGTREPSVHTLPDALNNLRLQDGAGDGPSSSADWNHPEDGDSRSIYVIPAPSIYAREDWLIQVLF SEQ ID NO:4 Human DLL1 full length protein (Uniprot, O00548): MGSRCALALAVLSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCD EHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGK ICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEGQGGP FPWVAVCAGVILVLMLLLGCAAVVVCVRLRLQKHRPPADPCRGETETMNNLANCQREKDISVSIIGATQIKNTNKKADFHGDHSADKNGFKARYPAVDYNLVQDLKGDDTAVRDAHSKRDTKCQPQGSSGEEKGTPTTLRGGEASERKRPDSGCSTSKDTKYQSVYVISEEKDECVIATEV SEQ ID NO:5 Human DLL4 full length protein (Uniprot, Q9NR61): MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSY RVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQ EDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFV GSRCEFPVGLPPSFPWVAVSLGVGLAVLLVLLGMVAVAVRQLRLRRPDDGSREAMNNLSDFQKDNLIPAAQLKNTNQKKELEVDCGLDKSNCGKQQNHTLDYNLAPGPLGRGTMPGKFPHSDKSLGEKAPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNECVIATEV Sequence number 6.

[0301] 1.1 Construction of cell lines highly expressing DLL3, DLL1, and DLL4 pCDH lentivirus expression vector plasmids (synthesized by GENEWIZ) containing SEQ ID NO: 1 to 6, and pCDH plasmids were transfected into 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17) with pVSVG and pCMV lentivirus packaging vectors, respectively, using Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent, and the culture supernatant containing the virus was collected, filtered, centrifuged at ultra-high speed, and the supernatant was discarded and resuspended in 0.2 mL of sterile PBS. The concentrated viruses were infected into Chinese hamster ovary cells CHO-s (Invitrogen, R80007), DMS53 (ATCC, CRL-2062) and H82 (ATCC, HTB-175), respectively, and screened with puromycin for 2 to 3 weeks, followed by FACS single cell sorting. The selected monoclonal cell lines were expanded and cryopreserved.

[0302] 1.2 Antigen preparation Using human DLL3 (Uniprot, Q9NYJ7), cynomolgus DLL3 (Uniprot, A0A2K5WSR4) and mouse DLL3 (Uniprot, O88516) sequences as templates, DLL3 ECD fusion proteins containing different tags were designed and cloned into pTT5 vector, respectively, and expressed in 293E cells to obtain antigens. The amino acid sequences of the relevant proteins are as follows: 1)His-hDLL3(ECD): [ka] SEQ ID NO:7 Note: dotted is the signal peptide sequence, single underlined is the his tag and linker, double underlined is the DLL3 extracellular domain.

[0303] 2) Fc-hDLL3(ECD): [ka] SEQ ID NO:8 Note: dotted is the signal peptide sequence, single underlined is the Fc tag and linker, double underlined is the DLL3 extracellular domain.

[0304] 3) hDLL3(ECD)-strep twin: [ka] SEQ ID NO:9 Note: The dotted part is the signal peptide sequence, the double underlined part is the DLL3 extracellular domain, and the single underlined part is the strep twin tag.

[0305] 4) cynoDLL3(ECD)-strep twin: [ka] SEQ ID NO:10 Note: The dotted part is the signal peptide sequence, the double underlined part is the DLL3 extracellular domain, and the single underlined part is the strep twin tag.

[0306] 5) mouDLL3(ECD)-strep twin: [ka] SEQ ID NO:11 Note: The dotted part is the signal peptide sequence, the double underlined part is the DLL3 extracellular domain, and the single underlined part is the strep twin tag.

[0307] Example 2: Preparation of mouse anti-human DLL3 monoclonal antibodies 1. Immunity Anti-human DLL3 monoclonal antibodies were produced by immunized mice. Female 6-8 week-old SJL mice (Shanghai SLAC Laboratory Animal Co., Ltd., Animal Production License Number: SCXK(Hu)2017-0005) were used in the experiment. Breeding environment: SPF grade. After purchasing the mice, they were kept in a laboratory environment for one week, with a 12 / 12 hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. The mice that had become accustomed to the environment were immunized according to the following schedule.

[0308] Immunization Plan: The immunization antigen for the first group of mice was His-hDLL3 (ECD) (SEQ ID NO: 7). Cross-immunization was performed with the adjuvant TiterMax® Gold Adjuvant (Sigma Cat No. T2684) and Thermo Imject® Alum (Thermo Cat No. 77161). The ratio of antigen to adjuvant TiterMax® Gold Adjuvant was 1:1, and the ratio of antigen to adjuvant Thermo Imject® Alum was 3:1, 50 μg / mouse / time (primary immunization), 25 μg / mouse / time (booster immunization). Inoculation was performed after emulsification of the antigen, and the immunization times were 0, 7, 14, and 21 days. Blood was collected on the 7th and 21st days, and the antibody titer in the mouse serum was determined by the ELISA method. After the fourth immunization, mice with a tendency to have a high and stable antibody titer in the serum were selected and splenocyte fusion was performed. Three days before splenocyte fusion, booster immunization was performed by intraperitoneally (ip) injection of an antigen solution prepared in physiological saline at 25 μg / mouse.

[0309] The immunization antigens for the second group of mice were DLL3 CHO-s and Fc-hDLL3 (ECD) (SEQ ID NO: 8), and the immunization method was alternating immunization with cells and protein antigens. Before the first immunization with DLL3 CHO-s cells, TiterMax® Gold Adjuvant (Sigma Cat No. T2684) was injected intraperitoneally into the mice at 0.1 mL per mouse, and after half an hour, the mice were immunized with 10 mL of saline. 80.1 mL of cell solution diluted to a concentration of 100 μg / mL was injected intraperitoneally into each mouse. After the cells were uniformly dispersed, they were inoculated on days 0, 14, 28, and 42. The Fc-hDLL3(ECD) antigen was cross-immunized with the adjuvant TiterMax® Gold Adjuvant (Sigma Cat No. T2684) and Thermo Imject® Alum (Thermo Cat No. 77161). The ratio of antigen to adjuvant TiterMax® Gold Adjuvant was 1:1, and the ratio of antigen to adjuvant Thermo Imject® Alum was 3:1, 50 μg / mouse / time (primary immunization) and 25 μg / mouse / time (booster immunization). The immunization times of hDLL3-Fc were days 7, 21, 35, and 49. Blood was collected on days 14, 35, and 49, and the antibody titers in the mouse serum were determined by ELISA. After the eighth immunization, mice with high and stable serum antibody titers were selected for splenocyte fusion. Three days before splenocyte fusion, a booster was administered and the hDLL3-Fc protein antigen solution prepared in saline was injected intraperitoneally (ip) at 50 μg / mouse.

[0310] 2. Fusion of splenocytes The splenic lymphocytes were fused with myeloma cells Sp2 / 0 cells (ATCC® CRL-8287®) by an optimized electrofusion method to obtain hybridoma cells.

[0311] The fused hybridoma cells were cultured at 3–4 × 10 5 The cells were resuspended in complete medium (IMDM medium containing 20% ​​FBS, 1x HAT, and 1x OPI) at a density of 100 / mL and inoculated into a 96-well plate at 150 μL / well. After incubation at 37°C and 5% CO2 for 3 to 4 days, the supernatant was removed and 200 μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1x HT, and 1x OPI) was added. After incubation at 37°C and 5% CO2 for 3 days, screening detection was performed.

