Anti-DLL3 antibody, antibody-drug conjugate thereof and medical application thereof

Abstract

Provided are an anti-DLL3 antibody, an antibody-drug conjugate thereof, and a medical use thereof. In particular to an anti-DLL3 antibody-avitecan drug conjugate shown as a general formula Pc-L-Y-D. Pc is an anti-DLL3 antibody, and L, Y and n are defined as in the specification.

Description

Anti-DLL3 antibodies, antibody-drug conjugates thereof and medical uses thereof Technical Field

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

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Small cell lung cancer (SCLC) is a relatively malignant type of lung cancer, accounting for 10%-15% of all lung cancer cases. SCLC tumors grow rapidly and are prone to metastasis, with a 5-year survival rate of less than 7%. Platinum / etoposide combination chemotherapy is often used to treat SCLC. SCLC patients respond well to chemotherapy initially, but are very prone to developing drug resistance and relapse. In recent years, immunotherapy, such as PD-L1 antibodies and PD1 antibodies, has had some effect on SCLC patients, but the efficacy is approximately 15%. Currently, no specific targeted therapy drugs have been developed.

[0004] DLL3 is a ligand that inhibits Notch. Under normal conditions, DLL3 is located on the Golgi apparatus. In cancer cells (such as small cell lung cancer cells), DLL3 goes to the cell surface and binds to Notch in a cis manner, hindering cell-cell binding and Notch endocytosis in target cells, thereby inhibiting the Notch signaling pathway and promoting tumor cell growth. DLL3 is mainly expressed in neural or neuroendocrine tumors, including SCLC, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumors, small cell bladder cancer, glioblastoma multiforme, metastatic castration-resistant prostate cancer, melanoma, etc. SCLC is particularly important, with more than 80% of SCLC expressing DLL3, while it is not expressed in normal lung cancer tissue and adjacent tissues. This difference in expression makes DLL3 a highly promising therapeutic target for the treatment of SCLC.

[0005] Exitecan is a camptothecin derivative that inhibits topoisomerase I, selectively inhibiting DNA replication in proliferating tumor cells. Furthermore, exitecan exhibits excellent membrane permeability, allowing it to penetrate killed cancer cells and continue to kill adjacent cancer cells, demonstrating a clear bystander effect in clinical practice.

[0006] Antibodies are linked to biologically active drugs via linkers to create antibody-drug conjugates (ADCs). ADCs leverage the specificity of antibodies for binding to surface antigens on both normal and tumor cells and the high efficacy of drugs (such as cytotoxic agents), while avoiding the drawbacks of antibodies, such as low efficacy and excessive toxic side effects. Compared to traditional chemotherapy drugs, antibody-drug conjugates can more precisely kill tumor cells while minimizing their effects on normal cells.

[0007] Summary of the Invention

[0008] The present disclosure relates to anti-DLL3 antibodies, antibody-drug conjugates thereof, and their medical uses. More specifically, the present disclosure provides ADCs of anti-DLL3 antibodies with novel sequences conjugated to a toxoid called exitecan.

[0009] In some embodiments, the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof having a structure as shown in the general formula Pc-LYD:

[0010] in:

[0011] Pc is an anti-DLL3 antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0012] i) the heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 57; and

[0013] 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] Wherein, SEQ ID NO: 57 is represented by PLYX1YGRSYNX2VAY, wherein X1 is Y or H; X2 is A or G; or

[0015] ii) the heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 16; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 17; and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; and

[0016] 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;

[0017] Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-; wherein R a and R b are the same or different and are each independently selected from hydrogen atoms, deuterium atoms, halogens and C 1-6 alkyl;

[0018] R 1 Halogenated C 1-6 Alkyl or C 3-6 Cycloalkyl;

[0019] R 2 Selected from hydrogen atoms, halogenated C 1-6 Alkyl and C 3-6 Cycloalkyl;

[0020] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0021] m is an integer from 0 to 4;

[0022] n is 1 to 10 (including integers and decimals);

[0023] L is a connector.

