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

Abstract

The present disclosure relates to anti-DLL3 antibodies, their antibody-drug conjugates, and pharmaceutical uses thereof. Specifically, the present disclosure relates to anti-DLL3 antibody-exatecan-based drug conjugates represented by the general formula Pc-LYD, where Pc is an anti-DLL3 antibody, and L, Y, and n are as defined herein. [Formula 1] JPEG2026508154000125.jpg57133

Description

[Technical Field]

[0001] The present disclosure relates to anti-DLL3 antibodies, antibody-drug conjugates thereof, and pharmaceutical uses thereof. [Background technology]

[0002] This discussion is not necessarily constitutive of prior art, but merely provides background information relevant to the present disclosure.

[0003] Small cell lung cancer (SCLC) is a relatively aggressive type of lung cancer, accounting for 10% to 15% of all lung cancer cases. SCLC has a rapid tumor growth rate, a high tendency to metastasize, and a 5-year survival rate of less than 7%. Platinum-based / etoposide combination chemotherapy is commonly used to treat SCLC. Although SCLC patients respond well to chemotherapy initially, they are highly susceptible to drug resistance and recurrence. Recent immunotherapies, such as PD-L1 antibodies and PD1 antibodies, have shown some efficacy in patients with SCLC, but the efficacy rate is approximately 15%. Currently, specific targeted therapeutic drugs have not yet been developed.

[0004] DLL3 is a ligand that inhibits Notch. Under normal conditions, DLL3 is located in the Golgi apparatus. 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 primarily expressed in neural or neuroendocrine tumors, including SCLC, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumors, small cell bladder cancer, glioblastoma multiforme, metastatic castrated prostate cancer, and melanoma. In particular, with regard to SCLC, over 80% of SCLCs express DLL3, but normal lung cancer tissue and paracancerous tissues do not. This differential expression makes DLL3 a highly potential therapeutic target for treating SCLC.

[0005] Exatecan toxins are camptothecin derivatives that can inhibit topoisomerase I and selectively inhibit DNA replication in proliferating tumor cells. In addition, exatecan toxins have good membrane permeability, allowing them to penetrate into killed cancer cells and continue to kill neighboring cancer cells, resulting in a clinically significant bystander effect.

[0006] An antibody is linked to a biologically active drug via a linker to form an antibody-drug conjugate (ADC). ADCs take full advantage of the specificity of antibodies to bind to normal cell and tumor cell surface antigens and the high efficiency of drugs (e.g., cytotoxic agents), while avoiding the drawbacks of antibodies, such as the relatively low therapeutic efficacy and excessive drug 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

[0007] The present disclosure relates to anti-DLL3 antibodies, antibody-drug conjugates thereof, and pharmaceutical uses thereof. More specifically, the present disclosure provides ADCs coupling multiple anti-DLL3 antibodies of novel sequences with an exatecan-based toxin.

[0008] In some embodiments, the disclosure provides an antibody-drug conjugate having a structure represented by the general formula Pc-LYD, or a pharmaceutically acceptable salt thereof. [C1] [ka] Among them, Pc is an anti-DLL3 antibody containing a heavy chain variable region and a light chain variable region, of which: 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; Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, of which R a and R b are the same or different and each independently represent a hydrogen atom, a deuterium atom, a halogen atom, and C 1-6 selected from alkyl groups, R 1 is halogenated C 1-6 Alkyl group or C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, halogenated C 1-6 Alkyl groups and C 3-6 cycloalkyl groups, Or, R 1 and R 2 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, m is an integer from 0 to 4, n is 1 to 10 (including integers and decimals), L is a linker.

[0009] In some embodiments, in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, n is the average number of drug modules of each antibody, which may be an integer or a decimal. In some embodiments, n is 1 to 10, or 2 to 10, or 3 to 10, or 4 to 10, or 5 to 10, or 6 to 10, or 7 to 10, or 8 to 10, or 1 to 9, or 2 to 9, or 3 to 9, or 4 to 9, or 5 to 9, or 6 to 9, or 7 to 9, or 1 to 8, or 2 to 8, or 3 to 8, or 4 to 8, or 5 to 8, or 6 to 8, or 1 to 7, or 2 to 7, or 3 to 7, or 4 to 7, or 5 to 7, or 1 to 6, or 2 to 6, or 3 to 6, or 4 to 6, or 1 to 5, or 2 to 5, or 3 to 5, or 1 to 4, or 2 to 4, or 1 to 3. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0010] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein 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 light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:25, LCDR2 comprising the amino acid sequence of SEQ ID NO:26, and LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

[0011] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein 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 light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:25, LCDR2 comprising the amino acid sequence of SEQ ID NO:26, and LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

[0012] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein 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 light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:25, LCDR2 comprising the amino acid sequence of SEQ ID NO:26, and LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

[0013] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein 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 light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:25, LCDR2 comprising the amino acid sequence of SEQ ID NO:26, and LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

[0014] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0015] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody is a humanized antibody.

[0016] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a human immunoglobulin framework region (FR region).

[0017] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein 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: 57, wherein SEQ ID NO: 57 is represented by PLYX1YGRSYNX2VAY, wherein X1 is Y or H and X2 is A or G, 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.

[0018] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein 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 backmutations selected from 1E, 27Y, 30T, 38K, 43K, 48I, 67A, 68A, 69L, 71V, 73K, 75S, 76N, and 93A; 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, 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.

[0019] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein 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 backmutations of 36L, 46G, 69A, 71Y, and 85D. The backmutation sites follow the Kabat numbering convention.

[0020] In a specific embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein 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 backmutations of 1E and 94S; and The light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 27. The backmutation sites follow the Kabat numbering convention.

[0021] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein 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.

[0022] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein 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; 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.

[0023] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein 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; 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.

[0024] In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein 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.

[0025] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, i) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence 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% sequence 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% sequence 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 includes a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13, 45, 46, 47, 48 or 49.

[0026] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises: i) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence 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% sequence 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% sequence 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% sequence 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% sequence 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% sequence 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% sequence 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% sequence identity to SEQ ID NO: 45, 46, 47, 48 or 49.

[0027] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises: i) the heavy chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence 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% sequence 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.

[0028] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, 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 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.

[0029] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises: 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; or 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.

[0030] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein 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.

[0031] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody is an antibody fragment, wherein the antibody fragment is Fab, Fab', F(ab')2, Fab'-SH, Fd, Fv, scFv, dsFv, a bibody, or a domain antibody.

[0032] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a light chain constant region and a heavy chain constant region.

[0033] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises an IgG1, IgG2, IgG3, or IgG4 constant region.

[0034] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a λ or κ chain constant region.

[0035] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises an IgG1 heavy chain constant region and a κ light chain constant region.

[0036] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises: 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.

[0037] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein 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% sequence 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% sequence 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% sequence 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% sequence identity to SEQ ID NO:59.

[0038] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises: 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 shown in SEQ ID NO:58 and / or the light chain comprises the sequence shown in SEQ ID NO:59.

[0039] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein the anti-DLL3 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 binds to H1184 cells expressing DLL3 with 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, or EC50≦0.06 nM, and the EC50 is detected by FACS; c) the anti-DLL3 antibody is capable of being endocytosed by cells expressing DLL3; and d) the anti-DLL3 antibody has an EC50 for binding to DLL3 or an epitope thereof of ≦0.1 nM, ≦0.09 nM, ≦0.08 nM, ≦0.07 nM, ≦0.06 nM, ≦0.05 nM, or ≦0.04 nM, and the EC50 is detected by ELISA; e) The above anti-DLL3 antibody and the positive antibody (e.g., BI-764532) recognize different DLL3 epitopes.

[0040] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein Y is [ka] Selected from The O-terminus of Y is linked to L.

[0041] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein Y is [ka] wherein the O-terminus of Y is linked to L.

[0042] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L is -L 1 -L 2 -L 3 -L 4 - and among them, L 1 -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein 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 1-6 Alkylene-C 3-6each cycloalkyl group is independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl; L 2 is -NR 4 (CH2CH2O) p CH2CH2C(O)-, -NR 4 (CH2CH2O) p CH2C(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, wherein 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 halogen, hydroxy group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; L 4 is -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 are 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 are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0043] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L is -L1 -L 2 -L 3 -L 4 - and among them, L 1 teeth, [ka] and s 1 is an integer from 2 to 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(CH)t—, where t is 1 or 2; Among them, the above L 1 The termini are ligated to Pc.

[0044] In some embodiments, the antibody-drug conjugate or pharmaceutically acceptable salt thereof, wherein L is [ka] It has the structure shown below.

[0045] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] Pc is any one of the anti-DLL3 antibodies described above, and n is 1-10.

[0046] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] The compound has a structure selected from the group consisting of: Pc is an anti-DLL3 antibody containing a heavy chain variable region and a light chain variable region, of which: 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; n is 1 to 10, preferably 3 to 8, and more preferably 6 to 8.

[0047] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] The compound has a structure selected from the group consisting of: Pc is an anti-DLL3 antibody containing a heavy chain variable region and a light chain variable region, of which: 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; n is 1 to 10, preferably 3 to 8, and more preferably 6 to 8.

[0048] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] The compound has a structure selected from the group consisting of: Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 60 and the light chain comprises the amino acid sequence of SEQ ID NO: 61; or the heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the light chain comprises the amino acid sequence of SEQ ID NO: 59; n is 1 to 10, preferably 3 to 8, and more preferably 6 to 8.

[0049] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] The compound has a structure selected from the group consisting of: Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61; n is 1 to 10, preferably 3 to 8, and more preferably 6 to 8.

[0050] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] The compound has a structure selected from the group consisting of: Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61; n is 4.43.

[0051] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] The compound has a structure selected from the group consisting of: Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61; n is 7.32.

[0052] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] The compound has a structure selected from the group consisting of: Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61; n is 6.12.

[0053] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] The compound has a structure selected from the group consisting of: Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the light chain comprises the amino acid sequence of SEQ ID NO: 59; n is 3.53.

[0054] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is [ka] The compound has a structure selected from the group consisting of: Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the light chain comprises the amino acid sequence of SEQ ID NO: 59; n is 7.43.

[0055] The present disclosure further provides a method for preparing an antibody-drug conjugate, the method comprising reducing any one of the anti-DLL3 antibodies described above and coupling the reduced antibody with a compound represented by (LYD) to obtain the antibody-drug conjugate described in the present disclosure.

[0056] In another aspect, the present disclosure provides a pharmaceutical composition comprising any one of the above-described antibody-drug conjugates or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients, diluents, or carriers. In some embodiments, the unit dose of the pharmaceutical composition comprises 0.1 to 3000 mg or 1 to 1000 mg of the antibody-drug conjugate.

[0057] In another aspect, the present disclosure provides use of any one of the above-described antibody-drug conjugates or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the same, as a medicament. In some embodiments, any one of the above-described antibody-drug conjugates or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the same, is used as a medicament for treating cancer or tumor. In some embodiments, any one of the above-described antibody-drug conjugates or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the same, is used as a medicament for treating cancer or tumor that expresses DLL3.

[0058] In another aspect, the present disclosure provides use of any one of the above-described antibody-drug conjugates or pharmaceutically acceptable salts thereof, or the above-described pharmaceutical compositions, 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 DLL3-expressing disease or condition.

[0059] In another aspect, the present disclosure provides use of any one of the above-described antibody-drug conjugates or pharmaceutically acceptable salts thereof, or the above-described pharmaceutical compositions, in preparing a medicament for treating or preventing a tumor or cancer, preferably, wherein the tumor or cancer is The tumor or cancer may be 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, preferably, the lung cancer is small cell lung cancer. In some embodiments, the tumor or cancer expresses DLL3.