[0312] 3. Screening of Hybridoma Cells and Sequencing of Antibodies Depending on the growth density of the hybridoma cells, the hybridoma culture supernatant was detected by ELISA method binding to DLL3 protein and FACS method binding to DLL3 CHO-s cells. Clones that bind to human DLL3 protein, monkey DLL3 protein, and DLL3 CHO-s cells but do not bind to wild-type CHO-s cells were selected, and cryopreservation, amplification, breeding conservation, and 1 to 2 subcloning were performed at appropriate times until single cell clones were obtained. Screening by the above experiment yielded hybridoma clones mAb100 and mAb6.

[0313] Hybridoma clones were expanded, RNA was extracted, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using degenerate primers for mouse Ig to obtain the antibody variable region sequences.

[0314] mAb6 heavy chain variable region: [ka] SEQ ID NO:12 mAb6 light chain variable region: [ka] SEQ ID NO:13 mAb100 heavy chain variable region: [ka] SEQ ID NO:14 mAb100 light chain variable region: [ka] Sequence number 15.

[0315] [Table 4] Note: The amino acid residues of the VH / VL CDRs are determined and annotated according to the Kabat numbering system.

[0316] The heavy and light chain variable regions of the mouse antibody were each cloned into a pTT5 vector plasmid containing the human IgG1 heavy chain constant region shown in sequence number 28 and the κ light chain constant region shown in sequence number 29, and then transfected into HEK293 cells to obtain the anti-DLL3 chimeric antibodies M6CHI and M100CHI.

[0317] Human IgG1 heavy chain constant region: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:28 Human kappa light chain constant region: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 29.

[0318] Example 3: Humanization of a mouse anti-DLL3 monoclonal antibody The Kabat human antibody heavy and light chain variable region germline gene database was aligned, and the highly homologous heavy and light chain variable region germline genes were selected as templates, and the CDRs of the mouse antibody were grafted onto the corresponding human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Amino acids in the variable regions were back-mutated and further recombined with constant regions (e.g., with the human IgG1 heavy chain constant region shown in SEQ ID NO:28 and the human κ light chain constant region shown in SEQ ID NO:29) to obtain full-length antibodies.

[0319] The human germline light chain template for the mAb6 antibody is IGKV1-16*01, and the human germline heavy chain templates are IGHV1-3*01, IGHV7-4-1*02, and IGHV3-73*01. The human germline light chain template for the H100 antibody is IGKV1-27*01, and the human germline heavy chain template is IGHV3-11*01.

[0320] Furthermore, the fourth amino acid residue in HCDR3: PLYYYGRSYNAVAY (SEQ ID NO: 24) of the heavy chain variable region of mAb100 was mutated from Y to H, and the eleventh residue was mutated from A to G to form a new [ka] HCDR3:PLY H YGRSYNAVAY (SEQ ID NO:30) and [ka] PLYYYGRSYN G VAY (SEQ ID NO:31) obtained.

[0321] [Table 5-1] [Table 5-2] Note: In the table, A100G is used as an example to indicate that the 100Eth A in the Kabat numbering has been mutated to G.

[0322] The resulting sequences of the humanized antibody variable regions are as follows: hAb6 VH1(Q1E, R71V, T73K) [ka] SEQ ID NO:32 hAb6 VH2(Q1E, I69L, R71V, T73K, S76N) [ka] SEQ ID NO:33 hAb6 VH3(Q1E, M48I, V67A, I69L, R71V, T73K, S76N) [ka] SEQ ID NO:34 hAb6 VH4(Q1E, Q43K,I69L, R71V, T73K, S76N) [ka] SEQ ID NO:35 hAb6 VH5(Q1E, L71V, T73K, F69L) [ka] SEQ ID NO:36 hAb6 VH6(Q1E, F69L, L71V, T73K, V75S, S76N) [ka] SEQ ID NO:37 hAb6 VH7(F27Y, S30T, I69L, R71V, D73K,T93A) [ka] SEQ ID NO:38 hAb6VH8 (Graft IGHV1-3*01) [ka] SEQ ID NO:39 hAb6VH9 (Graft IGHV7-4-1*02) [ka] SEQ ID NO:40 hAb6VH10 (Graft IGHV3-73*01) [ka] SEQ ID NO:41 hAb6VH11(Q1E, F69L, L71V, T73K,V75S, S76N,R38K) [ka] SEQ ID NO:42 hAb6VH12(Q1E,V68A,F69L, L71V, T73K, V75S, S76N) [ka] SEQ ID NO:43 hAb6VH13(Q1E, R38K,V68A,F69L, L71V, T73K, V75S, S76N) [ka] SEQ ID NO:44 hAb6 VL1(F36L, S46G) [ka] hAb6 VL2 (F36L, S46G, T69A, F71Y) [ka] hAb6 VL3(F36L, A43S, P44F, S46G, T69A, F71Y) [ka] SEQ ID NO:47 hAb6 VL4(F36L, S46G, T69A, F71Y, T85D) [ka] SEQ ID NO:48 hAb6VL5 (Graft IGKV1-16*01) [ka] SEQ ID NO:49 hAb100 VH1(Q1E,R94S) [ka] SEQ ID NO:50 hAb100 VH2(Q1E, S49A, R94S) [ka] SEQ ID NO:51 hAb100 VH3(Q1E, S49A, R94S,Y98H) [ka] SEQ ID NO:52 hAb100 VH4(Q1E, S49A, R94S, A100eG) [ka] SEQ ID NO:53 hAb100VH5 (Graft IGHV3-11*01) [ka] SEQ ID NO:54 hAb100 VL1 (Graft IGKV1-27*01) [ka] SEQ ID NO:55 hAb100 VL2(V43I) [ka] Sequence number 56.

[0323] Note: Single underlined regions are CDR regions and double underlined regions are mutation sites.

[0324] [Table 6] wherein X1 is Y or H, and X2 is A or G.

[0325] Exemplary combinations of heavy and light chain variable regions of humanized antibodies are as follows: [Table 7] Note: hAb6L1H1 indicates that the antibody comprises the heavy chain variable region hAb6VH1 and the light chain variable region hAb6VL1, and the sequence of the heavy chain constant region is SEQ ID NO: 28 and the sequence of the light chain constant region is SEQ ID NO: 29, and the rest are inferred accordingly.

[0326] [Table 8] Note: hAb100L1H1 indicates that the antibody comprises the heavy chain variable region hAb100VH1 and the light chain variable region hAb100VL1, and that the sequence of the heavy chain constant region is SEQ ID NO:28 and the sequence of the light chain constant region is SEQ ID NO:29, and the rest are inferred accordingly.