[0024] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described above, wherein n is the average number of drug moieties per antibody, which can be an integer or a decimal. In some embodiments, n is 1-10, or 2-10, or 3-10, or 4-10, or 5-10, or 6-10, or 7-10, or 8-10, or 1-9, or 2-9, or 3-9, or 4-9, or 5-9, or 6-9, or 7-9, or 1-8, or 2-8, or 3-8, or 4-8, or 5-8, or 6-8, or 1-7, or 2-7, or 3-7, or 4-7, or 5-7, or 1-6, or 2-6, or 3-6, or 4-6, or 1-5, or 2-5, or 3-5, or 1-4, or 2-4, or 1-3. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0025] In some embodiments, the aforementioned 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:

[0026] The heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, 30 or 31; and

[0027] 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.

[0028] In some embodiments, the aforementioned 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:

[0029] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 24; and

[0030] 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.

[0031] In some embodiments, the aforementioned 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:

[0032] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 30; and

[0033] 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.

[0034] In some embodiments, the aforementioned 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:

[0035] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 31; and

[0036] 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.

[0037] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0038] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody is a humanized antibody.

[0039] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a human immunoglobulin framework region (FR region).

[0040] In one embodiment, the aforementioned 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:

[0041] The heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and a 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 FRs of the heavy chain variable region comprise one or more back mutations selected from 1E, 49A, and 94S; and

[0042] 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; and the FRs of the light chain variable region comprise a 43I backmutation. The backmutation sites are numbered according to the Kabat convention.

[0043] In one embodiment, the aforementioned 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:

[0044] The heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 16; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 17; and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; and the FRs of the heavy chain variable region comprise one or more back mutations selected from 1E, 27Y, 30T, 38K, 43K, 48I, 67A, 68A, 69L, 71V, 73K, 75S, 76N and 93A; and

[0045] 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 FRs of the light chain variable region comprise one or more back mutations selected from 36L, 43S, 44F, 46G, 69A, 71Y, and 85D. The back mutation sites are numbered according to the Kabat convention.

[0046] In one embodiment, the aforementioned 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:

[0047] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 16; HCDR2 comprising the amino acid sequence of SEQ ID NO: 17; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; and the FRs of the heavy chain variable region comprise back mutations of 1E, 68A, 69L, 71V, 73K, 75S, and 76N; and

[0048] 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 FRs of the light chain variable region comprise backmutations at 36L, 46G, 69A, 71Y, and 85D. The backmutation sites are numbered according to the Kabat convention.

[0049] In a specific embodiment, the aforementioned 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:

[0050] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 24; and the FRs of the heavy chain variable region comprise back mutations of 1E and 94S; and

[0051] 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 back mutation sites are numbered according to the Kabat convention.

[0052] In one embodiment, the aforementioned 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:

[0053] The heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 24, 30 or 31; and the FRs of the heavy chain variable region comprise one or more back mutations selected from 1E, 49A and 94S; and

[0054] 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; and the FRs of the light chain variable region comprise a 43I backmutation. The backmutation sites are numbered according to the Kabat convention.

[0055] In one embodiment, the aforementioned 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:

[0056] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 24; and the FRs of the heavy chain variable region comprise one or more back mutations selected from 1E, 49A and 94S; and

[0057] 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; and the FRs of the light chain variable region comprise a 43I backmutation. The backmutation sites are numbered according to the Kabat convention.

[0058] In one embodiment, the aforementioned 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:

[0059] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 30; and the FRs of the heavy chain variable region comprise one or more back mutations selected from 1E, 49A and 94S; and

[0060] 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; and the FRs of the light chain variable region comprise a 43I backmutation. The backmutation sites are numbered according to the Kabat convention.

[0061] In one embodiment, the aforementioned 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:

[0062] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 31; and the FRs of the heavy chain variable region comprise one or more back mutations selected from 1E, 49A and 94S; and

[0063] 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; and the FRs of the light chain variable region comprise a 43I backmutation. The backmutation sites are numbered according to the Kabat convention.

[0064] In some embodiments, the aforementioned 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:

[0065] i) the heavy chain variable region comprises a sequence that has 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

[0066] 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

[0067] ii) the heavy chain variable region comprises a sequence that has 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

[0068] 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: 13, 45, 46, 47, 48 or 49.