[0060] 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 antibody-drug conjugates described above, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition. In some embodiments, the tumor or cancer is a DLL3-expressing tumor or cancer.

[0061] 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 antibody-drug conjugates or pharmaceutically acceptable salts thereof described above, or the pharmaceutical composition, wherein the tumor or cancer is The cancer may be 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 carcinoma), 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, and preferably, the lung cancer is small cell lung cancer.

[0062] The 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 characteristics. [Brief explanation of the drawings]

[0063] [Figure 1] Figure 1A shows the binding of Hu6 and Hu100 antibodies to DLL3 from different species. Figure 1A shows the FACS results for the binding of Hu6 and Hu100 antibodies to H1184 cells, Figure 1B shows the FACS results for the binding of Hu6 and Hu100 antibodies to cynoDLL3 / CHO-s cells, and Figure 1C shows the FACS results for the binding of Hu6 and Hu100 antibodies to ratDLL3 / CHO-s cells. [Figure 2] Figure 1 shows the results of a competitive binding experiment using different anti-DLL3 antibodies. The results show that Hu6 and Hu100 do not compete with BI-764532, suggesting that the antibodies Hu6 and Hu100 bind to different epitopes than BI-764532. [Figure 3] The results show that Hu6 and Hu100 antibodies are endocytosed by cells, and as shown by the results, both Hu6 and Hu100 can be endocytosed by cells. [Figure 4]Figure 4 shows the results of binding of ADC-1, ADC-2, and ADC-3 to human and monkey DLL3 cells, where Figure 4A shows the results of binding of ADC-1, ADC-2, and ADC-3 to human DLL3 cells, and Figure 4B shows the results of binding of ADC-1, ADC-2, and ADC-3 to monkey DLL3 cells. [Figure 5] Figure 5 shows the inhibitory effects of ADC-1, ADC-2, and ADC-3 on the growth of different cells. Figure 5A shows the inhibitory effect of ADC-1, ADC-2, and ADC-3 on the growth of H1184 cells, which highly express DLL3. Figure 5B shows the inhibitory effect of ADC-1, ADC-2, and ADC-3 on the growth of DMS53 cells, which highly express DLL3. Figure 5C shows the inhibitory effect of ADC-1, ADC-2, and ADC-3 on the growth of SK-MEL3 cells, which lowly express DLL3. Figure 5D shows the inhibitory effect of ADC-1, ADC-2, and ADC-3 on the growth of CHO-K1 cells, which do not express DLL3. [Figure 6] The results of the bystander cytotoxicity effects of ADC-1, ADC-2, and ADC-3 are shown. [Figure 7] 1 shows the results of ADC-1, ADC-2, ADC-4, and ADC-5 inhibiting the growth of subcutaneously transplanted DMS53 cell tumors in mice. [Figure 8] 1 shows the results of ADC-1 and ADC-3 inhibiting the growth of subcutaneously transplanted DMS53 cell tumors in mice. [Figure 9] Figure 1 shows the results of ADC-2, ADC-3, ADC-6, and ADC-7 on the inhibition of the growth of subcutaneously transplanted H1184 cell tumors in mice. [Figure 10] Pharmacokinetic results of ADC-1, ADC-2, and ADC-3 in rats are shown, where Figure 10A shows the pharmacokinetic results of ADC-1 in rats, Figure 10B shows the pharmacokinetic results of ADC-2 in rats, and Figure 10C shows the pharmacokinetic results of ADC-3 in rats. [Figure 11] Pharmacokinetic results of ADC-2 in cynomolgus monkeys are shown. DETAILED DESCRIPTION OF THE INVENTION

[0064] 1. Terminology In order that the present disclosure may be more readily understood, the following provides an explanation of some technical and scientific terms. Unless expressly defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0065] Unless specifically stated to the contrary, terms used in the specification and claims have the following meanings.

[0066] As used in the specification and claims, the singular forms "a," "an," and "said" include plural referents unless the context clearly dictates otherwise.

[0067] Unless the context clearly indicates otherwise, in the patent specification and claims, the words "comprises," "has," "includes," and the like are to be understood in the sense of "including, but not limited to," rather than in the exclusive or exhaustive sense.

[0068] The term "and / or" is intended to be inclusive. 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, it is intended to include formulations of products under that trade name, drugs, and active drug moieties of products under that trade name.

[0069] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0070] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, γ-carboxyglutamate, 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 bonded 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 a manner similar to a naturally occurring amino acid.

[0071] 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 used to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced binding to Fc receptors. Deletions and insertions in the amino acid sequence include deletions and insertions 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 naturally occurring 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 for modifying amino acid side groups other than genetic engineering, such as chemical modification, may also be utilized. Herein, various names may be used to refer to the same amino acid mutation. Herein, the amino acid residue at a specific site may be designated by the format 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 with W.

[0072] The term "antibody" is used in the broadest sense and covers a variety of 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 portions thereof), provided they exhibit the desired antigen-binding activity. Intact antibodies typically contain two light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain contains one variable region (VH), also called a variable heavy domain or 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 contains one variable region (VL), also called a variable light domain or light chain variable domain, followed by one constant light domain (light chain constant region, CL).

[0073] The terms "full-length antibody," "complete antibody," and "whole antibody" may be used interchangeably herein and refer to antibodies having a structure substantially similar to that of a naturally occurring antibody or having an Fc region in the heavy chain. The light chain of a naturally occurring complete antibody comprises a light chain variable region VL and a constant region CL, with VL at the amino terminus of the light chain and the light chain constant region comprising a kappa chain and a lambda chain. The heavy chain comprises a variable region VH and constant regions (CH1, CH2, and CH3), with VH at the amino terminus of the heavy chain and the constant region at the carboxy terminus, of which CH3 is closest to the carboxy terminus. 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.

[0074] 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 light chain variable region (VL) of an antibody each contain four conserved framework regions (FR) and three complementarity-determining regions (CDR). The term "complementarity-determining region" or "CDR" refers to the region in the variable domain that primarily mediates antigen binding, while "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, while 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 referred to as the N-terminus) to the carboxy terminus (also referred to as the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0075] The boundaries of the amino acid sequences of 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). The correspondence between various numbering systems is well known to those skilled in the art and is shown, for example, in Table 1 below.

[0076] [Table 1]

[0077] Unless otherwise stated, both variable region and CDR sequences in this disclosure follow the Kabat numbering convention.

[0078] The term "antibody fragment" refers to a molecule distinct from an intact antibody and includes a portion of the intact antibody that binds to the same antigen as the intact antibody. 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.

[0079] The term "Fc region" or "fragment crystallizable region" is intended to define the C-terminal region of an antibody heavy chain, and includes 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 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 of the Fc region (residue 447 according to the EU numbering system) or deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise specified, the numbering convention for the Fc region is the EU numbering system, also known as the EU index.

[0080] 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.

[0081] The term "humanized antibody" refers to an antibody that retains the reactivity of a non-human antibody while having relatively 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 their human counterparts (i.e., the constant and variable region framework region portions).

[0082] 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 derived from human genes but with sequences that, for example, reduce potential immunogenicity, increase affinity, or remove cysteines or glycosylation sites that may cause undesired folding. The terms also cover 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 containing human immunoglobulin heavy and light chain loci. The definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0083] 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 usually be expressed as a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0084] As used herein, the terms "kassoc" or "ka" refer to the association rate of a particular antibody-antigen interaction, and the terms "kdis" or "kd" refer 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 can be measured by a biosensing system such as a surface plasmon resonance system, or by solution equilibrium titration (SET).

[0085] The term "effector function" refers to a biological activity that can be attributed to an antibody Fc region (a native sequence Fc region or an amino acid sequence mutated Fc region) and that varies depending on 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.

[0086] 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 possible minor natural mutations. In contrast, a polyclonal antibody preparation typically contains a plurality of different antibodies with different amino acid sequences in their variable domains, which are generally specific for different epitopes. "Monoclonal" should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0087] 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 can interact with different antigen-binding proteins (e.g., antibodies).

[0088] The term "epitope" refers to an area or region on an antigen capable of specific binding by an antibody or antigen-binding fragment thereof. An epitope may be formed by a contiguous string of amino acids (linear epitope) or may include non-contiguous amino acids that are spatially adjacent, for example, due to folding (i.e., tertiary folding) of the antigen (conformational epitope). Conformational and linear epitopes differ in that antibody binding to a conformational epitope is undetectable 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 conventional methods in the art, including, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0089] 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 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 measured by BIACORE® surface plasmon resonance assays.

[0090] 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 antigens with a greater affinity than other antigens or epitopes. -7 M or less (e.g., about 1 × 10 -8An antibody binds to an antigen or its epitope with an equilibrium dissociation constant (KD) of 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 for binding to a non-specific antigen (e.g., BSA, casein) of the antibody. KD can be measured by known methods, for example, by BIACORE® surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or its epitope may be cross-reactive with other related antigens, for example, with corresponding antigens from other species (homologs) (e.g., humans, or monkeys such as Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes (chimpanzee, chimp)) or common marmoset (Callithrix jacchus, marmoset).

[0091] The term "nucleic acid" may be used interchangeably with the term "polynucleotide" herein 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. Such nucleic acids may be synthetic, naturally occurring, or non-naturally occurring, and have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell, as defined below, which typically 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 implicitly covers its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly stated sequence. Specifically, as detailed below, degenerate codon substitutions can be obtained by substituting mixed-base and / or deoxyinosine residues in the third position of one or more selected (or all) codons to produce sequences.

[0092] The terms "polypeptide" and "protein" may be used interchangeably herein and 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 specified, a particular polypeptide sequence also implicitly covers conservatively modified variants thereof.

[0093] The term "sequence identity" refers to the degree (percentage) of homology between two sequences at equivalent amino acid / nucleic acid positions. When optimally aligning two sequences, gaps may be introduced, if necessary, to maximize the percentage of identity. Conservative substitutions are not considered part of the sequence identity. To measure the percentage of sequence identity, alignment can be achieved using 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 and align sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0094] 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 loop 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 are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell after introduction and thereby replicate 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, when present, RNA splicing of the coding region operably linked to it.

[0095] 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, including "transformants" and "transformed cells," include the original transformed cell and its derived progeny, regardless of the number of passages. 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 (COS) cells, 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, and Trichoderma reesei.

[0096] 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.

[0097] A "drug" (abbreviation: D) is any substance with biological or detectable activity (e.g., therapeutic agents, detectable labels, binding agents, etc.) and any prodrug that is metabolized to an active drug in the body. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cell growth inhibitory agents, 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 inhibitory agents.

[0098] A cytotoxic effect refers to the deletion, elimination, and / or killing of targeted cells. A cytotoxic agent refers to an agent that has a cytotoxic and / or cell growth inhibitory effect on cells. A cell growth inhibitory effect refers to the inhibition of cell proliferation. A cell growth inhibitory agent refers to an agent that has a cell growth inhibitory effect on cells, thereby inhibiting the growth and / or proliferation of a specific subpopulation of cells.

[0099] Additional exemplary 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 a therapeutic agent can be described by one or more of the above terms. For example, the radioisotope selected may also be a cytotoxin. The therapeutic agent may be prepared as a pharmaceutically acceptable salt, acid, or derivative of any one of the above. Typically, conjugates having a radioisotope as the agent are called radioimmunoconjugates, and conjugates having a chemotherapeutic agent as the agent are called chemoimmunoconjugates.

[0100] Examples of cytotoxic agents include, but are not limited to, exatecan, 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, and photosensitizers (i.e., for photodynamic therapy) may also be used.