[0327] The antibodies were cloned, expressed, and purified, and humanized antibodies with good activity were selected through protein binding experiments (Test Example 1), cell binding experiments (Test Example 2), and Biacore (Test Example 4). The heavy and light chain amino acid sequences of exemplary humanized antibodies are as follows: Hu6 (also called hAb6L4H12) heavy chain: [ka] Note: The underlined parts in the sequences are variable regions and the italicized parts are constant regions. SEQ ID NO:58

[0328] Hu6 (also called hAb6L4H12) light chain: [ka] Note: The underlined parts in the sequences are variable regions and the italicized parts are constant regions. SEQ ID NO:59

[0329] Hu100 (also called hAb100L1H1) heavy chain: [ka] Note: The underlined parts in the sequences are variable regions and the italicized parts are constant regions. SEQ ID NO:60

[0330] Hu100 (also called hAb100L1H1) light chain: [ka] Note: The underlined parts in the sequences are variable regions and the italicized parts are constant regions. Sequence number 61.

[0331] The positive control molecule used in this disclosure is BI-764532 (constructed with reference to WO2019234220A1) and the negative control is C25, whose sequences are respectively as follows: BI-764532 heavy chain: [ka] Note: The underlined parts in the sequences are variable regions and the italicized parts are constant regions. SEQ ID NO:62

[0332] BI-764532 light chain: [ka] Note: The underlined parts in the sequences are variable regions and the italicized parts are constant regions. SEQ ID NO:63

[0333] C25 heavy chain: [ka] SEQ ID NO:64

[0334] C25 light chain: [ka] Sequence number 65. Note: The underlined parts in the sequences are variable regions and the italicized parts are constant regions.

[0335] II. Preparation of Compounds Experimental methods in the examples of this disclosure for which specific conditions are not specified generally follow common conditions or conditions recommended by the manufacturers of raw materials or products. Reagents for which specific sources are not specified are common commercially available reagents.

[0336] The structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). For the NMR measurement, a nuclear magnetic device Bruker AVANCE-400 is used, the measurement solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), the internal standard is tetramethylsilane (TMS), and the chemical shift is 10 -6 Expressed in ppm.

[0337] For the MS measurements, a mass spectrometer FINNIGAN LCQAd(ESI) (Thermo, model number: Finnigan LCQ advantage MAX) was used.

[0338] For UPLC measurements, a liquid chromatograph mass spectrometer Waters Acquity UPLC SQD was used.

[0339] For the HPLC measurements, a high performance liquid chromatograph Agilent 1200DAD (Sunfire C18 150×4.6 mm column) and a high performance liquid chromatograph Waters 2695-2996 (Gimini C18 150×4.6 mm column) were used.

[0340] For UV-HPLC measurements, a Thermo NanoDrop 2000 ultraviolet spectrophotometer was used.

[0341] As the silica gel plate for thin layer chromatography, Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate is used. The specification of the silica gel plate used for thin layer chromatography (TLC) is 0.15-0.2 mm, and the specification of the silica gel plate used for separation and purification of products by thin layer chromatography is 0.4-0.5 mm.

[0342] Column chromatography generally used Yantai Huanghai 200-300 mesh silica gel as the vector.

[0343] Known starting materials according to the present disclosure may be synthesized by employing or following methods known in the art, or may be purchased from companies such as ABCR GmbH & Co. KG, Acros Organnics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), and Darui Chemical.

[0344] Unless otherwise specified in the examples, all reactions were carried out in an argon or nitrogen atmosphere.

[0345] An argon or nitrogen atmosphere refers to an argon or nitrogen balloon of approximately 1 L volume attached to the reaction flask.

[0346] Hydrogen atmosphere refers to a hydrogen balloon of approximately 1 L volume connected to the reaction flask.

[0347] For the pressurized hydrogenation reaction, a Parr 3916EKX hydrogenation apparatus and a Seiran QL-500 hydrogen generator or an HC2-SS hydrogenation apparatus were used.

[0348] The hydrogenation reaction was generally carried out by repeating the process of evacuating and refilling with hydrogen three times.

[0349] A CEM Discover-S 908860 microwave reactor was used for microwave reactions.

[0350] Unless otherwise specified in the examples, the solutions used in the reactions refer to aqueous solutions.

[0351] Unless otherwise specified in the examples, the reaction temperature is room temperature, and the temperature range is 20 to 30°C.

[0352] Preparation of PBS buffer solution with pH=6.5 in the embodiment: 8.5 g of KH2PO4, 8.56 g of K2HPO4.3H2O, 5.85 g of NaCl, and 1.5 g of EDTA were taken and placed in a flask, and the volume was adjusted to 2 L. It was completely dissolved by ultrasonication, and the solution was obtained by shaking.

[0353] The eluent system of column chromatography and the developing solvent system of thin layer chromatography used in purifying the compound include A: dichloromethane and isopropyl alcohol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system, and the volume ratio of the solvents may be adjusted according to the polarity of the compound, or by adding a small amount of triethylamine and an acidic or basic reagent, etc.

[0354] Some of the compounds of the present disclosure were characterized by Q-TOF LC / MS using an Agilent 6530 accurate mass quadrupole-time of flight mass spectrometer and an Agilent 1290-Infinity ultra-performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1 mm×75 mm column).

[0355] The drug moiety of the antibody-drug conjugates of the present disclosure may be selected from cytotoxic drugs known in the art, with exemplary drugs being the ecteinascidin derivatives described in WO2021218896A1 (Application No.: PCT / CN2021089838), which is incorporated herein by reference in its entirety.

[0356] An exemplary synthetic route for an antibody-drug conjugate according to the present disclosure is as follows.

[0357] Example 4-1. Synthesis of Compound 7 (Linker) [ka] Step 1 [ka] Compound 7-1 (1.3 g, prepared by the method disclosed in WO2013106717A1) was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.2 g), benzyl bromide (1.35 mL), and tetrabutylammonium iodide (415 mg) were added in that order. The mixture was stirred at room temperature, filtered, concentrated, and purified by silica gel column chromatography using petroleum ether / ethyl acetate as a developing solvent to obtain compound 7-2.

[0358] Compounds 7-2 (121 mg) and 7-3 (180 mg) were placed in a reaction flask, and 4 mL of tetrahydrofuran was added. Under a nitrogen atmosphere, the temperature was lowered to about 0°C in an ice-water bath, potassium t-butoxide (109 mg, 0.98 mmol) was added, the temperature was raised to room temperature, and the mixture was stirred for 40 minutes. 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL x 2) and chloroform (10 mL x 5), and the organic phases were combined and concentrated. The resulting residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL x 3), and the organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The compound 7-4 was obtained by purification using silica gel column chromatography with petroleum ether / ethyl acetate as a developing solvent. MS m / z(ESI): 515.0[M+1]+ + .It was.

[0359] Compound 7-4 (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V=2:1), palladium carbon (12 mg, content 10%, dry) was added, hydrogen gas was substituted three times, and the reaction was carried out at room temperature for 1 hour with stirring. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude product, the title compound 7-5 (13 mg). The product was used in the next reaction without purification. MS m / z (ESI): 424.9 [M+1].

[0360] Step 2 [ka] Compound 7-5 (8.00 g, 18.9 mmol, 1 eq) was placed in a 250 mL three-neck flask, and dry dichloromethane (100 mL) was added under nitrogen protection. The mixture was dissolved with stirring to obtain 0. oCool the mixture to 30°C, add 4-dimethylaminopyridine (250 mg, 2.05 mmol), 2,4-dimethoxybenzyl alcohol (4.45 g, 26.5 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (5.04 g, 28.6 mmol) in that order, and then add 0 o C and reacted with stirring for 4 hours, then added water (50 mL) to quench the reaction, warmed to room temperature, extracted twice with methyl tert-butyl ether (100 mL), combined organic phases, washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to dryness to give the crude product of compound 7-6 (13.1 g). MS Calc: 574.2, Observed: 575.0 [M+H] + .