[0069] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:

[0070] i) the heavy chain variable region comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14; and / or

[0071] the light chain variable region comprising 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

[0072] ii) the heavy chain variable region comprises a sequence that has 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

[0073] 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

[0074] iii) the heavy chain variable region comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12; and / or

[0075] 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

[0076] iv) the heavy chain variable region comprises a sequence that has 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

[0077] 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.

[0078] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:

[0079] i) the heavy chain variable region comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 50; and / or

[0080] 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

[0081] ii) the heavy chain variable region comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 43; and / or

[0082] The light chain variable region comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:48.

[0083] In some embodiments, the aforementioned 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:

[0084] 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

[0085] 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 55 or 56; or

[0086] 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

[0087] 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.

[0088] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:

[0089] 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

[0090] 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.

[0091] In some embodiments, the aforementioned 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.

[0092] In some embodiments, the aforementioned 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, diabody or domain antibody.

[0093] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a light chain constant region and a heavy chain constant region.

[0094] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises the constant region of IgG1, IgG2, IgG3 or IgG4.

[0095] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a constant region of a λ chain or a κ chain.

[0096] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises an IgG1 heavy chain constant region and a κ chain light chain constant region.

[0097] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:

[0098] 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.

[0099] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a heavy chain and a light chain, wherein:

[0100] the heavy chain comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:60, and / or the light chain comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:61; or

[0101] The heavy chain comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 58, and / or the light chain comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 59.

[0102] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:

[0103] The heavy chain comprises the sequence shown in SEQ ID NO: 60, and / or the light chain comprises the sequence shown in SEQ ID NO: 61; or

[0104] 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.

[0105] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody has at least one of the following properties:

[0106] a) the anti-DLL3 antibody binds to human DLL3 or an epitope thereof with a KD value 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, as determined by Biacore;

[0107] 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 determined by FACS;

[0108] c) the anti-DLL3 antibody is capable of being internalized by a cell expressing DLL3; and

[0109] d) the anti-DLL3 antibody binds to DLL3 or an epitope thereof with an EC50 of ≤0.1 nM, ≤0.09 nM, ≤0.08 nM, ≤0.07 nM, ≤0.06 nM, ≤0.05 nM, or ≤0.04 nM, as measured by ELISA;

[0110] e) The anti-DLL3 antibody recognizes a different DLL3 epitope than the positive antibody (eg, BI-764532).

[0111] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein said Y is selected from:

[0112] The O-end of Y is connected to L.

[0113] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein said Y is selected from: (include ); wherein the O-end of Y is connected to L.

[0114] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the L is -L 1 -L 2 -L 3 -L 4 -,in:

[0115] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein W is selected from C 1-6 Alkylene, C 1-6 Alkylene-C 3-6 Cycloalkyl, wherein the C 1-6 Alkylene, C 1-6 Alkylene-C 3-6The cycloalkyl groups are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

[0116] L 2 Selected from-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;

[0117] 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, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0118] L 4 Selected from-NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - and chemical bonds, wherein t is an integer from 1 to 6;

[0119] 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 halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0120] 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 halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0121] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the L is -L 1 -L 2 -L 3 -L 4 -,in:

[0122] L 1 for s 1 is an integer from 2 to 8;

[0123] L 2is a chemical bond;

[0124] L 3 is a tetrapeptide residue; preferably, L 3 is a tetrapeptide residue comprising glycine-glycine-phenylalanine-glycine;

[0125] L 4 -NH(CH2)t-, t is 1 or 2;

[0126] The L 1 The end is connected to the PC.

[0127] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the L has the structure shown below:

[0128] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has the following structure:

[0129] Pc is the anti-DLL3 antibody as described in any of the preceding items; n is 1 to 10.

[0130] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure selected from the following:

[0131] in:

[0132] Pc is an anti-DLL3 antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0133] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 24; and

[0134] 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;

[0135] n is 1 to 10; preferably, n is 3 to 8; more preferably, n is 6 to 8.

[0136] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure selected from the following:

[0137] in:

[0138] Pc is an anti-DLL3 antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0139] 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;

[0140] n is 1 to 10; preferably, n is 3 to 8; more preferably, n is 6 to 8.