[0101] The term "label," as used herein, refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody to produce a "labeled" antibody. The label may be itself detectable (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.

[0102] The terms "linker unit" and "linker" refer 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 another linker before being linked to the antibody or drug. Linking of the linker to the antibody may be achieved 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 tags containing acyl donor glutamines or endogenous glutamines made reactive by modification of the polypeptide in the presence of transglutaminase and amines. Several ADC linkage systems are known in the art and include hydrazone-, disulfide-, and peptide-based linkages.

[0103] The linker may comprise 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").

[0104] The linker may be selected from the group consisting of an extender, a spacer, and an amino acid unit, 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 drug release 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.

[0105] Linker elements include, but are not limited to: [ka] MC=6-maleimidocaproyl, 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 was modified to prevent cleavage 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.

[0106] "LD" is the linker-drug moiety produced by linking a drug (D) to a linker (L).

[0107] The "drug loading," also known as the drug-to-antibody ratio (DAR), is the average number of drugs coupled to each antibody in an ADC. It can range, for example, from about 1 to about 10 drugs coupled to each antibody, and in some embodiments, from about 1 to about 8 drugs coupled to each antibody, preferably from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 1 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 5 to 6, 5 to 7, 5 to 8, and 6 to 8. The general formula of an ADC of the present disclosure includes a collection of antibody-drug conjugates within the above-mentioned range. In embodiments of the present disclosure, the drug loading may be represented by n, which may be a decimal or an integer. The drug loading can be measured by common methods such as UV / visible spectroscopy, mass spectrometry, ELISA, HIC, and RP-HPLC.

[0108] In one embodiment of the present disclosure, the drug is coupled to a reactive group (eg, a sulfhydryl group) of the antibody via a linker.

[0109] The drug loading of the ADC is (1) controlling the molar ratio of the linking reagent to the monoclonal antibody; (2) controlling the 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:

[0110] 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 1-20The alkyl group is an alkyl group having 1 to 12 carbon atoms (i.e., C 1-12 alkyl groups) are preferred, but alkyl groups having 1 to 6 carbon atoms (i.e., C 1-6alkyl groups) are more preferred. 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, Examples of 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 point of attachment, 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.

[0111] The term "alkylene group" refers to a divalent alkyl group, wherein alkyl 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) are preferred, but alkylene groups having 1 to 6 carbon atoms (i.e., C 1-6 Alkylene groups are more preferred. Non-limiting examples include -CH-, -CH(CH)-, -CH(CH)-, -CHCH-, -CH(CH)-, -CHCH-, -CH(CHCH)-, -CHCH(CH)-, -CHCHCH(CH)-, -CHCHCH-, -CHCHCHCH-, and the like. The alkylene group can be substituted or unsubstituted, and if substituted, it can be substituted at any available point of attachment, and the substituent is preferably one or more selected from a deuterium atom, a halogen, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxy group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0112] The term "alkenyl group" refers to an alkyl group containing at least one carbon-carbon double bond in the molecule, where alkyl is as defined above, and having 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 substituent is preferably one or more selected from a deuterium atom, an alkoxy group, a halogen atom, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxy group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0113] The term "alkynyl group" refers to an alkyl group containing at least one carbon-carbon triple bond in the molecule, wherein alkyl is as defined above, and having 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, and the like. The alkynyl group may be substituted or unsubstituted, and if substituted, it may be substituted at any available point of attachment, and the substituent is preferably one or more selected from a deuterium atom, an alkoxy group, a halogen atom, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxy group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0114] The term "alkoxy group" refers to an -O-(alkyl group), wherein alkyl is defined above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy groups. The alkoxy group may be substituted or unsubstituted, and if substituted, may be substituted at any available point of attachment, and the substituent is preferably one or more selected from a deuterium atom, a halogen, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxy group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0115] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic all-carbon 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 even more preferably a cycloalkyl group having 3 to 6 ring atoms (i.e., a 3- to 6-membered cycloalkyl group, C 3-6 Cycloalkyl groups are most preferred.

[0116] The monocyclic cycloalkyl groups include, by way of non-limiting example, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups.

[0117] The polycyclic cycloalkyl groups include spirocycloalkyl groups, fused cycloalkyl groups and bridged cycloalkyl groups.

[0118] The term "spirocycloalkyl group" refers to a polycyclic ring system in which the rings share one carbon atom (referred to as a spiro atom), and 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 nitrogen may optionally be oxidized, i.e., to form nitrogen oxides, and which sulfur may optionally be substituted with an oxo group, i.e., to form sulfoxides or sulfones, but which does 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 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 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), and more preferably a spirocycloalkyl group having 7 to 10 ring atoms (i.e., a 7- to 10-membered spirocycloalkyl group). The spirocycloalkyl group includes monospirocycloalkyl groups and polyspirocycloalkyl groups (e.g., bisspirocycloalkyl groups), and is preferably a monospirocycloalkyl group or a bisspirocycloalkyl group, and 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 include: [ka] The connection point may be located in any position.

[0119] The term "fused cycloalkyl group" refers to a polycyclic ring system in which rings share two adjacent carbon atoms, such as a monocyclic cycloalkyl group fused with one or more monocyclic cycloalkyl groups, or a monocyclic cycloalkyl group fused with one or more heterocyclyl groups, aryl groups, or heteroaryl groups, where the point of attachment is on the monocyclic cycloalkyl group, which may contain one or more double bonds within 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- to 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- to 14-membered fused cycloalkyl group), and more preferably a fused cycloalkyl group having 7 to 10 ring atoms (i.e., a 7- to 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: [ka] The connection point may be located in any position.

[0120] 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 linked to each other, and may contain one or more double bonds within 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- to 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- to 14-membered bridged cycloalkyl group), and more preferably a bridged cycloalkyl group having 7 to 10 carbon atoms (i.e., a 7- to 10-membered bridged cycloalkyl group). The bridged cycloalkyl group includes bicyclic bridged cycloalkyl groups and polycyclic bridged cycloalkyl groups (e.g., tricyclic bridged cycloalkyl groups, tetracyclic bridged cycloalkyl groups, etc.), and is preferably a bicyclic bridged cycloalkyl group or a tricyclic bridged cycloalkyl group. Non-limiting examples include: [ka] Including, The connection point may be located in any position.

[0121] The cycloalkyl group may be substituted or unsubstituted, and when 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, oxo groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups and heteroaryl groups.

[0122] 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 is optionally oxidized, i.e., to form a nitrogen oxide, and the sulfur is 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), still 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).

[0123] Examples of the monocyclic heterocyclyl group include, but are not limited to, pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl groups.

[0124] The polycyclic heterocyclyl groups include spiroheterocyclyl groups, fused heterocyclyl groups and bridged heterocyclyl groups.

[0125] 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) heteroatom selected from nitrogen, oxygen, and sulfur within the ring (wherein the nitrogen is optionally oxidized, i.e., to form a nitrogen oxide, and the sulfur is 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, 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 spiroheterocyclyl group). The 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 (e.g., bisspiroheterocyclyl groups), preferably a monospiroheterocyclyl group or a bisspiroheterocyclyl 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 monospiroheterocyclyl group. Non-limiting examples are: [ka] Includes:

[0126] 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) heteroatom selected from nitrogen, oxygen, and sulfur within the ring, wherein the nitrogen is optionally oxidized, i.e., to form a nitrogen oxide, and the sulfur is optionally substituted with an oxo group, i.e., to form a sulfoxide or sulfone, but is not limited to -OO-, -OS-, or -SS-. (excluding the following), 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, in which the connection 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- to 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- to 14-membered fused heterocyclyl group), and more preferably a fused heterocyclyl group having 7 to 10 ring atoms (i.e., a 7- to 10-membered fused heterocyclyl group). The fused heterocyclyl group includes bicyclic and polycyclic fused heterocyclyl groups (e.g., tricyclic fused heterocyclyl groups, tetracyclic fused heterocyclyl groups, etc.), preferably a bicyclic fused heterocyclyl group or a tricyclic fused heterocyclyl 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 bicyclic fused heterocyclyl group. Non-limiting examples are: [ka] Includes:

[0127] The term "bridged heterocyclyl group" refers to a polycyclic heterocyclic ring system in which the rings share two atoms that are not directly linked, and which optionally contains one or more double bonds within the ring, and which contains at least one (e.g., 1, 2, 3, or 4) heteroatom selected from nitrogen, oxygen, and sulfur within the ring (wherein the nitrogen is optionally oxidized, i.e., to form a nitrogen oxide, and the sulfur is optionally 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 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] Includes:

[0128] The heterocyclyl group may be substituted or unsubstituted, and when substituted, it may be substituted at any available point of attachment, and the substituents are preferably one or more selected from deuterium 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 "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 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 6- to 14-membered aryl group). The aryl group is preferably an aryl group having 6 to 10 ring atoms (i.e., a 6- to 10-membered aryl group). An example of the monocyclic aryl group is a phenyl group. Non-limiting examples of the polycyclic aryl group include a naphthyl group, an anthryl group, and a phenanthryl group. The polycyclic aryl groups further include a phenyl group fused to one or more heterocyclyl or cycloalkyl groups, or a naphthyl group fused to one or more heterocyclyl or cycloalkyl groups, 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 are: [ka] Includes:

[0130] The aryl group may be substituted or unsubstituted, and when 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, oxo groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups.

[0131] 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 (the nitrogen may optionally be oxidized, i.e., to form a nitrogen oxide, and the 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 having 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 5- to 14-membered heteroaryl group). The heteroaryl group is preferably a heteroaryl group having 5 to 10 ring atoms (i.e., a 5- to 10-membered heteroaryl group), and more preferably a heteroaryl group having 5 or 6 ring atoms (i.e., a 5- or 6-membered heteroaryl group).

[0132] Non-limiting examples of the monocyclic heteroaryl group include furanyl, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furazanyl, pyrrolyl, N-alkylpyrrolyl, pyridyl, pyrimidine, pyridonyl, and N-alkylpyridone (e.g., [ka] etc.), pyrazinyl group, pyridazinyl group, etc.

[0133] Non-limiting examples of the polycyclic heteroaryl group include indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalyl, phthalazinyl, benzimidazolyl, benzothienyl, quinazolinyl, benzothiazolyl, carbazolyl, etc. The polycyclic heteroaryl group also includes a monocyclic heteroaryl group fused with 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 group also includes a monocyclic heteroaryl group fused with 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 include: [ka] Includes:

[0134] The heteroaryl group may be substituted or unsubstituted, and when substituted, it may be substituted at any available point of attachment, and the substituent is preferably one or more selected from a deuterium atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxy group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0135] The term "amino-protecting group" refers to a group that is easily removed and introduced into an amino group to prevent the amino group from being altered when other parts of the molecule react. 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.

[0136] The term "hydroxy-protecting group" refers to a labile group 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.

[0137] The term "cycloalkyloxy" refers to a cycloalkyl-O- group, in which the cycloalkyl group is as defined above.

[0138] The term "heterocyclyloxy" refers to a heterocyclyl-O- group, wherein the heterocyclyl group is as defined above.

[0139] The term "aryloxy group" refers to an aryl-O- group, in which the aryl group is as defined above.

[0140] The term "heteroaryloxy" refers to a heteroaryl-O-, wherein the heteroaryl group is as defined above.

[0141] The term "alkylthio" refers to alkyl-S-, in which the alkyl group is as defined above.

[0142] The term "haloalkyl group" refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above.

[0143] The term "haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein the alkoxy group is as defined above.

[0144] The term "deuterated alkyl group" refers to an alkyl group substituted with one or more deuterium atoms, wherein the alkyl group is as defined above.