[0361] Step 3 [ka] The crude product of compound 7-6 (13.1 g) was placed in a 500 mL reaction flask, DCM (160 mL) was added, and the mixture was dissolved under stirring. Diethylamine (80 mL) was added, and the temperature was raised to 15-18 °C. o C for 3 hours, and then concentrated under reduced pressure until dry to obtain a crude product. The crude product was separated by silica gel column chromatography, and the desired component was collected and then evaporated to dryness (water temperature <35°C). o C) Compound 7-7 was obtained as an oil (5.47 g), with a two-step yield of 82.2%. MS Calc: 352.2, Observed: 353.1 [M+H] + .

[0362] Step 4 [ka] Compound 7-7 (4.36 g, 12.4 mmol) and compound 7-8 (7.03 g, 14.9 mmol, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)caproyl)glycylglycyl-L-phenylalanine, prepared by the method in Example 73 of patent application "EP2907824B1") were placed in a 250 mL reaction flask, and under nitrogen protection, dry N,N-dimethylformamide (50 mL) was added and dissolved with stirring to obtain 0.01 g of dimethylformamide. o Cool to 30°C, add 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (4.80 g, 16.3 mmol), and o The mixture was heated to 100° C. and reacted with stirring for 1 h. The mixture was cooled in an ice-water bath, and water (150 mL) was added to quench the reaction. The mixture was extracted twice with ethyl acetate (300 mL). The combined organic phase was washed once with saturated aqueous sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to dryness. The crude product was purified by silica gel column chromatography to give impure pale yellow solid 7-9 (6.02 g). The solid was purified by slurrying with methyl tert-butyl ether (60 mL) to give compound 7-9 (5.08 g) as an off-white solid in a yield of 50.8%. MS Calc: 806.4, Observed: 807.2 [M+H] + .

[0363] Step 5 [ka] Compound 7-9 (150 mg, 0.186 mmol) was placed in a 50 mL one-neck flask, and dry dichloromethane (6 mL) and anisole (60 mg, 0.558 mmol) were added under nitrogen protection, and the mixture was stirred to obtain a 0 o Cool to 0°C, add dichloroacetic acid (0.24 mL, 2.9 mmol) to dissolve the substrate, and o C and react for 3 to 4 hours with stirring. Methyl tert-butyl ether (18 mL) was added to the reaction solution, and the oC was slurried and stirred for 30 min, filtered and sucked dry, and the solid was added to methyl tert-butyl ether (18 mL), slurried and stirred at room temperature for 30 min, filtered and sucked dry to give compound 7 (120 mg, 0.183 mmol), 98% yield. 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.56 (brs, 1H), 8.50 (t, J = 6.8 Hz, 1H), 8.28 (t, J = 6.0 Hz, 1H), 8.20-8.05 (m, 2H), 8.00 (t, J = 5.6 Hz, 1H), 7.30-7.15 (m, 5H), 6.99 (s, 2H), 4.65-4.40 (m, 3H), 3.80-3.55 (m, 7H), 3.45 (d, J = 7.6 Hz, 2H), 3.10-3.00 (m, 1H), 2.85-2.75 (m, 1H), 2.11 (t, J = 7.6 Hz, 2H), 1.55-1.40 (m, 4H), 1.25-1.15 (m, 2H), 1.05-0.95 (m, 1H), 0.55-0.40 (m, 2H), 0.40-0.30 (m, 2H). MS Calc: 656.3, Observed: 679.2 [M+Na] + .

[0364] Example 4-2. Synthesis of L-9 [ka] Step 1 [ka] Compound 8-6 (50 mg, 0.063 mmol, synthesized according to the method described in Example 8 in WO2021218896A1) and compound 7 (85 mg, 0.13 mmol) were weighed and placed in a reaction flask, and the system was protected with nitrogen. A solution of N-methylimidazole (21 mg, 0.25 mmol) in dry MeCN (4 mL) was added. The system was cooled in an ice-water bath, and then a solution of TCFH (45 mg, 0.16 mmol, N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate) in dry MeCN (1 mL) was added dropwise. The reaction was carried out in an ice-water bath for 80 minutes, and the raw material was completely consumed by sampling and LCMS. The reaction solution was suction filtered with a sand core funnel. The filter cake was washed with dry MeCN. The filtrate was combined and concentrated under reduced pressure to give the crude product, which was purified by column chromatography (methanol / dichloromethane=1:25) to give about 83 mg of white solid L-8-6, with a yield of 92.22%. MS Calc: 1430.6, Observed: 1431.4 [M+H].

[0365] Step 2 [ka] Compound L-8-6 (81 mg, 0.057 mmol) was weighed and dissolved in dry MeCN (7 mL). A solution of AgNO3 (781 mg, 4.60 mmol) in water (4.7 mL) was added. The reaction was carried out at room temperature under nitrogen protection in the dark for 21 hours, and the raw material was completely consumed by sampling and LCMS. Saturated aqueous NaHCO3 solution (11.7 mL) and saturated NaCl solution (11.7 mL) were added to the reaction solution, which was stirred vigorously for 30 minutes and filtered through diatomaceous earth. The filtrate was extracted with (MeOH / CHCl3=1 / 10, 20 mL x 3). The organic phases were combined, dried by adding Na2SO4, filtered, and concentrated under reduced pressure to dryness to obtain the crude product, which was purified by prep-HPLC to obtain about 52 mg of white solid L-9, with a yield of 64.6%. 1H NMR (400 MHz, CD3OD) δ 7.32-7.18 (m, 7H), 6.97 (d, J = 8.4 Hz, 1H), 6.75 (s, 2H), 6.66 (s, 1H), 6.25 (s, 1H), 6.07 (s, 1H), 5.23 (d, J = 11.2 Hz, 1H), 4.77 (s, 1H), 4.62-4.56 (m, 4H), 4.50 (dd, J = 6.0, 8.4 Hz, 1H), 4.41 (s, 1H), 4.18 (d, J = 10.8 Hz, 1H), 3.87-3.55 (m, 12H), 3.45 (t, J = 6.8 Hz, 2H), 3.27 (s, 3H), 3.22-3.17 (m, 2H), 3.05-2.88 (m, 3H), 2.79 (d, J = 15.2 Hz, 1H), 2.65-2.59 (m, 2H), 2.39 (s, 3H), 2.37 (s, 3H), 2.29 (s, 3H), 2.24 (t, J = 7.6 Hz, 2H), 2.12 (d, J = 15.9 Hz, 1H), 2.03 (s, 4H), 1.63-1.50 (m, 4H), 1.31-1.23 (m, 2H), 1.01-0.95 (m, 1H), 0.44-0.22 (m, 3H), -0.09--0.14 (m, 1H).

[0366] 3. Preparation of ADC Example 5-1. Preparation of ADC-1 [ka] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP hydrochloride) (10 mM, 67.5 μL, 675 nmol) was added to an aqueous solution of antibody Hu6 in PBS buffer (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 3.6 mL, 243.1 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0367] Compound L-9 (3.46 mg, 2431 nmol) was dissolved in 170 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 ° C for 3 hours to stop the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH 6.5) to obtain the title product ADC-1 in PBS buffer (2.83 mg / mL, 16.0 mL), which was stored at 4 ° C.