[0141] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure selected from the following:

[0142] in:

[0143] Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:

[0144] 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

[0145] 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;

[0146] n is 1 to 10; preferably, n is 3 to 8; more preferably, n is 6 to 8.

[0147] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure selected from the following:

[0148] in:

[0149] Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:

[0150] 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;

[0151] n is 1 to 10; preferably, n is 3 to 8; more preferably, n is 6 to 8.

[0152] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure selected from the following:

[0153] in:

[0154] Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:

[0155] 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;

[0156] n is 4.43.

[0157] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure selected from the following:

[0158] in:

[0159] Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:

[0160] 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;

[0161] n is 7.32.

[0162] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure selected from the following:

[0163] in:

[0164] Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:

[0165] 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;

[0166] n is 6.12.

[0167] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure selected from the following:

[0168] in:

[0169] Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:

[0170] 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;

[0171] n is 3.53.

[0172] In some embodiments, the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure selected from the following:

[0173] in:

[0174] Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:

[0175] 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;

[0176] n is 7.43.

[0177] The present disclosure further provides a method for preparing an antibody-drug conjugate, comprising reducing the anti-DLL3 antibody as described in any of the preceding items, and then performing a coupling reaction with the compound represented by (LYD) to obtain the antibody-drug conjugate described in the present disclosure.

[0178] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate as described in any of the preceding items, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, diluents, or carriers. In some embodiments, a unit dose of the pharmaceutical composition contains 0.1-3000 mg or 1-1000 mg of the antibody-drug conjugate as described above.

[0179] In another aspect, the present disclosure provides the use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, as described in any of the preceding items as a medicament. In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, as described in any of the preceding items is used as a medicament for treating cancer or tumors. In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, as described in any of the preceding items is used as a medicament for treating cancer or tumors that express DLL3.

[0180] In another aspect, the present disclosure provides use of the antibody-drug conjugate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, as described in any of the preceding items, in the preparation of a medicament for treating a DLL3-mediated disease or condition. In some embodiments, the DLL3-mediated disease or condition is a tumor or cancer. In some embodiments, the DLL3-mediated disease or condition is a disease or condition in which DLL3 is expressed.

[0181] In another aspect, the present disclosure provides use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition, as described in any of the preceding items, in the preparation of a medicament for treating or preventing a tumor or cancer; preferably, the tumor or cancer is selected from:

[0182] 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, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer (e.g., colon cancer and rectal cancer), kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer, glioblastoma, skin cancer, and melanoma; preferably, wherein the lung cancer is small cell lung cancer. In some embodiments, the tumor or cancer is a tumor or cancer that expresses DLL3.

[0183] In another aspect, the present disclosure further relates to a method for treating and / or preventing tumors or cancers, comprising administering to a subject in need thereof a therapeutically effective dose of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in any of the preceding items. In some embodiments, the tumor or cancer is one that expresses DLL3.

[0184] In another aspect, the present disclosure further relates to a method for treating or preventing a tumor or cancer, comprising administering to a subject in need thereof a therapeutically effective dose of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition; wherein the tumor or cancer is selected from:

[0185] Lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, large cell lung cancer), head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer (e.g., medullary thyroid cancer), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer (e.g., colon cancer and rectal cancer), kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer, glioblastoma, skin cancer, and melanoma; preferably, wherein the lung cancer is small cell lung cancer.

[0186] The antibody-drug conjugate provided by the present disclosure has good affinity with cell surface antigens, can be effectively internalized by cells expressing DLL3, and has a strong effect of inhibiting tumor growth while having good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0187] Figures 1A to 1C show the binding of Hu6 and Hu100 antibodies to DLL3 from different species. Figure 1A shows the FACS results of Hu6 and Hu100 antibody binding to H1184 cells; Figure 1B shows the FACS results of Hu6 and Hu100 antibody binding to cynoDLL3 / CHO-S cells; and Figure 1C shows the FACS results of Hu6 and Hu100 antibody binding to ratDLL3 / CHO-S cells.