[0145] The term "hydroxyalkyl group" refers to an alkyl group substituted with one or more hydroxy groups, wherein the alkyl group is as defined above.

[0146] The term "methylene group" refers to =CH2.

[0147] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0148] The term "hydroxy group" refers to -OH.

[0149] The term "mercapto" refers to -SH.

[0150] The term "amino group" refers to -NH2.

[0151] The term "cyano" refers to -CN.

[0152] The term "nitro group" refers to -NO2.

[0153] The term "oxo" or "oxo group" refers to "=O".

[0154] The term "carbonyl group" refers to C=O.

[0155] The term "carboxy" refers to -C(O)OH.

[0156] 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 alkyl and cycloalkyl are as defined above.

[0157] The abbreviation "Me" in the chemical formula is the methyl group.

[0158] The abbreviation "Ph" in the chemical formula stands for the phenyl group.

[0159] The term "THF" refers to tetrahydrofuran.

[0160] The term "EtOAc" refers to ethyl acetate.

[0161] The term "MeOH" refers to methanol.

[0162] The term "DMF" refers to N,N-dimethylformamide.

[0163] The term "DIPEA" refers to diisopropylethylamine.

[0164] The term "TFA" refers to trifluoroacetic acid.

[0165] The term "MeCN" refers to acetonitrile.

[0166] The term "DMA" refers to N,N-dimethylacetamide.

[0167] The term "Et2O" refers to diethyl ether.

[0168] The term "DCE" refers to 1,2-dichloroethane.

[0169] The term "DIPEA" refers to N,N-diisopropylethylamine.

[0170] The term "NBS" refers to N-bromosuccinimide.

[0171] The term "NIS" refers to N-iodosuccinimide.

[0172] The term "Cbz-Cl" refers to benzyl chloroformate.

[0173] The term "Pd2(dba)3" refers to tris(dibenzylideneacetone)dipalladium.

[0174] The term "Dppf" refers to 1,1'-bisdiphenylphosphinoferrocene.

[0175] The term "HATU" refers to 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0176] The term "KHMDS" refers to potassium hexamethyldisilazide.

[0177] The term "LiHMDS" refers to lithium bis(trimethylsilyl)amide.

[0178] The term "MeLi" refers to methyllithium.

[0179] The term "n-BuLi" refers to n-butyllithium.

[0180] The term "NaBH(OAc)3" refers to sodium triacetoxyborohydride.

[0181] The term "DCM" refers to dichloromethane.

[0182] The term "DMAP" refers to 4-dimethylaminopyridine.

[0183] The term "DMBOH" refers to 2,4-dimethoxybenzyl alcohol.

[0184] The term "EDCI" refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0185] The term "MTBE" is methyl tert-butyl ether.

[0186] The term "DMF" refers to N,N-dimethylformamide.

[0187] The term "DMTMM" refers to 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride.

[0188] The term "EtOAc" refers to ethyl acetate.

[0189] JPEG2026508154000028.jpg74154

[0190] JPEG2026508154000029.jpg25154

[0191] 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 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]

[0192] For example, a reference to a pyrazolyl group should be understood to include a mixture of any one or two tautomers of the following two structures: [ka]

[0193] All tautomeric forms are within the scope of the disclosure, and the naming of a compound does not exclude any tautomeric form.

[0194] 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, and deuterium is preferred.

[0195] 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 of 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 may be independently replaced with a deuterium atom, where deuterium substitution may be partial or complete, and partial deuterium substitution refers to the replacement of at least one hydrogen with at least one deuterium.

[0196] 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 (which is 0.015%) (i.e., at least 15% deuterium incorporated). 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 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 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 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 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 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 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 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 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 incorporated).

[0197] "Substituted" or "substituted" refers to one or more hydrogen atoms, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms in a group, being independently replaced with a corresponding number of substituents. Those 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).

[0198] The present disclosure further includes various deuterated forms of antibody-drug conjugates of formula (Pc-LYD). Each available hydrogen atom linked to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of antibody-drug conjugates of formula (Pc-LYD) by referring to relevant literature. When preparing deuterated forms of antibody-drug conjugates of formula (Pc-LYD), commercially available deuterated starting materials may be used, or they may be synthesized using common techniques with deuterated reagents, including, but not limited to, deuterated borane, tritiated borane in tetrahydrofuran, lithium aluminum deuterated hydride, deuterated iodoethane, and deuterated iodomethane.

[0199] "Optionally" or "optionally" means that the event or circumstance described thereafter may or may not occur, and includes two cases: when the event or circumstance occurs and when it does not occur. For example, "an alkyl group optionally (arbitrarily) substituted with a halogen or a cyano group" includes a case where the alkyl group is substituted with a halogen or a cyano group, and a case where the alkyl group is not substituted with a halogen or a cyano group.

[0200] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or physiologically / medicinally acceptable salts or prodrugs thereof and other chemical components, together with other components such as physiologically / medicinally acceptable carriers and excipients, to facilitate administration to the body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0201] The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Acceptable solvents and vehicles include water, Ringer's solution, and isotonic sodium chloride solution. Sterile injectable preparations may also be sterile injectable oil-in-water microemulsions in which the active ingredient is dissolved in the oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol and processed to form a microemulsion. The injectable solutions or microemulsions can be infused into the subject's bloodstream via localized bolus injection. Alternatively, solutions and microemulsions can be administered in a manner that maintains a constant, cyclical concentration of the compounds of the present disclosure. To maintain such a constant concentration, a continuous intravenous administration device can be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous pump.

[0202] The pharmaceutical compositions may be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. Such suspensions can be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents, as described above. Sterile injectable preparations may also be sterile injectable solutions or suspensions prepared in non-toxic, gastrointestinal-acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Sterile fixed oils are also conveniently used as solvents or suspending media. For this purpose, any suitable fixed oil, including synthetic monoglycerides or diglycerides, can be used. Fatty acids, such as oleic acids, can also be used to prepare injectables.

[0203] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of an antibody-drug conjugate of the present disclosure, which has safety and effectiveness when used in a subject, as well as the desired biological activity. By way of example, the antibody-drug conjugate of the present disclosure contains at least one amino group and can therefore form salts with acids, and non-limiting examples of medicinal salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, 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.

[0204] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, stabilizers, or preservatives.

[0205] The term "excipient" refers to an additive other than an active ingredient in a drug formulation, and may also be called an additive. For example, binders, fillers, disintegrants, and lubricants in tablets, base materials in semi-solid preparations such as ointments and creams, and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure adjusters, and coloring agents in liquid preparations may all be called excipients.

[0206] The term "diluent," also known as a filler, is primarily used to increase the weight and volume of tablets. The addition of a diluent not only ensures a consistent volume, but also reduces the dosage deviation of the active ingredient and improves the compressibility of the drug. When a tablet contains an oily ingredient, an absorbent must be added to absorb the oily substance in order to maintain a "dry" state and facilitate tablet preparation. Examples include starch, lactose, inorganic calcium salts, and microcrystalline cellulose.

[0207] 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" or "subject" may be used interchangeably herein. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

[0208] "Administration" or "giving," when used in reference to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

[0209] The term "sample" refers to a collection of fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are biological fluids, such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected from bronchial washings, synovial fluid, liquid solutions that have come into contact with a subject or biological source, such as cell and organ media (including cell- or organ-conditioned media), lavage fluids, tissue biopsies, fine needle aspirates, surgically resected tissues, organ cultures, or cell cultures.

[0210] "Treatment" and "treatment" (and grammatical variations thereof) refer to clinical intervention that seeks to alter the pathological process in the individual being treated, and can be performed prophylactically or during clinical pathological processes. 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 consequences of the disease, prevention of metastasis, slowing 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 slow the progression of disease.

[0211] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or their underlying causes, prevent the onset of symptoms and / or their underlying causes, and / or ameliorate 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 in any way with the disease state. 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 with a single dose, but may occur after a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or multiple doses. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on several 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 indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

[0212] II. Description of Specific Embodiments A. Antibody Structure in Antibody-Drug Conjugates In certain embodiments, the antibodies provided herein are full-length antibodies.

[0213] In certain embodiments, the antibodies provided herein are antibody fragments.

[0214] 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 the VL, VH, CL, and CH1 domains. "Fab fragments" may be produced by lysing an antibody with papain. "Fab'" contains the VL, CL, and VH and CH1 domains, and also contains the region between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains of two Fab' fragments to form F(ab')2 molecules. "Fab'-SH" is a Fab' fragment in which cysteine ​​residues in the constant regions have free sulfhydryl groups. "F(ab')2" is a bivalent fragment containing two Fab fragments linked by disulfide bonds at the hinge region.

[0215] In another embodiment, the antibody fragment is a diabody, a triabody, or a tetrabody. A diabody is an antibody fragment having 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 between the two domains on the same chain, forcing them to pair with complementary domains on another chain, thereby generating two antigen-binding sites, where the two antigens may be the same or different.

[0216] 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, wherein the domains and 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 native disulfide bond between CL and CH1. Additionally, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., position 44 in the heavy chain variable region and position 100 in the light chain variable region, according to Kabat numbering) to create interchain disulfide bonds.

[0217] 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, wherein the light chain variable region and the heavy chain variable region are contiguously linked by a short flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv maintains the specificity of the intact antibody from which it is derived. Unless otherwise specified herein, an scFv may have the VL and VH variable regions in any one order; for example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv may comprise VL-linker-VH or VH-linker-VL.

[0218] In another embodiment, the antibody fragment is an Fd fragment consisting of the VH and CH1 domains.

[0219] In another embodiment, the antibody fragment is an Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0220] 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 substituted with a cysteine ​​residue via a disulfide bond between the cysteine ​​residues. The amino acid residue substituted with the cysteine ​​residue can be selected based on prediction of the three-dimensional structure of an antibody by a known method (Protein Engineering. 7:697 (1994)).

[0221] 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.

[0222] 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 corresponding 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 lines. dAbs are also highly stable and retain activity even under harsh conditions, such as 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.

[0223] In certain 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 mouse, rat, hamster, rabbit, or non-human primate such as a 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.

[0224] 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. Generally, a humanized antibody comprises one or more variable regions in which CDRs or portions thereof are derived from a non-human antibody and FRs or portions thereof are derived from a human antibody. Optionally, a humanized antibody may further comprise a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody may be replaced with corresponding residues from a non-human antibody (e.g., the antibody providing the CDR sequences).

[0225] Humanized antibodies and methods for their production are generally described in, for example, 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 "resurfacing"), 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 "guided selection" methods of FR shuffling).

[0226] 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 derived from consensus sequences of human antibodies of a particular subclass of light or heavy chain variable region (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. Immunol. 151:2623 (1993)). al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0227] B. Antibody Modification in Antibody-Drug Conjugates In some embodiments, amino acid sequence variants of the antibodies in the antibody conjugates provided herein are carved out. For example, these variants are expected to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions, insertions, and / or substitutions of residues in the antibody amino acid sequence. The final construct can be obtained by any combination of deletions, insertions, and substitutions, provided that the final construct possesses the desired properties, such as antigen-binding properties.

[0228] a) Substitutions, insertions and deletions of mutants In one embodiment, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions." More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions," and are 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.

[0229] [Table 2]

[0230] 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.

[0231] Non-conservative substitutions involve substituting a member of one of these classes for a member of another class.

[0232] 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 antibody's ability to bind to antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) can be made to a CDR that do not substantially reduce binding affinity. Such changes may, for example, be outside of the antigen-contacting 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.

[0233] A method that can be used to identify potential target sites for mutagenesis in antibodies 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 a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Additionally, contact points between the antibody and antigen can be identified by examining crystal structures of antigen-antibody complexes. These contact residues and adjacent residues can be targeted or removed as substitution candidates. Mutants can be screened to determine whether they contain desired properties.