[0368] The drug loading amount of ADC-1 in each batch was calculated by the HIC method, and the DAR value was n=3.73-4.27.

[0369] Example 5-2. Preparation of ADC-2 [ka] Under the condition of 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP hydrochloride) (10 mM, 75.1 μL, 751 nmol) was added to an aqueous solution of antibody Hu100 in PBS buffer (0.05 M aqueous solution of PBS buffer with pH=6.5, 10.0 mg / mL, 4.0 mL, 270.1 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0370] Compound L-9 (3.84 mg, 2701 nmol) was dissolved in 190 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 ° C. for 3 hours to stop the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH 6.5) to obtain the title product ADC-2 in PBS buffer (3.52 mg / mL, 16.0 mL), which was stored at 4 ° C.

[0371] The drug loading amount of ADC-2 in each batch was calculated by the HIC method, and the DAR value was n=3.72-4.88.

[0372] Analysis of drug loading in ADC stock solutions ADC is an antibody-drug conjugate whose therapeutic mechanism for disease is to deliver drugs to cells by targeting them with antibodies, thereby killing the cells or inhibiting their growth. The amount of drug loaded plays a crucial role in the efficacy of the drug.

[0373] In this disclosure, the drug loading is analyzed by HIC method, and the process is basically as follows: Reagents and equipment: Ammonium sulfate: Kokuyo Pharmaceutical, 500mg / bottle, Isopropyl alcohol: LC grade, 4L / bottle, Thermo Fisher, Anhydrous sodium dihydrogen phosphate: Kokuyo Pharmaceutical, 500mg / bottle.

[0374] High performance liquid chromatograph: Agilent 1260.

[0375] Preparation of solutions: Mobile phase A (50 mM anhydrous sodium dihydrogen phosphate solution, pH 7.0): Measure 1000 mL of purified water in a measuring cylinder, add 6.49 g of anhydrous sodium dihydrogen phosphate solid, stir to mix evenly, adjust the pH to 7.0, and then use it for filtration and store at 2-8°C for 14 days.

[0376] Mobile phase B (2 M ammonium sulfate dissolved in 50 mM anhydrous sodium dihydrogen phosphate solution, pH 7.0): 264.3 g of ammonium sulfate solid was added to the prepared 50 mM anhydrous sodium dihydrogen phosphate solution, stirred well to mix uniformly, filtered and used, and stored at 2-8 °C for 14 days.

[0377] Mobile phase C (IPA): 1 L isopropyl alcohol.

[0378] Naked antibody and samples awaiting testing (concentration 1 mg / mL, approximately 200 μL) were prepared for injection use.

[0379] Chromatography conditions: Column: TSKgel Butyl-NPR 4.6mm x 10cm 2.5μm, Column temperature: 30°C, DAD detector, detection wavelength 280nm (370nm), Sample chamber temperature: 4°C, flow rate: 0.5mL / min, injection volume: 40μL, Chromatographic gradient: A% (start 35%-15min 75%-20min 75%), C% (start 5%-15min 25%-20min 25%).

[0380] [Table 9]

[0381] Analysis of data: By comparing the spectrum of the sample with that of the naked antibody, the positions of DAR0, DAR2, DAR4, DAR6, and DAR8 were distinguished, and the spectrum of the detected sample was integrated to calculate the DAR value by the peak area. The calculation formula is as follows: DAR = Σ (number of linked drugs × percentage of peak area) / sum of peak areas.

[0382] The activities of the antibodies and ADCs of the present disclosure were verified by biochemical test methods described below.

[0383] Test Example 1: Protein-level ELISA binding experiment Plates were coated with streptavidin (abcam, ab136200, 1 μg / mL) at 100 μL / well and incubated overnight at 4°C. Plates were washed 3 times with 250 μL / well with PBST solution (PBS containing 0.1% Tween 20). Plates were blocked with 250 μL / well with 5% milk for 2 hours at 37°C. Plates were washed 3 times with 250 μL / well with PBST solution. Biotinylated DLL3 antigen (1 μg / mL) (SEQ ID NO: 9) was added and incubated for 1 hour at 37°C. Plates were washed 3 times with 250 μL / well with PBST solution. Antibodies Hu6, Hu100, BI-764532 and C25 (negative antibody) (maximum concentration 100 nM, 4-fold serial dilution) were prepared and incubated for 1 hour at 37°C. The plate was washed six times with PBST solution at 250 μL / well. Working concentration of human IgG(H+L)-HRP (Jackson, 109-035-003, diluted 1:4000) antibody was added at 100 μL / well and incubated at 37°C for 1 h. The plate was washed six times with PBST solution at 250 μL / well. TMB (KPL, 5120-0077) color development solution was added at 100 μL / well and developed at room temperature for 5-10 min. 1M H2SO4 was added at 100 μL / well to stop the color development, and the plate was read at 450 nm using a plate reader (Molecular Devices, VERSA max).

[0384] [Table 10]

[0385] As shown by the results, both antibodies Hu6 and Hu100 have excellent binding ability to DLL3.

[0386] Test Example 2: FACS binding experiment at the cell level DLL3-expressing small cell lung cancer cell line H1184 (ATCC, product number CRL-5858), DLL3 / H82, cynoDLL3 / CHO-s, and RatDLL3 / CHO-s cells were diluted in FACS buffer (1% BSA + PBS pH 7.4) at 1 × 106 The cells were prepared in a 100 μL / mL cell suspension and added to a 96-well round-bottom plate (Corning, 3795) at 100 μL / well. The supernatant was removed by centrifugation at 300 g for 5 minutes. Antibodies to be measured at different concentrations were added at 100 μL / well. The plates were incubated in the dark in a 4°C refrigerator for 1 hour. After three centrifugation washes at 300 g, working concentrations of APC anti-human IgG Fc (BioLegend, 410712) or PE F(ab')2-sheep anti-human IgG (invitrogen, H10104) were added and incubated in the dark in a 4°C refrigerator for 40 minutes. After three centrifugation washes at 300 g, the geometric mean fluorescence intensity was detected with an Invitrogen flow cytometer, and the binding EC50 value of the antibody to DLL3-expressing cells was calculated. The results are shown in Table 11-1, Table 11-2, Table 11-3, and Figures 1A to 1C.

[0387] [Table 11]

[0388] [Table 12]

[0389] [Table 13]

[0390] As shown by the results, all of the antibodies in the present disclosure can specifically bind to DLL3 expressed in cells, among which Hu6 has high binding ability to both DLL3 cells expressing different species. Hu100 has excellent binding ability to both human and cynomolgus monkey DLL3-expressing cells, but Hu100 does not bind to rat DLL3-expressing cells. BI-764532 has binding ability only to human and cynomolgus monkey DLL3-expressing cells, and its binding activity is weaker than Hu6 and Hu100.