[0188] Figure 2 shows the results of a competition binding experiment with different anti-DLL3 antibodies. The results show that Hu6 and Hu100 do not compete with BI-764532, indicating that antibodies Hu6 and Hu100 bind to different epitopes than BI-764532.

[0189] FIG3 shows the results of cellular endocytosis of Hu6 and Hu100 antibodies; the results show that both Hu6 and Hu100 can be endocytosed by cells.

[0190] Figures 4A and 4B show the binding results of ADC-1, ADC-2, and ADC-3 to human and monkey DLL3 cells. Figure 4A shows the binding results of ADC-1, ADC-2, and ADC-3 to human DLL3 cells; Figure 4B shows the binding results of ADC-1, ADC-2, and ADC-3 to monkey DLL3 cells.

[0191] Figures 5A to 5D show the growth inhibitory effects of ADC-1, ADC-2, and ADC-3 on different cell lines. Figure 5A shows the growth inhibition results of ADC-1, ADC-2, and ADC-3 on H1184 cells that overexpress DLL3; Figure 5B shows the growth inhibition results of ADC-1, ADC-2, and ADC-3 on DMS53 cells that overexpress DLL3; Figure 5C shows the growth inhibition results of ADC-1, ADC-2, and ADC-3 on SK-MEL3 cells that underexpress DLL3; and Figure 5D shows the growth inhibition results of ADC-1, ADC-2, and ADC-3 on CHO-K1 cells that do not express DLL3.

[0192] FIG6 shows the results of the bystander cytotoxic effects of ADC-1, ADC-2, and ADC-3.

[0193] FIG7 shows the results of ADC-1, ADC-2, ADC-4 and ADC-5 inhibiting the growth of subcutaneously transplanted DMS53 cell tumors in mice.

[0194] FIG8 shows the results of ADC-1 and ADC-3 inhibiting the growth of subcutaneously transplanted DMS53 cell tumors in mice.

[0195] FIG9 shows the results of ADC-2, ADC-3, ADC-6 and ADC-7 inhibiting the growth of H1184 cell subcutaneously transplanted tumors in mice.

[0196] Figures 10A to 10C show the pharmacokinetic results of ADC-1, ADC-2, and ADC-3 in rats. 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.

[0197] FIG11 shows the pharmacokinetic results of ADC-2 in cynomolgus monkeys. DETAILED DESCRIPTION

[0198] 1. Terminology

[0199] In order to make the present disclosure more easily understood, certain technical and scientific terms are described below. Unless otherwise specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0200] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0201] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0202] Unless the context clearly requires otherwise, in the patent specification and claims, the words "comprising," "having," "including," and the like should be construed in the sense of "including but not limited to," rather than in an exclusive or exhaustive sense.

[0203] The term "and / or" is intended to encompass both "and" and "or." For example, the phrase "A, B, and / or C" is intended to encompass each of the following: 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 a trade name is used in this disclosure, it is intended to include the formulation of the trade name product, the drug substance, and the active drug portion of the trade name product.

[0204] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0205] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as 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 (i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as naturally occurring amino acids, 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 naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0206] The term "amino acid mutation" includes amino acid substitutions (also known as amino acid replacements), deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to achieve the final construct, as long as the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of the polypeptide chain. Specific amino acid mutations can be amino acid substitutions. 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 with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position can be represented by position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 is replaced by W instead of T.

[0207] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. A complete antibody typically comprises two light chains and two heavy chains. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or a light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0208] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to antibodies with a structure substantially similar to that of a native antibody or whose heavy chain has an Fc region. A native intact antibody light chain comprises a light chain variable region (VL) and a constant region (CL). The VL is located at the amino terminus of the light chain, and the light chain constant region comprises a kappa chain and a lambda chain. The heavy chain comprises a variable region (VH) and constant regions (CH1, CH2, and CH3). The VH is located at the amino terminus of the heavy chain, and the constant region is at the carboxyl terminus, with CH3 closest to the carboxyl terminus. The heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0209] The term "variable region" or "variable domain" of an antibody refers to the domain of an antibody heavy or light chain that participates in antigen binding. Herein, the heavy chain variable region (VH) and light chain variable region (VL) of an antibody each comprise four conserved framework regions (FRs) and three complementarity determining regions (CDRs). The term "complementarity determining region" or "CDR" refers to the region within the variable domain that primarily contributes to antigen binding; "framework" or "FR" refers to the variable domain residues excluding the CDR residues. The VH comprises three CDR regions: HCDR1, HCDR2, and HCDR3; the VL comprises three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus), in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0210] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc. The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified as shown in Table 1 below.