[0234] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions of polypeptides ranging in length from one residue to 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is 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 of an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0235] 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.

[0236] In some embodiments, the one or more amino acid modifications reduce Fc binding to an Fc receptor, e.g., its binding to an Fcγ receptor, and can 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, or 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., by introducing M252Y / S254T / T256E mutations into the Fc region. In some embodiments, the altered 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 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 substitution of amino acid residues 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, as 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.

[0237] 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.

[0238] In some embodiments, the Fc domain of an 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). Generally, this technique involves introducing a protrusion ("knob") into the interface of a first polypeptide (e.g., the first CH3 domain in a first antibody heavy chain) and a cavity ("hole") into the interface of a second polypeptide (e.g., the second CH3 domain in a second antibody heavy chain) so 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 a first antibody heavy chain) with a relatively large side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan). A complementary cavity having the same or similar size as 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 the 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 containing the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc domain containing the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues allows for 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-into-hole mutations include, but are not limited to, those listed in Table 3.

[0239] [Table 3] 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).

[0240] The C-terminus of the Fc region may be a complete C-terminus ending with amino acid residues PGK, or may be a truncated C-terminus, e.g., one or two C-terminal amino acid residues have already been removed. In one preferred embodiment, the C-terminus of the heavy chain is a truncated C-terminus ending with PG. Thus, in some embodiments, a composition of whole antibodies may include antibodies in which any of the K447 and / or G446+K447 residues have been removed. In some embodiments, a composition of whole antibodies may include antibodies in which the K447 and / or G446+K447 residues have not been removed. In some embodiments, a composition of whole antibodies includes a mixture of antibodies with and without the K447 and / or G446+K447 residues.

[0241] C. 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.

[0242] In one embodiment, the present disclosure provides isolated nucleic acids encoding the above-described antibodies. Such nucleic acids can each independently encode any one of the above-described polypeptide chains. In another aspect, the present disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In another aspect, the present disclosure provides host cells comprising such nucleic acids. In one embodiment, a method for preparing an anti-DLL3 antibody is provided, the method comprising culturing a host cell comprising 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).

[0243] 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 acids can be readily isolated and sequenced using standard procedures, or produced by recombinant methods, or obtained by chemical synthesis.

[0244] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly 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.

[0245] D. Measurement The antibody-drug conjugates 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 antibody-drug conjugates of the present disclosure is measured by known methods, such as ELISA, Western blotting, etc.

[0246] E. Treatment Methods and Routes of Administration Any of the antibody-drug conjugates or pharmaceutically acceptable salts thereof provided herein can be used to treat diseases.

[0247] In one aspect, the present disclosure provides the use of an anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a tumor or cancer, hi some embodiments, the present disclosure provides the use of an anti-DLL3 antibody-drug conjugate or a pharmaceutically 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 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 gland cancer, glioblastoma, skin cancer, and melanoma; preferably, the lung cancer is small cell lung cancer.

[0248] In some embodiments, the present disclosure provides a use of an anti-DLL3 antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a DLL3-associated disease.

[0249] 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).

[0250] In a further aspect, a pharmaceutical composition is provided comprising the anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof, e.g., for use in any of the pharmaceutical uses or therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises any of the polypeptides or drug conjugates provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0251] The anti-DLL3 antibody-drug conjugates or pharmaceutically acceptable salts thereof of the present disclosure may be used for therapy alone or in combination with other agents, for example, the anti-DLL3 antibody-drug conjugates or pharmaceutically acceptable salts thereof of the present disclosure may be administered with at least one additional therapeutic agent.

[0252] The anti-DLL3 antibody-drug conjugates or pharmaceutically acceptable salts thereof of the present disclosure can be administered by any suitable means, including parenteral, pulmonary, and intranasal administration, and, when localized treatment is required, intralesional administration. Parenteral infusions include 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 administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0253] The anti-DLL3 antibody-drug conjugates or pharmaceutically acceptable salts thereof of the present disclosure are 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 condition of the individual patient, the cause of the condition, the delivery site of the reagent, the method of administration, the administration schedule, and other factors known to medical professionals. 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 by the same routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at different dosages and by any appropriate route as empirically / clinically determined.

[0254] The appropriate dose of an anti-DLL3 antibody-drug conjugate of the present disclosure, or a pharmaceutically acceptable salt thereof (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 treating physician. The therapeutic molecule is administered to the patient at one time or over a series of treatments, as appropriate.

[0255] F.Product In another aspect of the present disclosure, an article of manufacture (e.g., a kit) is provided containing materials that can be used to treat, prevent, and / or diagnose the above-described 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.

[0256] The container contains the anti-DLL3 antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, either 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-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof. The label or package insert indicates that the composition is used for treating the condition of choice.

[0257] The article of manufacture may also include (a) a first container containing the anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof, and (b) a second container containing a composition comprising another cytotoxic agent or other therapeutic agent.

[0258] Alternatively, or in addition, the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer. From a business and user standpoint, it may further comprise other materials necessary, including other buffers, diluents, filters, needles and syringes. [Example]

[0259] 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.

[0260] In the examples or test examples of this disclosure, experimental methods for which specific conditions are not specified generally follow common conditions or conditions recommended by raw material or product manufacturers. Reagents for which specific sources are not specified are commonly available commercially.

[0261] 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 cells 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): [Case 2] [ka] Cynomolgus monkey DLL3 full-length protein (Uniprot, A0A2K5WSR4): [C3] [ka] Rat DLL3 full-length protein (Uniprot, O88671): [C4] [ka] Mouse DLL3 full-length protein (Uniprot, O88516): [5] [ka] Human DLL1 full-length protein (Uniprot, O00548): [6] [ka] Human DLL4 full-length protein (Uniprot, Q9NR61): [7] [ka]

[0262] 1.1 Construction of cell lines highly expressing DLL3, DLL1, and DLL4 pCDH lentiviral expression vector plasmids (synthesized by GENEWIZ) containing SEQ ID NOs: 1–6 and pCDH plasmids were transfected into 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17) using Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent along with pVSVG and pCMV lentiviral packaging vectors, respectively. The virus-containing culture supernatant was collected, filtered, and centrifuged at ultrahigh speed. The supernatant was discarded and resuspended in 0.2 mL of sterile PBS. The concentrated virus was then infected into Chinese hamster ovary cells CHO-s (Invitrogen, R80007), DMS53 (ATCC, CRL-2062), and H82 (ATCC, HTB-175), respectively. The cells were screened with puromycin for 2–3 weeks and then subjected to FACS single-cell sorting. Selected monoclonal cell lines were expanded and cryopreserved.

[0263] 1.2 Antigen preparation Using the human DLL3 (Uniprot, Q9NYJ7), cynomolgus monkey DLL3 (Uniprot, A0A2K5WSR4), and mouse DLL3 (Uniprot, O88516) sequences as templates, DLL3 ECD fusion proteins containing different tags were designed and cloned into pTT5 vectors. After expression in 293E cells, antigens were obtained. The amino acid sequences of the relevant proteins are as follows: 1) His-hDLL3(ECD): [8] [ka] NOTE: The dotted part is the signal peptide sequence, the single underlined part is the his-tag and linker, and the double underlined part is the DLL3 extracellular region.

[0264] 2) Fc-hDLL3(ECD): [9] [ka] NOTE: The dotted part is the signal peptide sequence, the single underlined part is the Fc tag and linker, and the double underlined part is the DLL3 extracellular region.

[0265] 3) hDLL3(ECD)-strep twin: [C10] [ka] NOTE: The dotted part is the signal peptide sequence, the double underlined part is the DLL3 extracellular region, and the single underlined part is the strep twin tag.

[0266] 4) cynoDLL3(ECD) - strep twin: [C11] [ka] NOTE: The dotted part is the signal peptide sequence, the double underlined part is the DLL3 extracellular region, and the single underlined part is the strep twin tag.

[0267] 5) mouDLL3(ECD) -strep twin: [C12] [ka] NOTE: The dotted part is the signal peptide sequence, the double underlined part is the DLL3 extracellular region, and the single underlined part is the strep twin tag.

[0268] Example 2: Preparation of mouse anti-human DLL3 monoclonal antibody 1.Immunity Anti-human DLL3 monoclonal antibodies were produced in immunized mice. Female SJL mice, 6-8 weeks old, were used in the experiment (Shanghai SLAC Laboratory Animal Co., Ltd., Animal Production License Number: SCXK(Hu)2017-0005). The breeding environment was SPF grade. After purchasing, the mice were housed 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%. Once acclimated, the mice were immunized according to the following schedule.

[0269] Immunization Plan: The immunization antigen for Group 1 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, with 50 μg / mouse / injection (primary immunization) and 25 μg / mouse / injection (booster immunization). After antigen emulsification, mice were immunized on days 0, 7, 14, and 21. Blood samples were collected on days 7 and 21, and antibody titers in the mouse serum were determined by ELISA. After the fourth immunization, mice with high and stable serum antibody titers were selected and subjected to splenocyte fusion. Three days before splenocyte fusion, booster immunization was performed by intraperitoneally (ip) injection of 25 μg / mouse of an antigen solution prepared in physiological saline.

[0270] 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, mice were intraperitoneally injected with 0.1 mL of TiterMax® Gold Adjuvant (Sigma Cat No. T2684) per mouse. After half an hour, mice were immunized with 10 mL of saline. 8Each mouse was intraperitoneally injected with 0.1 mL of cell suspension diluted to a concentration of 1 / mL. After uniformly dispersing the cells, 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, with 50 μg / mouse / injection (primary immunization) and 25 μg / mouse / injection (booster immunization). Immunization with hDLL3-Fc was performed on days 7, 21, 35, and 49. Blood samples were 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 immunization was performed, and 50 μg of hDLL3-Fc protein antigen solution prepared in saline was injected intraperitoneally (ip) into the mice.

[0271] 2. Splenocyte Fusion Splenic lymphocytes were fused with myeloma cells Sp2 / 0 (ATCC® CRL-8287) by an optimized electrofusion method. TM ) to obtain hybridoma cells.

[0272] 3-4 x 10 fused hybridoma cells 5 The cells were resuspended in complete medium (IMDM medium containing 20% ​​FBS, 1x HAT, and 1x OPI) at a density of 1 / mL and seeded at 150 μL / well into a 96-well plate. After incubation at 37°C and 5% CO2 for 3-4 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.

[0273] 3. Screening of Hybridoma Cells and Sequencing of Antibodies Depending on the hybridoma cell growth density, hybridoma culture supernatants were detected by ELISA for binding to DLL3 protein and FACS for binding to DLL3 CHO-s cells. Clones that bound to human DLL3 protein, monkey DLL3 protein, and DLL3 CHO-s cells but did not bind to wild-type CHO-s cells were selected. These clones were then cryopreserved, expanded, stockpiled, and subcloned one or two times until single-cell clones were obtained. Screening using the above experiments yielded hybridoma clones mAb100 and mAb6.

[0274] 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.

[0275] mAb6 heavy chain variable region: [C13] [ka] mAb6 light chain variable region: [C14] [ka] mAb100 heavy chain variable region: [C15] [ka] mAb100 light chain variable region: [C16] [ka]

[0276] [Table 4]

[0277] The heavy and light chain variable regions of the mouse antibody were cloned into a pTT5 vector plasmid containing the human IgG1 heavy chain constant region shown in SEQ ID NO: 28 and the κ light chain constant region shown in SEQ ID NO: 29, respectively, and then transfected into HEK293 cells to obtain the anti-DLL3 chimeric antibodies M6CHI and M100CHI.