[0391] Test Example 3: DLL1 and DLL4 binding experiment Stably transfected human DLL1 / CHO-s and human DLL4 / CHO-s cells were cultured at 1 × 10 6 A cell suspension of 100 μL / mL was prepared and added to a 96-well round plate (Corning, 3795) at 100 μL / well. The supernatant was removed by centrifugation at 300 g for 5 min. Antibodies to be detected were added at 100 μL / well and incubated in the dark in a 4°C refrigerator for 1 h. After centrifugation and washing three times at 300 g, a working concentration of PE F(ab')2-sheep anti-human IgG Fc biantibody (invitrogen, H10104) was added and incubated in the dark in a 4°C refrigerator for 40 min. After centrifugation and washing three times at 300 g, the geometric mean fluorescence intensity was detected on an Invitrogen flow cytometer.

[0392] The results show that neither antibody Hu6 nor Hu100 binds to human DLL1 or DLL4.

[0393] Test Example 4: Measurement of Biacore antibody affinity The antibodies to be measured were affinity captured by a Protein A biosensor chip (Cat. # 29127556, Cytiva) for 18 seconds, and the antigens human DLL3 (ACRO, DLL3-H52H4), cynomolgus monkey DLL3 (KACTUS, DLL-RM103) and mouse DLL3 (KACTUS, DLL-MM103) were flowed over the surface of the chip for 180 seconds, followed by dissociation for 600 seconds. The reaction signals were detected in real time by a Biacore 8K (Cytiva) instrument to obtain binding-dissociation curves. After the cycle dissociation of each experiment was completed, the biosensor chip was washed and regenerated with 10 mM glycine-HCl solution (pH 1.5) (Cat. # BR-1003-54, Cytiva). The data fitting model was a 1:1 model. The results are shown in Table 12-1, Table 12-2 and Table 12-3.

[0394] [Table 14]

[0395] [Table 15]

[0396] [Table 16]

[0397] As shown by the results, the humanized and chimeric antibodies mAb100 and mAb6 can specifically bind to human DLL3 and have high affinity. Here, the antibody Hu6 has high affinity to all of human, cynomolgus monkey, and mouse DLL3, and Hu100 has high affinity to human and cynomolgus monkey DLL3 but does not bind to mouse DLL3.

[0398] Test Example 5: Antibody epitope competitive binding experiment Plates were coated with BI-764532 antibody (1 μg / mL) at 100 μL / well and incubated overnight at 4°C. Plates were washed 3 times with 250 μL / well with PBST solution. Blocked with 5% milk at 250 μL / well for 2 hours at 37°C. Plates were washed 3 times with 250 μL / well with PBST solution. Biotinylated DLL3-Strep (0.1 μg / mL, SEQ ID NO: 9) was added. Competing antibodies, BI-764532, Hu6 and Hu100 (maximum concentration 100 μg / mL, 4-fold serial dilutions) were prepared and incubated at 37°C for 1 hour. Plates were washed 6 times with 250 μL / well with PBST solution. Streptavidin-peroxidase (diluted 1:2000) (Jackson Immuno Research, 016-030-084) was added at 100 μL / well and incubated at 37°C for 1 hour. The plate was washed 6 times with 250 μL / well of PBST solution. TMB (KPL, 5120-0077) color development solution was added at 100 μL / well and color development was carried out at room temperature for 5-10 min. 1M H2SO4 was added at 100 μL / well to stop color development, and the values ​​were read at 450 nm using a plate reader. The results are shown in Figure 2.

[0399] As shown by the results, the antibodies Hu6 and Hu100 do not compete with BI-764532, suggesting that the antibodies Hu6 and Hu100 bind to a different epitope than BI-764532.

[0400] Test Example 6: Detection of endocytosis activity of anti-DLL3 antibody DT3C is a recombinantly expressed fusion protein that is composed of the diphtheria toxin fragment A (toxin portion only) and the G group streptococcus 3C fragment (IgG binding portion). This protein has a good affinity with the IgG portion of the antibody, and when the antibody is endocytosed, it enters the cell together with the antibody, and releases the toxic DT under the action of intracellular furin. DT inhibits the activity of EF2-ADP ribosylation, blocks the protein translation process, and ultimately causes cell death. DT3C that has not entered the cell does not have the activity of killing the cell. The activity of the antibody to be endocytosed by the cell was evaluated according to the cell killing situation.

[0401] Experimental Procedure a. DMS53 / DLL3 cell suspension was prepared in fresh cell culture medium RPMI1640 (GE, SH30809.01) containing 20% ​​FBS, and added to a 96-well cell culture plate at 2000 cells / 50 μL / well. 50 μL of medium was added to columns 1 and 12 without seeding cells, and the plate was cultured at 37°C with 5% carbon dioxide for 16 hours.

[0402] b. DT3C (9600nM, expressed and purified by Shanghai Panchao Biotechnology Co., Ltd.) solution was prepared at 4x concentration in serum-free medium and filtered through a 0.22μm filter. Antibody (1600nM) was prepared at 4x concentration in serum-free medium, and 80μL of DT3C solution and 80μL of antibody solution were mixed uniformly at a 1:1 volume and incubated at room temperature for 30 minutes.

[0403] c. The mixture was serially diluted 5-fold in serum-free medium to give a total of 9 concentrations, with the 10th point being pure medium.

[0404] d. 50 μL of the diluted antibody was added to the cells and incubated in an incubator for 3 days.

[0405] e. 50 μL of CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, G7573) was added to each well, incubated at room temperature in the dark for 10 minutes, and chemiluminescence was read on a Victor3.

[0406] The results are shown in Table 13 below and in FIG.

[0407] [Table 17]

[0408] The results show that both antibodies Hu6 and Hu100 can be endocytosed. Test Example 7: Cytotoxicity experiment of ADC with different DLL3 expression levels The experimental procedure is as follows: A. A cell suspension was prepared using fresh cell culture medium containing 10% FBS and added to a 96-well cell culture plate (Corning, 3903) at 135 μL / well. 135 μL of medium alone was added to columns 1 and 12 without seeding cells, and the plate was cultured at 37°C with 5% carbon dioxide for 16 hours.

[0409] b) ADC samples were prepared in PBS as a working solution (10x concentration) in the first well, and serially diluted with PBS to the corresponding initial concentration. 15 μL of 10x ADC solution was added to each well and incubated at 37°C with 5% carbon dioxide for 6 days.

[0410] c. 70 μL of CTG (Promega, G7573) was added to each well and incubated at room temperature in the dark for 10 minutes. Chemiluminescence was read using Victor3, data was processed using GraphPad Prism5, and antibody or ADC concentration was plotted on the X-axis against light intensity on the Y-axis.

[0411] The seeding density of different cells on the plate, the working concentration of the first well (10x concentration), and the dilution ratio are shown in Table 14.

[0412] [Table 18] The cells used in the experiments are as follows: DMS53(+), CRL-2062, purchased from ATCC; H1184(+++), CRL-5858, purchased from ATCC; U-2OS(-), HTB-96, purchased from ATCC; HT-29(-), HTB-38, purchased from ATCC; CHO-K1(-), CCL-61 purchased from ATCC.

[0413] Among them, "+" indicates the expression level of DLL3, and "-" indicates no expression of DLL3.

[0414] The results are shown in Table 15 and Figures 4A to 4E.

[0415] [Table 19]

[0416] The results show that ADC-1 and ADC-2 have strong target cell killing activity and can kill DLL3-expressing DMA53 and H1184 cells, whereas U-2OS, HT-29 and CHO-K1 cells are negative for DLL3.