[0211] Table 1. Relationships between CDR numbering systems

[0212] Unless otherwise indicated, the variable region and CDR sequences disclosed herein are numbered using the Kabat numbering convention.

[0213] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. 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 formed from antibody fragments.

[0214] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and reconstructed Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable Fc regions for 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 can also vary, such as by deleting the C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) or by deleting the C-terminal glycine and lysine in the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise indicated, the numbering convention for the Fc region is the EU numbering system, also known as the EU index.

[0215] 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 a different source or species.

[0216] The term "humanized antibody" is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the rest of the antibody with their human counterparts (i.e., the constant region and the framework region portion of the variable region).

[0217] The terms "human antibody," "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably to refer to antibodies whose variable and constant regions are human sequences. The term encompasses antibodies that are derived from human genes but have sequences that, for example, reduce potential immunogenicity, increase affinity, or eliminate cysteine ​​or glycosylation sites that may cause undesirable folding. The term encompasses antibodies that are recombinantly produced in non-human cells (which may confer glycosylation that is not characteristic of human cells). The term also encompasses antibodies that have been raised in transgenic mice containing human immunoglobulin heavy and light chain loci. The meaning of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0218] The term "affinity" refers to the overall strength of the 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 indicated, as used herein, binding "affinity" refers to internal binding affinity, which reflects the interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0219] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific 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 using methods well known in the art. For example, a biosensor system such as a system measures surface plasmon resonance, or affinity in solution is measured by solution equilibrium titration (SET).

[0220] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence mutated Fc region) and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0221] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for possible natural mutations that may be present in small amounts. In contrast, polyclonal antibody preparations are typically comprised of a variety of different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes. "Monoclonal" should not be interpreted as requiring the production of antibodies by any ad hoc method. In some embodiments, the antibody provided by the present disclosure is a monoclonal antibody.

[0222] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent such as an antigen binding protein (including, for example, an antibody). An antigen may have one or more epitopes that can interact with different antigen binding proteins (e.g., antibodies).

[0223] The term "epitope" refers to an area or region on an antigen that is capable of specific binding to an antibody or antigen-binding fragment thereof. An epitope can be formed by a continuous string of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, brought into spatial proximity by folding of the antigen (i.e., by tertiary folding). Conformational epitopes differ from linear epitopes 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 using routine methods in the art, such as, 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.

[0224] The terms "anti-DLL3 antibody" and "antibody that binds to DLL3" refer to an antibody that binds to DLL3 or an epitope thereof with sufficient affinity. In one embodiment, the extent of binding of the anti-DLL3 antibody to an unrelated protein is less than at least about 10% of the binding of the antibody to DLL3, and the binding can be Surface plasmon resonance measurements.

[0225] The terms "capable of specifically binding", "specific binding" or "binding" refer to an antibody that binds to an antigen or epitope with a higher affinity than to other antigens or epitopes. -7 M or less (e.g., about 1×10 -8 In some embodiments, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, for example, by However, an antibody that specifically binds to an antigen or an epitope thereof may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset).

[0226] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this article, and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections. The nucleic acids are synthetic, naturally occurring, and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). "Isolated" nucleic acid refers to a nucleic acid molecule separated from the components of its natural environment. Isolation nucleic acid is included in the nucleic acid molecule contained in the following cells, which cells typically contain the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or is present at a location different from its natural chromosome position. The isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or a separate vector, and such one or more nucleic acid molecules present in one or more positions in a host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences. Specifically, as described in detail below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0227] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and apply to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0228] The term sequence "identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions; when two sequences are optimally aligned, gaps are introduced, if necessary, to obtain the maximum percent identity, and any conservative substitutions are not considered part of the sequence identity. To determine percent sequence identity, alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0229] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be connected to the viral genome. Some vectors can be autonomously replicated (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins) in the host cell in which they are introduced. Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. The term "expression vector" or "expression construct" refers to a vector that can transform a host cell and contains a nucleic acid sequence for guiding and / or controlling the expression of one or more heterologous coding regions operably connected thereto. Expression constructs can include but are not limited to affecting or controlling transcription, translation and affecting the sequence of RNA splicing of the coding region operably connected thereto when there are introns.