[0278] Human IgG1 heavy chain constant region: [C17] [ka] human kappa light chain constant region, [C18] [ka]

[0279] Example 3: Humanization of a murine anti-DLL3 monoclonal antibody The Kabat database of human antibody heavy and light chain variable region germline genes was aligned, and highly homologous heavy and light chain variable region germline genes were selected as templates. The CDRs of the mouse antibody were grafted onto the corresponding human templates to form the variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Amino acids in the variable regions were backmutated and further recombined with constant regions (e.g., 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.

[0280] The human germline light chain variable region FR1, FR2, and FR3 of the mAb6 antibody are derived from IGKV1-16*01, the light chain FR4 region is derived from IGKJ4*01, the human germline heavy chain variable region FR1, FR2, and FR3 are derived from IGHV1-3*01, IGHV7-4-1*02, or IGHV3-73*01, and the heavy chain FR4 region is derived from IGHJ6*01. The human germline light chain variable region FR1, FR2, and FR3 of the mAb100 antibody are derived from IGKV1-27*01, the light chain FR4 region is derived from IGKJ4*01, the human germline heavy chain variable region FR1, FR2, and FR3 are derived from IGHV3-11*01, and the heavy chain FR4 region is derived from IGHJ6*01.

[0281] In addition, 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 HCDR3:PLY H YGRSYNAVAY (SEQ ID NO: 30) and PLYYYGRSYN G VAY (SEQ ID NO: 31) was obtained.

[0282] [Table 5-1] [Table 5-2]

[0283] The resulting humanized antibody variable region sequences are as follows: hAb6 VH1(Q1E, R71V, T73K) [C19] [ka] hAb6 VH2(Q1E, I69L, R71V, T73K, S76N)

[20] [ka] hAb6 VH3(Q1E, M48I, V67A, I69L, R71V, T73K, S76N) [Chemistry 21]

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[38] [ka] hAb100 VH3(Q1E, S49A, R94S, Y98H)

[39] [ka] hAb100 VH4(Q1E, S49A, R94S, A100EG)

[40] [ka] hAb100VH5(Graft IGHV3-11*01)

[41] [ka] hAb100 VL1(Graft IGKV1-27*01)

[42] [ka] hAb100 VL2(V43I)

[43] [ka]

[0284] Note: The single underlined parts are CDR regions, and the double underlined parts are mutation sites.

[0285] [Table 6-1] [Table 6-2] wherein X1 is Y or H, and X2 is A or G.

[0286] Exemplary combinations of heavy and light chain variable regions of humanized antibodies are as follows: [Table 7]

[0287] [Table 8]

[0288] The above antibodies were cloned, expressed, and purified, and then subjected to protein binding experiments (Test Example 1), cell binding experiments (Test Example 2), and Biacore (Test Example 4). As a result, humanized antibodies with relatively good activity were selected. The heavy and light chain amino acid sequences of exemplary humanized antibodies are as follows: Hu6 (also called hAb6L4H12) heavy chain:

[44] [ka] Note: The underlined parts in the sequences are variable regions, and the italicized parts are constant regions.

[0289] SEQ ID NO:58 Hu6 (also called hAb6L4H12) light chain:

[45] [ka] Note: The underlined parts in the sequences are variable regions, and the italicized parts are constant regions.

[0290] SEQ ID NO:59 Hu100 (also called hAb100L1H1) heavy chain:

[46] [ka] Note: The underlined parts in the sequences are variable regions, and the italicized parts are constant regions.

[0291] SEQ ID NO: 60

[47] [ka] Note: The underlined parts in the sequences are variable regions, and the italicized parts are constant regions. Sequence number 61.

[0292] The positive control antibody used in the present disclosure is BI-764532 (constructed with reference to WO2019234220A1), and the negative control is C25 (of which the VH / VL sequences are from patent US6114143A), whose sequences are as follows: BI-764532 heavy chain:

[48] [ka] Note: The underlined parts in the sequences are variable regions, and the italicized parts are constant regions.

[0293] SEQ ID NO: 62 BI-764532 light chain:

[49] [ka] Note: The underlined parts in the sequences are variable regions, and the italicized parts are constant regions.

[0294] SEQ ID NO: 63 C25 heavy chain:

[50] [ka] C25 light chain:

[51] [ka]

[0295] Note: The underlined parts in the sequences are variable regions, and the italicized parts are constant regions.

[0296] II. Preparation of ADC Example 4. Preparation of ADC-1

[52] [ka] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 50.7 μL, 507 nmol) was added to an aqueous solution of antibody Hu100 in PBS buffer (0.05 M PBS buffer solution with pH 6.5, 10.0 mg / mL, 3 mL, 203 nmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0297] Compound 9-A (2.18 mg, 2030 nmol, prepared with reference to Example 9 of Patent Application WO2020063676A1) was dissolved in 150 μL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and reacted at 25 °C for 3 hours with shaking to terminate the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-1 in PBS buffer (1.61 mg / mL, 15.1 mL), which was then stored refrigerated at 4 °C.

[0298] The mean value was calculated by RP-HPLC: n=4.43.

[0299] Example 5. Preparation of ADC-2

[53] [ka] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 121.7 μL, 1217 nmol) was added to an aqueous solution of antibody Hu100 in PBS buffer (0.05 M PBS buffer solution with pH 6.5, 10.0 mg / mL, 3 mL, 203 nmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0300] Compound 9-A (3.27 mg, 3042 nmol) was dissolved in 150 μL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and reacted at 25 °C for 3 hours with shaking. The reaction mixture was then desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-2 in PBS buffer (1.42 mg / mL, 15.8 mL). This product was then stored refrigerated at 4 °C.

[0301] The mean value was calculated by RP-HPLC: n=7.32.

[0302] Example 6. Preparation of ADC-3

[54] [ka] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 159.4 μL, 1594 nmol) was added to an aqueous solution of antibody Hu100 in PBS buffer (0.05 M PBS buffer solution with pH 6.5, 10.0 mg / mL, 6.5 mL, 440 nmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0303] Compound 9-A (5.66 mg, 5265 nmol) was dissolved in 330 μL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and reacted at 25 °C for 3 hours with shaking. The reaction mixture was then desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-3 in PBS buffer (2.72 mg / mL, 19.2 mL). This product was then stored refrigerated at 4 °C.

[0304] The mean value was calculated by RP-HPLC: n = 6.12.

[0305] Example 7. Preparation of ADC-4

[55] [ka] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 1.14 mL, 11.4 μmol) 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, 76 mL, 5140 nmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0306] Compound 9-A (49.0 mg, 45.6 μmol) was dissolved in 3.8 mL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and reacted at 25°C for 3 hours with shaking to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-4 in PBS buffer (4.07 mg / mL, 167 mL). This was then stored refrigerated at 4°C.

[0307] The mean value was calculated by RP-HPLC: n=3.53.

[0308] Example 8. Preparation of ADC-5

[56] [ka] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 2.74 mL, 27.4 μmol) 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, 68 mL, 4595 nmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0309] Compound 9-A (82.75 mg, 77.04 μmol) was dissolved in 3.4 mL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and reacted at 25°C for 3 hours with shaking to terminate the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-5 in PBS buffer (3.12 mg / mL, 174.1 mL), which was then stored refrigerated at 4°C.

[0310] The mean value was calculated by RP-HPLC: n=7.43.

[0311] Example 9. Preparation of ADC-6

[57] [ka] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 35.6 μL, 356 nmol) was added to an aqueous solution of antibody C25 in PBS buffer (0.05 M PBS buffer solution with pH 6.5, 10.0 mg / mL, 1.6 mL, 108 nmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0312] Compound 9-A (1.39 mg, 1.294 μmol) was dissolved in 80 μL of dimethyl sulfoxide and added dropwise to the reaction mixture. The mixture was placed in a water bath shaker and reacted at 25 °C for 3 hours with shaking. The reaction mixture was then desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-6 in PBS buffer (1.06 mg / mL, 12.3 mL). This product was then stored refrigerated at 4 °C.

[0313] The mean value was calculated by RP-HPLC: n=6.76.

[0314] Example 10. Preparation of ADC-7

[58] [ka] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 64.8 μL, 648 nmol) was added to an aqueous solution of antibody C25 in PBS buffer (0.05 M PBS buffer solution with pH 6.5, 10.0 mg / mL, 1.6 mL, 108 nmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0315] Compound 9-A (1.74 mg, 1.62 μmol) was dissolved in 80 μL of dimethyl sulfoxide and added to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-7 in PBS buffer (1.03 mg / mL, 11.9 mL). This was then stored refrigerated at 4 °C.

[0316] The mean value was calculated by RP-HPLC: n=7.75.

[0317] Analysis of drug loading in ADC stock solutions ADCs are antibody-drug conjugates whose therapeutic mechanism is to deliver drugs to cells via antibody targeting, thereby killing the cells or inhibiting their growth. The amount of drug loaded plays a crucial role in the efficacy of the drug.

[0318] In the present disclosure, the drug loading is analyzed by RP-HPLC method, and the process is basically as follows: Reagents and equipment: Trifluoroacetic acid (TFA): Sigma, 100 mL / bottle; acetonitrile: LC grade, 4 L / bottle, Thermo Fisher; DTT: Sigma, 1 g / bottle.

[0319] High-performance liquid chromatograph: Agilent 1200.

[0320] Preparation of solutions: 1) 0.25M DTT solution: Preparation example: 5.78 mg of DTT was taken and thoroughly dissolved in 150 μL of purified water, after which a 0.25 M DTT solution was prepared and stored at -20°C.

[0321] 2) Mobile phase A (0.1% TFA in water): Preparation example: 1000 mL of purified water was measured out using a measuring cylinder, 1 mL of TFA was added, and the mixture was mixed thoroughly and uniformly before use. The mixture was then stored at 2 to 8°C for 14 days.

[0322] 3) Mobile phase B (0.1% TFA in acetonitrile): Preparation example: 1000 mL of acetonitrile was measured out using a measuring cylinder, 1 mL of TFA was added, and the mixture was thoroughly mixed uniformly before use. The mixture was then stored at 2 to 8°C for 14 days.

[0323] The naked antibody and the sample to be tested (concentration 1 mg / mL, approximately 200 μL) were reduced with 4 μL of DTT and placed in a water bath at 37°C for 1 hour. After completion, the sample was removed and placed in an inner tube, ready for injection.

[0324] Chromatography conditions: Chromatography column: Agilent PLRP-S 1000A 8μm 4.6*250mm, column temperature: 80℃, DAD detector: detection wavelength 280 nm, sample chamber temperature: 4°C, flow rate: 1 mL / min, Injection volume: 40μL, The chromatographic gradient is shown in Table 9-1.

[0325] [Table 9] Data analysis: The positions of the light and heavy chains were identified by comparing the spectra of the sample and the naked antibody, and the DAR value was calculated by integrating the spectrum of the detected sample. The calculation formula is as follows: [Table 10] The activities of the antibodies and ADCs of the present disclosure were verified using biochemical test methods as follows.

[0326] Test Example 1: ELISA detection experiment of antibody binding at the protein level The plate was coated with streptavidin (Abcam, ab136200, 1 μg / mL) at 100 μL / well and incubated overnight at 4°C. The plate was washed three times with 250 μL / well of PBST solution (PBS containing 0.1% Tween 20). Blocking was performed with 250 μL / well of 5% milk at 37°C for 2 hours. The plate was washed three times with 250 μL / well of PBST solution. Biotinylated DLL3 antigen (1 μg / mL) (SEQ ID NO: 9) was added and incubated at 37°C for 1 hour. The plate was washed three times with 250 μL / well of PBST solution. Antibodies Hu6 and Hu100 (maximum concentration 100 nM, 4-fold serial dilutions) were prepared and incubated at 37°C for 1 hour. The plate was washed six times with 250 μL / well of PBST solution. 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 hour. The plate was washed six times with 250 μL / well of PBST solution. 100 μL / well of TMB (KPL, 5120-0077) color development solution was added and developed at room temperature for 5–10 minutes. 100 μL / well of 1M H2SO4 was added to stop the color development, and the plate was read at 450 nm using a plate reader (Molecular Devices, VERSA max).