[0417] Test Example 8: Bypass killing activity experiment DMS53 / DLL3high (DMS53 cells stably transfected with DLL3) and U-2OS (ATCC, HTB-96) cells were cultured in RPMI1640 + 20% FBS + 1x Glutamax and McCoy's 5A + 10% FBS, respectively, and the cells were digested with pancreatin, neutralized with fresh medium, and centrifuged at 1000 rpm for 3 min. The supernatant was discarded and the cells were resuspended in RPMI1640 + 20% FBS + 1x Glutamax. After cell counting, the DMS53 / DLL3high cell density was adjusted to 9 x 10 4 The U-2OS cell density was adjusted to 3 × 10 cells / mL. 4The concentration was adjusted to 1 / mL. 500 μL of DMS53 / DLL3high cells and 500 μL of U-2OS cells were added to each of the corresponding wells of the 12-well plate. 500 μL of U-2OS cells and 500 μL of RPMI1640+20%FBS+1×Glutamax culture medium were added to the corresponding wells of the 12-well plate. The cells were cultured at 37°C with 5% carbon dioxide for 24 hours. The samples were prepared as 40× intermediate solutions (200 nM). 25 μL of the above ADC samples were taken and added to the corresponding wells of the 12-well plate. A solvent control group was set up. The cells were cultured at 37°C with 5% carbon dioxide for 6 days. The cells in the 12-well plate were digested with pancreatin, neutralized with fresh medium, 20 μL of cells were taken, 20 μL of trypan blue was added, and counted. The cells were centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. The cells were washed once with 100 μL of FACS buffer, centrifuged at 1500 rpm for 3 min, and the supernatant was discarded. The cells were resuspended in 100 μL of FACS buffer, and 2 μg / mL of anti-DLL3 positive antibody was added and incubated on ice for 60 min. The cells were washed once with FACS buffer, centrifuged at 1500 rpm for 3 min, and incubated with secondary antibody APC anti-human IgG Fc (100x) for 30 min, and washed once with FACS buffer. The cells were resuspended with 200 μL of FACS buffer and detected by FACS. The streaming data was analyzed by FlowJo to obtain the DMS53 / DLL3high ratio, and the total amount of DMS53 / DLL3high and U-2OS was calculated. The data was plotted by GraphPad Prism5, with different samples as the X-axis and the calculated cell number as the Y-axis, and the results are shown in Figure 5.

[0418] As shown by the results, ADC-1 and ADC-2 have a clear bystander cytotoxic effect.

[0419] Biological evaluation of in vivo activity Test Example 9: Evaluation of in vivo efficacy in DMS53 cell CDX mouse model Human small cell lung cancer DMS53 cells (5 × 10 6Balb / c mice were subcutaneously inoculated with 200 μL of 50% Matrigel (ATCC, CRL-2062) per mouse into the right armpit. After 21 days of inoculation, the tumor volume was approximately 200 mm 3 When the mice reached the age of 10, those with body weight and tumors that were too large or too small were removed, and the mice were randomly divided into groups of 8 mice per group according to tumor volume, and treatment began on the same day. ADC was injected intraperitoneally at a dose of 1.8 mg / kg, once a week for a total of two doses. Tumor volume and body weight were measured twice a week, and the data were recorded. Data were recorded using Excel statistical software, plotted using GraphPad Prism software, and statistically analyzed by Two-way ANOVA or One-way ANOVA, so that the average value was calculated as avg, the SD value was calculated as STDEV, and the SEM value was calculated as STDEV / SQRT (number of animals in each group).

[0420] Tumor volume (V) calculation formula: V = 1 / 2 × L long × L short 2 The relative tumor growth rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment and control groups at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0421] Tumor inhibition rate TGI(%)=1-T / C(%).

[0422] The results are shown in Table 16 and FIG.

[0423] [Table 20]

[0424] As shown by the results, at a dose of 1.8 mpk, both ADC-1 and ADC-2 can significantly inhibit the growth of subcutaneously implanted DMS53 cell tumors.

[0425] Test Example 10: Evaluation of in vivo efficacy in H1184 cell CDX mouse model Human small cell lung cancer NCI-H1184 cells (5 × 10 6NDG mice were subcutaneously inoculated with 200 μL of 50% Matrigel (ATCC, CRL-5858) at the right armpit. After 18 days of inoculation, the tumor volume was 180 mm 3 When the mice reached the age of 10, those with excessive or excessively small tumors were removed, and those with excessive or excessively large tumors were randomly divided into groups of 8 mice per group according to tumor volume, and treatment began on the same day. ADC was injected intraperitoneally at a dose of 1.8 mg / kg. The mice were administered once a week for three days. Tumor volume was measured and body weight was measured twice a week, and the data was recorded.

[0426] Data were recorded using Excel statistical software, plotted using GraphPad Prism software, and statistically analyzed by Two-way ANOVA or One-way ANOVA such that the mean was calculated as avg, the SD as STDEV, and the SEM as STDEV / SQRT (number of animals in each group).

[0427] Tumor volume (V) calculation formula: V = 1 / 2 × L long × L short 2 The relative tumor growth rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment and control groups at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0428] Tumor inhibition rate TGI(%)=1-T / C(%).

[0429] The results are shown in Table 17 and FIG.

[0430] [Table 21]

[0431] As shown by the results, at a dose of 1.8 mpk, both ADC-1 and ADC-2 can significantly inhibit the growth of H1184 xenograft tumors.

Claims

1. An anti-DLL3 antibody comprising a heavy chain variable region and a light chain variable region, i) The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 22, HCDR2 containing the amino acid sequence of SEQ ID NO: 23, and HCDR3 containing the amino acid sequence of SEQ ID NO: 57, The light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 25, LCDR2 containing the amino acid sequence of SEQ ID NO: 26, and LCDR3 containing the amino acid sequence of SEQ ID NO:

27. Of these, sequence number 57 is PLYX １ YGRSYNX ２ It is shown as VAY, and among them, X １ is Y or H, X ２ is A or G, or ii) The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 16, HCDR2 containing the amino acid sequence of SEQ ID NO: 17, and HCDR3 containing the amino acid sequence of SEQ ID NO: 18, and The light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 19, LCDR2 containing the amino acid sequence of SEQ ID NO: 20, and LCDR3 containing the amino acid sequence of SEQ ID NO:

21. Preferably, The heavy chain variable region includes HCDR1 containing the amino acid sequence of SEQ ID NO: 22, HCDR2 containing the amino acid sequence of SEQ ID NO: 23, and HCDR3 containing the amino acid sequence of SEQ ID NO: 24, 30, or 31, and The light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 25, LCDR2 containing the amino acid sequence of SEQ ID NO: 26, and LCDR3 containing the amino acid sequence of SEQ ID NO:

27. Anti-DLL3 antibody.

2. These are mouse antibodies, chimeric antibodies, or humanized antibodies. The anti-DLL3 antibody according to claim 1.