[0327] [Table 11] As shown by the results, both antibodies Hu6 and Hu100 have excellent binding ability to DLL3.

[0328] Test Example 2: Detection of antibody cell-level binding by FACS 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 cultured at 1 × 10 in FACS buffer (1% BSA + PBS, pH 7.4). 6 A cell suspension was prepared at 100 μL / well and added to a 96-well round-bottom plate (Corning, 3795). The cells were centrifuged at 300 g for 5 minutes and the supernatant was removed. Different concentrations of the antibody to be tested were added at 100 μL / well. The cells were then incubated in the dark at 4°C for 1 hour. After three centrifugations at 300 g and washing, 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 at 4°C for 40 minutes. After three centrifugations at 300 g and washing, the binding EC of the antibody to the DLL3-expressing cells was measured using an Invitrogen flow cytometer to detect the geometric mean fluorescence intensity. 50 The values ​​were calculated, and the results are shown in Tables 11-1, 11-2, and 11-3 and in Figures 1A to 1C.

[0329] [Table 12] [Table 13] [Table 14]

[0330] As shown by the results, all antibodies disclosed herein can specifically bind to DLL3 expressed in cells. Among them, Hu6 has relatively high binding ability to DLL3-expressing cells from 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 binds only to human and cynomolgus monkey DLL3-expressing cells, and its binding activity is weaker than that of Hu6 and Hu100.

[0331] Test Example 3: Detection experiment of antibody binding to DLL1 and DLL4 cells by FACS Stably transfected human DLL1 / CHO-s and human DLL4 / CHO-s cells were cultured at 1 × 10 in FACS buffer (containing 1% BSA and PBS, pH 7.4). 6 A cell suspension was prepared at 1 / mL and added at 100 μL / well to a 96-well round plate (Corning, 3795). The plate was centrifuged at 300 g for 5 minutes and the supernatant removed. Antibodies to be detected were added at 100 μL / well and incubated in the dark in a 4°C refrigerator for 1 hour. After washing by centrifugation three times at 300 g, a working concentration of PE F(ab')2-sheep anti-human IgG Fc antibody (Invitrogen, H10104) was added and incubated in the dark in a 4°C refrigerator for 40 minutes. After washing by centrifugation three times at 300 g, the geometric mean fluorescence intensity was detected using an Invitrogen flow cytometer.

[0332] As shown by the results, neither antibody Hu6 nor Hu100 binds to human DLL1 or DLL4.

[0333] Test Example 4: Antibody affinity detection experiment using Biacore The antibody to be measured was affinity-captured using a Protein A biosensor chip (Cat. #29127556, Cytiva) for 18 seconds. The antigens human DLL3 (ACRO, DLL3-H52H4), monkey DLL3 (KACTUS, DLL-RM103), and mouse DLL3 (KACTUS, DLL-MM103) were then flowed over the chip surface for 180 seconds, followed by 600 seconds of dissociation. The reaction signals were detected in real time using a Biacore 8K (Cytiva) instrument, and binding / dissociation curves were obtained. After the dissociation cycle for each experiment was completed, the biosensor chip was regenerated by washing with 10 mM glycine-HCl solution (pH 1.5) (Cat. #BR-1003-54, Cytiva). A 1:1 model was used for data fitting. The results are shown in Tables 12-1, 12-2, and 12-3.

[0334] [Table 15] [Table 16-1] [Table 16-2] [Table 17]

[0335] The results show that the humanized and chimeric antibodies mAb100 and mAb6 can specifically bind to human DLL3 with relatively high affinity. Among them, antibody Hu6 has relatively high affinity to human, monkey, and mouse DLL3, while Hu100 has relatively high affinity to human and monkey DLL3 but does not bind to mouse DLL3.

[0336] Test Example 5: Antibody epitope competitive binding experiment The plate was coated with BI-764532 antibody (1 μg / mL) at 100 μL / well and incubated overnight at 4°C. The plate was washed three times with 250 μL / well of PBST solution. Blocking was performed with 250 μL / well of 5% milk at 37°C for 2 hours. The plate was washed three times with 250 μL / well of 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. The plate was washed six times with 250 μL / well of 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 six times with 250 μL / well of PBST solution. TMB (KPL, 5120-0077) color development solution was added at 100 μL / well and allowed to develop at room temperature for 5-10 minutes. 1M H2SO4 was added at 100 μL / well to stop the color development, and the readings were read at 450 nm using a plate reader. The results are shown in Figure 2.

[0337] As shown by the results, the antibodies Hu6 and Hu100 do not compete with BI-764532, which explains that the antibodies Hu6 and Hu100 bind to epitopes different from those of BI-764532.

[0338] Test Example 6: Detection of endocytosis activity of anti-DLL3 antibody DT3C is a recombinantly expressed fusion protein composed of diphtheria toxin fragment A (toxin portion only) and group G streptococcus 3C fragment (IgG binding portion). This protein has a high affinity for the IgG portion of antibodies, and when antibodies are endocytosed, they enter cells together. Under the action of intracellular furin, they release the toxic DT, which inhibits EF2-ADP-ribosylation, blocking the protein translation process and ultimately causing cell death. DT3C that has not entered cells does not have cell-killing activity. The antibody's endocytosed activity was evaluated based on the cell-killing status.

[0339] 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. Columns 1 and 12 were seeded with 50 μL of medium alone, without cells, and cultured at 37°C in 5% carbon dioxide for 16 hours.

[0340] b. A 4x concentration of DT3C (9600 nM, expressed and purified by Shanghai Pancha Biotechnology Co., Ltd.) solution was prepared in serum-free medium and filtered through a 0.22 μm filter. A 4x concentration of antibody (1600 nM) was prepared in serum-free medium. 80 μL of the DT3C solution and 80 μL of the antibody solution were mixed uniformly at a 1:1 volume and incubated at room temperature for 30 minutes.

[0341] c. The mixture was serially diluted 5-fold with serum-free medium to a total of 9 concentrations, with the 10th point being pure medium.

[0342] d. 50 μL of the diluted antibody was added to the cells and incubated in an incubator for 3 days.

[0343] 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.

[0344] The results are shown in Table 13 below and in FIG.

[0345] [Table 18] The results show that both antibodies Hu6 and Hu100 can be endocytosed.

[0346] Test Example 7: Detection of cellular binding of ADC by FACS DLL3-expressing hDLL3 / CHO-s cells and cynoDLL3 / CHO-s cells were cultured at 1 × 10 in FACS buffer (1% BSA + pH 7.4 PBS). 6 A cell suspension was prepared at 100 μL / well and added to a 96-well round plate (Corning, 3795). The plate was centrifuged at 300 g for 5 minutes and the supernatant removed. Different concentrations of ADC to be assayed were added at 100 μL / well. The plate was then incubated in the dark at 4°C for 1 hour. After three centrifugations at 300 g and washing, working concentrations of APC anti-human IgG Fc (BioLegend, 410712) or PE F(ab')2-sheep anti-human IgG (Invitrogen, H10104) were added and the plate was incubated in the dark at 4°C for 40 minutes. After three centrifugations at 300 g and washing, the geometric mean fluorescence intensity was detected using an Invitrogen flow cytometer to determine the binding EC of the antibody to the DLL3-expressing cells. 50 The values ​​were calculated and the results are shown in Table 14 and Figures 4A and 4B.

[0347] [Table 19]

[0348] As shown by the results, the ADCs of the present disclosure can all specifically bind to human and monkey DLL3 expressed in cells, and the binding of the ADCs is essentially unaffected by increasing DAR values.

[0349] Test Example 8: Detection experiment of ADC affinity using Biacore The antibody to be measured was affinity-captured using a Protein A biosensor chip (Cat. #29127556, Cytiva) for 18 seconds. The antigens human DLL3 (ACRO, DLL3-H52H4) and monkey DLL3 (KACTUS, DLL-RM103) were then flowed over the chip surface for 180 seconds, followed by 600 seconds of dissociation. The reaction signal was detected in real time using a Biacore 8K (Cytiva) instrument, and binding / dissociation curves were obtained. After the dissociation cycle for each experiment was completed, the biosensor chip was regenerated by washing with 10 mM glycine-HCl solution (pH 1.5) (Cat. #BR-1003-54, Cytiva). A 1:1 model was used for data fitting. The results are shown in Table 15.

[0350] [Table 20-1] [Table 20-2]

[0351] The results show that the ADCs of the present disclosure can specifically bind to both human and monkey DLL3 proteins, and that increasing the DAR value does not substantially affect the binding of the ADCs.

[0352] Test Example 9: Cytotoxicity experiment of ADC against 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. Columns 1 and 12 were added with 135 μL of medium alone without seeding cells, and the plate was cultured at 37°C in 5% carbon dioxide for 16 hours.

[0353] b. The ADC sample was prepared in PBS to a working solution (10x concentration) in the first well, and serially diluted with PBS to the corresponding initial concentration. 15 μL of the 10x ADC solution was added to each well, and the wells were incubated at 37°C under 5% carbon dioxide for 6 days.

[0354] c. 70 μL of CTG (Promega, G7573) was added to each well and incubated in the dark at room temperature for 10 minutes. Chemiluminescence was read using Victor3, and the data was processed using GraphPad Prism5, with antibody or ADC concentration on the X axis and light intensity on the Y axis.

[0355] The seeding density of different cells onto the plate, the working concentration of the first well (10x concentration), and the dilution ratio are shown in Table 16.

[0356] [Table 21]

[0357] The cells used in the experiments were as follows: H1184(+++), CRL-5858, purchased from ATCC; DMS53(++), CRL-2062, purchased from ATCC; SK-MEL3(+), HTB-69, purchased from ATCC; CHO-K1(-), CCL-61 purchased from ATCC.

[0358] Among them, "+" indicates the expression level of DLL3, and "-" indicates no expression of DLL3.

[0359] The results are shown in Table 17 and Figures 5A to 5D.

[0360] [Table 22]

[0361] As shown by the results, ADC-1, ADC-2, and ADC-3 have relatively strong target cell-killing activity and can kill DLL3-expressing SK-MEL3, DMS53, and H1184 cells, but not DLL3-negative CHO-K1 cells.

[0362] Test Example 10: Bystander killing activity experiment DMS53 / DLL3high (DMS53 cells stably transformed with DLL3) and U-2OS (ATCC, HTB-96) cells were cultured in RPMI1640 + 20% FBS + 1x Glutamax and McCoy's 5A + 10% FBS, respectively. The cells were digested with pancreatin, neutralized with fresh medium, and centrifuged at 1000 rpm for 3 minutes. 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 9x10 4 The U-2OS cell density was adjusted to 3 x 10 cells / mL. 4The concentration was adjusted to cells / mL. 500 μL of DMS53 / DLL3high cells and 500 μL of U-2 OS cells were added to corresponding wells of a 12-well plate. 500 μL of U-2 OS cells and 500 μL of RPMI1640 + 20% FBS + 1× Glutamax medium were added to corresponding wells of the 12-well plate. The cells were cultured at 37°C under 5% carbon dioxide for 24 hours. The samples were prepared at a 40× intermediate concentration (200 nM). 25 μL of each ADC sample was added to corresponding wells of the 12-well plate. A solvent control group was also set up. The cells were cultured at 37°C under 5% carbon dioxide for 6 days. The cells in the 12-well plate were digested with pancreatin, neutralized with fresh medium, and 20 μL of cells were removed. 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 minutes, 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 minutes. The cells were washed once with FACS buffer, centrifuged at 1500 rpm for 3 minutes, and incubated with the secondary antibody APC anti-human IgG Fc (100x) for 30 minutes. The cells were then washed once with FACS buffer. 200 μL of FACS buffer was added, and the cells were resuspended and detected by FACS. The streaming data was analyzed using FlowJo to obtain the DMS53 / DLL3 high ratio, and the total amount of DMS53 / DLL3 high and U-2OS was calculated. The data were plotted using GraphPad Prism 5, with the different samples on the X axis and the calculated cell number on the Y axis. The results are shown in Figure 6.

[0363] As shown by the results, ADC-1, ADC-2 and ADC-3 have clear bystander cytotoxicity effects.

[0364] Biological evaluation of in vivo activity Test Example 11-1: Evaluation of in vivo drug efficacy in a 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 per mouse (ATCC, CRL-2062) into the right flank. After 7 days of inoculation, the tumor volume was approximately 220 mm. 3 At 4 days, mice with excessive or excessively large tumors were removed. They were randomly divided into groups of 8 mice per group based on tumor volume, and treatment began on the same day. ADC was injected intraperitoneally at a dose of 1.5 mg / kg, once a week for a total of two doses. After the second dose, drug administration was discontinued, and tumor growth was monitored. 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 using two-way ANOVA or one-way ANOVA, with the mean calculated as avg, the SD calculated as standard deviation, and the SEM calculated as standard deviation / square root of the number of animals in each group.

[0365] Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 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.

[0366] Tumor inhibition rate TGI(%)=1-T / C(%).

[0367] The results are shown in Table 18 and FIG.

[0368] [Table 23]

[0369] The results show that at a dose of 1.5 mpk, ADC-1 and ADC-4 both significantly inhibited the growth of subcutaneously implanted DMS53 cell tumors, while at doses of 0.5 mpk and 1.5 mpk, ADC-2 and ADC-5 both significantly inhibited the growth of subcutaneously implanted DMS53 cell tumors.

[0370] Test Example 11-2: Evaluation of in vivo drug efficacy in a DMS53 cell CDX mouse model Human small cell lung cancer DMS53 cells (5 × 10 6 Balb / c mice were subcutaneously inoculated with 200 μL of 30% matrigel per mouse (ATCC, CRL-2062) into the right flank. After 15 days of inoculation, the tumor volume was approximately 180 mm. 3 At 100°C, mice with excessive or excessively large tumors were removed. Mice were randomly divided into groups of 8 mice per group based on tumor volume, and treatment began on the same day. ADC was injected intraperitoneally at a dose of 1.5 mg / kg or 0.5 mg / kg, once a week for a total of two doses. After the second dose, drug administration was discontinued, and tumor growth was monitored. 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 using two-way ANOVA or one-way ANOVA, with the mean calculated as avg, the SD calculated as standard deviation, and the SEM calculated as standard deviation / square root of the number of animals in each group.

[0371] Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 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.

[0372] Tumor inhibition rate TGI(%)=1-T / C(%).

[0373] The results are shown in Table 19 and FIG.

[0374] [Table 24]

[0375] As shown by the results, ADC-3 at doses of 0.5mpk and 1.5mpk, and ADC-1 at a dose of 1.5mpk, can significantly inhibit the growth of subcutaneously transplanted DMS53 cell tumors.

[0376] Test Example 12: Evaluation of in vivo drug efficacy in H1184 cell CDX mouse model NCI-H1184 cells (6 × 10 6 80 NDG mice were subcutaneously inoculated with 200 μL of 50% MatrixGel per mouse (ATCC, CRL-5858) into the right flank. After 13 days of inoculation, the tumor volume was approximately 185 mm. 3 At this point, mice with overweight or undersized tumors were removed, and the mice were randomly divided into 7 groups (9 mice per group) based on tumor volume. Treatment began on the same day, as shown in Table 19. Antibodies were injected intraperitoneally for a total of 21 days. Tumor volume was measured twice a week, and the mice were weighed and recorded. Data were recorded using Excel statistical software, plotted using GraphPad Prism software, and statistically analyzed using two-way ANOVA or one-way ANOVA, with the mean calculated as avg, the SD calculated as standard deviation, and the SEM calculated as standard deviation / square root (number of animals in each group).

[0377] Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 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.

[0378] Tumor inhibition rate TGI(%)=1-T / C(%).

[0379] The results are shown in Table 20 and FIG.

[0380] [Table 25] The results show that both ADC-2 and ADC-3 can significantly inhibit the growth of NCI-H1184 tumors.

[0381] Test Example 13: PK study in rats The single-dose pharmacokinetics of the ADC molecules was studied in SD rats (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.).

[0382] The ADC molecules were intravenously injected at 3 mg / kg (n=4 / group), and blood samples were collected from the rats' fundus vein at 5 min, 8 h, 1 d, 2 d, 4 d, 7 d, 10 d, 14 d, 21 d, and 28 d after administration. The collected blood samples were left at room temperature for half an hour until coagulation occurred, and then centrifuged at 1000 g for 15 min at 4°C to collect serum. Bioanalytical measurements were performed using HTRF on the rat serum samples, biotin-labeled DLL3 capture monoclonal antibody, and biotin-labeled antitoxin antibody capture toxin. Quantitative analysis of the contents of samples waiting to be measured was performed using a four-parameter model curve of the standard. Pharmacokinetic parameters were analyzed using a noncompartmental model in WinNonlin software (6.4). The experimental results are shown in Table 21 and Figures 10A-10C.

[0383] [Table 26] As shown by the results, the AUCs of the whole antibody and the complete ADC measured in vivo were similar, and the complete ADC structures of all three ADC molecules were stable.

[0384] Example 14: PK study in monkeys The single-dose pharmacokinetics of the ADC molecules were studied in cynomolgus monkeys (Suzhou Xishan Zhongke Animal Experiment Co., Ltd.).

[0385] The ADC molecules were intravenously injected at 10 mg / kg (n=3 / group), and blood samples were collected 5 min, 8 h, 1 d, 1.5 d, 2 d, 3 d, 4 d, 7 d, 10 d, 14 d, 21 d, and 28 d after administration. The collected blood samples were left at room temperature for half an hour until coagulation occurred, then centrifuged (4°C, 2600 g, 10 min). The supernatant serum was collected and immediately stored at -60°C. Serum was collected for bioanalysis. Serum samples were assayed using the DELFIA method, and the contents of the samples awaiting assay were quantitatively analyzed using a four-parameter model curve of the standard. Pharmacokinetic parameters were analyzed using a non-compartmental model in WinNonlin software (6.4). The experimental results are shown in Table 22 and Figure 11.

[0386] [Table 27]

[0387] The results showed that after intravenous administration of ADC-2 10mpk to cynomolgus monkeys, the half-lives of the whole antibody and the complete ADC were 9.9±1.1 days and 8.5±0.1 days, respectively, and the AUCs of the whole antibody and the complete ADC were 27405μg / mL*h and 25702μg / mL*h, respectively. The half-lives of the whole antibody and the complete ADC were essentially the same in the bodies of cynomolgus monkeys, and the AUC ratio of the complete ADC to the whole antibody was 94%, indicating that ADC-2 has relatively good stability in the bodies of cynomolgus monkeys.

Claims

1. An antibody-drug conjugate having a structure represented by the general formula Pc-LY-D or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 Among them, Pc is 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; Among them, SEQ ID NO: 57 is PLYX １ YGRSYNX ２ VAY, of which X １ is Y or H, and X ２ 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; Preferably, 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; Y is —O—CR １ R ２ -C(O)-, in which R １ is halogenated C １－６ Alkyl group or C ３－６ is a cycloalkyl group, and R ２ is a hydrogen atom, halogenated C １－６ Alkyl group and C ３－６ cycloalkyl groups, or R １ and R ２ are C together with the carbon atoms connected to them. ３－６ forming a cycloalkyl group, n is 1 to 10; L is a linker; An antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

2. The anti-DLL3 antibody is a murine antibody, a chimeric antibody, or a humanized antibody, preferably a humanized antibody. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1.

3. The anti-DLL3 antibodies include: i) the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 50, 14, 51, 52, 53, or 54; and / or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 55, 15, or 56; or ii) the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 43, 12, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 44; and / or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 48, 13, 45, 46, 47, or 49; Preferably, i) 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 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 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 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 preferably, 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; or 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; The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2.

4. The anti-DLL3 antibody is an antibody fragment, and preferably, the antibody fragment is Fab, Fab', or F(ab'). ２ , Fab'-SH, Fd, Fv, scFv, dsFv, bibody or domain antibody, The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

5. the anti-DLL3 antibody comprises a heavy chain constant region and a light chain constant region, preferably wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 28 and / or the light chain constant region comprises the amino acid sequence of SEQ ID NO: 29; The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

6. The anti-DLL3 antibody comprises a heavy chain and a light chain, the heavy chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 60, and / or the light chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 61; or the heavy chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 58, and / or the light chain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 59; Preferably, the heavy chain comprises the amino acid sequence of SEQ ID NO: 60 and the light chain comprises the amino acid sequence of SEQ ID NO: 61; or the heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the light chain comprises the amino acid sequence of SEQ ID NO: 59; The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 5.

7. The Y is 【Chemistry 2】 Selected from wherein the O-terminus of Y is linked to L; The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6.

8. The L is -L １ -L ２ -L ３ -L ４ - and among them, L １ is -(succinimide-3-yl-N)-W-C(O)-, -CH ２ —C(O)—NR ３ -WC(O)- and -C(O)-WC(O)-, wherein W is C １－６ Alkylene group or C １－６ Alkylene-C ３－６ cycloalkyl groups, among which the C １－６ Alkylene group or C １－６ Alkylene-C ３－６ each cycloalkyl group is independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl; L ２ is -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)— 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, wherein 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 halogen, hydroxy group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group and cycloalkyl group; L ４ is -NR ５ (CR ６ R ７ ) ｔ -, -C(O)NR ５ , —C(O)NR ５ (CH ２ ) 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 are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; Preferably, L １ teeth, 【Transformation 3】 and s １ is an integer from 2 to 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(CH ２ ) t-, where t is 1 or 2; Among them, the above-mentioned L １ The end is linked to Pc, More preferably, the linker has the following structure: 【Chemistry 4】 The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7.

9. The antibody-drug conjugate comprises: 【Transformation 5】 and having a structure represented by Among them, Pc is the anti-DLL3 antibody of claim 1, n is 1 to 10, Preferably, the antibody-drug conjugate comprises: 【Transformation 6】 wherein: Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, of which: the heavy chain comprises the amino acid sequence of SEQ ID NO: 60 and the light chain comprises the amino acid sequence of SEQ ID NO: 61; or the heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the light chain comprises the amino acid sequence of SEQ ID NO: 59; n is 3 to 8, more preferably n is 6 to 8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8.

10. 1. A pharmaceutical composition comprising: The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, and one or more pharmaceutically acceptable excipients, diluents or carriers. Pharmaceutical compositions.

11. Use of the antibody-drug conjugate or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 10, in the preparation of a medicament for treating a tumor or cancer, preferably, wherein the tumor or cancer is The cancer is selected from 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, medullary thyroid cancer, malignant pleural mesothelioma, breast cancer, triple-negative breast cancer, liver cancer, hepatic and gallbladder 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 lung cancer is small cell lung cancer. use.

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