3. i) The heavy chain variable region includes an amino acid sequence having at least 90% identity with SEQ ID NOs: 50, 14, 51, 52, 53, or 54, and / or The light chain variable region includes an amino acid sequence having at least 90% identity with SEQ ID NOs. 55, 15, or 56, or ii) The heavy chain variable region includes an amino acid sequence having at least 90% identity with SEQ ID NOs: 43, 12, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 44, and / or The light chain variable region includes an amino acid sequence having at least 90% identity with SEQ ID NOs: 48, 13, 45, 46, 47, or 49. Preferably, i) The heavy chain variable region includes any one amino acid sequence selected from SEQ ID NOs: 50, 51, 52, 53, and 54, and / or the light chain variable region includes the amino acid sequence of SEQ ID NO: 55 or 56, or ii) The heavy chain variable region comprises any one amino acid sequence selected from SEQ ID NOs: 43, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 and 44, and / or the light chain variable region comprises any one amino acid sequence selected from SEQ ID NOs: 48, 45, 46, 47 and 49, or iii) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 14, and / or the light chain variable region includes the amino acid sequence of SEQ ID NO: 15, or iv) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13, more, The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 50, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 55, or The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 43, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

48. The anti-DLL3 antibody according to claim 1.

4. The antibody fragment is preferably Fab, Fab', F(ab') ２ , Fab'-SH, Fd, Fv, scFv, dsFv, biantibody or domain antibody, The anti-DLL3 antibody according to claim 1.

5. It comprises a heavy chain steady region and a light chain steady region, preferably the heavy chain steady region comprises the amino acid sequence of SEQ ID NO: 28, and / or the light chain steady region comprises the amino acid sequence of SEQ ID NO:

29. The anti-DLL3 antibody according to claim 1.

6. It includes heavy chains and light chains, of which, The heavy chain comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 60, and / or the light chain comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 61, or The heavy chain comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 58, and / or the light chain comprises an amino acid sequence having at least 85% identity with SEQ ID NO:

59. Preferably, The heavy chain contains the amino acid sequence of SEQ ID NO: 60, and the light chain contains the amino acid sequence of SEQ ID NO: 61, or The heavy chain contains the amino acid sequence of SEQ ID NO: 58, and the light chain contains the amino acid sequence of SEQ ID NO:

59. The anti-DLL3 antibody according to claim 5.

7. a) The anti-DLL3 antibody has a KD value of ≤3 nM, ≤2 nM, or ≤1 nM that binds to human DLL3 or its epitope, and the KD value is measured by Biacore. b) The anti-DLL3 antibody has an EC50 ≤ 3 nM that binds to H1184 cells expressing DLL3, and the EC50 is detectable by FACS. c) The anti-DLL3 antibody is endocytosizable by cells expressing DLL3, and d) The anti-DLL3 antibody has an EC50 ≤ 0.1 nM that binds to DLL3 or its epitope, and the EC ５０ Having at least one of the following characteristics: The anti-DLL3 antibody according to claim 1.

8. Encoding the anti-DLL3 antibody described in claim 1, Isolated nucleic acid.

9. A nucleic acid comprising the isolated nucleic acid described in claim 8, host cell.

10. A method for preparing an anti-DLL3 antibody, comprising culturing the host cells described in claim 9 under conditions suitable for the expression of the antibody, method.

11. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, The anti-DLL3 antibody specified in claim 1, Drugs, and Preferably, the drug is one or more selected from cytotoxic agents, radioactive markers, fluorophores, chromophores, developers, immunomodulators, angiogenesis inhibitors, cell growth inhibitors, apoptosis promoters, and cytolytic enzymes. Antibody-drug conjugates or pharmaceutically acceptable salts thereof.

12. It has a structure represented by the general formula (Pc-L-Da), 【Chemistry 1】 Eventually, Pc is the anti-DLL3 antibody as defined in claim 11, L is Linker, n is 1 to 10, preferably 3 to 5. The antibody-drug conjugate according to claim 11 or a pharmaceutically acceptable salt thereof.

13. It has a structure represented by the general formula (Pc-L-D), 【Chemistry 2】 Eventually, Pc is the anti-DLL3 antibody as defined in claim 11, L is Linker, n is 1 to 10, preferably 3 to 5. The antibody-drug conjugate according to claim 11 or a pharmaceutically acceptable salt thereof.

14. The linker is -L １ -L ２ -L ３ -L ４ -, wherein L １ is -(succinimido-3-yl-N)-W-C(O)-,-CH ２ -C(O)-NR ３ Selected from -W-C(O)- and -C(O)-W-C(O)-, where W is C １－６ Alkylene group, C １－６ Alkylene-C ３－６ Selected from cycloalkyl groups, of which the C １－６ Alkylene group, C １－６ Alkylene-C ３－６ Each cycloalkyl group is independently and optionally further substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ２ -NR ４ (CH ２ CH ２ O) ｐ CH ２ CH ２ C(O)-, -NR ４ (CH ２ CH ２ O) ｐ CH ２ C(O)-, -S(CH ２ ) ｐ Selected from C(O)- and chemical bonds, where p is an integer from 1 to 20. L ３ This is a peptide residue composed of 2 to 7 amino acid residues, of which the amino acids are selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally further substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ４ -NR ５ (CR ６ R ７ ) ｔ -, -C(O)NR ５ , -C(O)NR ５ (CH ２ ) ｔ - and selected from chemical bonds, where t is an integer from 1 to 6, R ３ , R ４ and R ５ They are identical or different, and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group. R ６ and R ７ They are the same or different, and each is independently selected from a hydrogen atom, halogen, alkyl group, haloalkyl group, deuterated alkyl group, and hydroxyalkyl group. Preferably, L １ teeth 【Transformation 3】 and s １ is an integer between 2 and 8, L ２ It is a chemical bond, L ３ is a tetrapeptide residue, preferably L ３ This is a tetrapeptide residue containing glycine-glycine-phenylalanine-glycine, L ４ NH(CH ２ ) t-, where t is 1 or 2, Of these, L １ The end is connected to PC, More preferably, the linker is shown in the following structure: 【Chemistry 4】 The antibody-drug conjugate according to claim 12 or a pharmaceutically acceptable salt thereof.

15. The antibody-drug conjugate is 【Transformation 5】 It has a structure selected from, Eventually, The antibody-drug conjugate according to claim 11, or a pharmaceutically acceptable salt thereof, wherein Pc is the anti-DLL3 antibody specified in claim 11, and n is 1 to 10.

16. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, The antibody-drug conjugate is 【Transformation 6】 It has the structure shown, and among them, Pc is the anti-DLL3 antibody described in claim 6, An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n is 1 to 8, more preferably 3 to 5.

17. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 15, wherein the drug is one or more selected from cytotoxic agents, radiomarkers, fluorophores, chromophores, developers, immunomodulators, angiogenesis inhibitors, cell growth inhibitors, apoptosis promoters, and cell lysing enzymes.

18. An anti-DLL3 antibody according to any one of claims 1 to 7, or an antibody-drug conjugate according to any one of claims 11 to 15, or a pharmaceutically acceptable salt thereof, A compound comprising one or more pharmaceutically acceptable excipients, diluents, or vectors, Pharmaceutical composition.

19. A pharmaceutical composition according to claim 18 for treating a tumor or cancer.

20. The aforementioned tumor or cancer is The pharmaceutical composition according to claim 19, wherein the tumor or cancer is selected from lung cancer, small cell lung cancer, large cell lung cancer, squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, medullary thyroid cancer, malignant pleural mesothelioma, breast cancer, triple-negative breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer, glioblastoma, skin cancer, and melanoma, and more preferably, the tumor or cancer is small cell lung cancer.

Images