[0230] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its derived progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Mutant progeny are included herein that have the same function or biological activity as the cells screened or selected from the initial transformed cell. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Exemplary host cells are as follows: Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells and HEK-293 cells, Pichia pastoris, Pichia finlandica, Candida albicans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei.

[0231] An “antibody-drug conjugate” (ADC) is a conjugate obtained by connecting an antibody (or its antigen-binding fragment) to a drug directly or through a linker.

[0232] "Drug" is any substance with biological or detectable activity (e.g., a therapeutic agent, a detectable label, a binding agent, etc.) and a prodrug that is metabolized in vivo to an active agent. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cell growth inhibitors, and immunomodulators. Chemotherapeutic agents are chemical compounds that can be used to treat cancer. Representative therapeutic agents include cytotoxins, cytotoxic agents, and cell growth inhibitors.

[0233] A cytotoxic effect refers to the deletion, elimination, and / or killing of target cells. A cytotoxic agent refers to an agent that has a cytotoxic and / or cytostatic effect on cells. A cytostatic effect refers to the inhibition of cell proliferation. A cytostatic agent refers to an agent that has a cytostatic effect on cells, thereby inhibiting the growth and / or proliferation of a specific subset of cells.

[0234] Additional representative therapeutic agents include radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, pro-apoptotic agents and cell lytic enzymes (e.g., RNAse). These drug descriptors are not mutually exclusive, and therefore, one or more of the above terms can be used to describe therapeutic agents. For example, selected radioisotopes are also cytotoxins. Therapeutic agents can be prepared as pharmaceutically acceptable salts, acids or derivatives of any of the above. Typically, conjugates with radioisotopes as drugs are referred to as radioimmunoconjugates, and those with chemotherapeutic agents as drugs are referred to as chemoimmunoconjugates.

[0235] Examples of cytotoxic agents include, but are not limited to, exitecan, anthracyclines, auristatins, CC-1065, dolastatin, duocarmycin, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, 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), radioactive isotopes, photosensitizers (i.e., for photodynamic therapy) may also be used.

[0236] The term "label" as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody to generate a "labeled" antibody. The label can be self-detectable (e.g., a radioisotope label or a fluorescent label), or in the case of an enzymatic label, the label can catalyze a detectable chemical change in the 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. A label can also be an undetectable entity, such as a toxin.

[0237] The terms "linker unit" and "linker" refer to a chemical structure fragment or bond that is connected to an antibody at one end and to a drug at the other end. Other linkers can also be connected to the antibody or drug. Attachment of the linker to the antibody can be accomplished in a variety of ways, such as 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 the acyl donor glutamine, or by engineering the polypeptide in the presence of transglutaminase and an amine to provide reactive endogenous glutamine. Various ADC linking systems are known in the art, including hydrazone-, disulfide-, and peptide-based linkages.

[0238] 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-1carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB").

[0239] The linker can be selected from the following elements or combinations thereof: an extender, a spacer, and an amino acid unit. Linkers can be synthesized by methods known in the art, such as those described in US20050238649A1. Linkers can be "cleavable linkers" that facilitate release of the drug in cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0240] Connector components include but are not limited to:

[0241] MC=6-maleimidocaproyl, the structure is as follows:

[0242] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker),

[0243] Citrulline = 2-amino-5-ureidopentanic acid,

[0244] PAB = p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker element),

[0245] Me-Val-Cit = N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to protect it from cleavage by cathepsin B),

[0246] MC(PEG)6-OH = Maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines),

[0247] SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate,

[0248] SPDP = N-succinimidyl 3-(2-pyridyldithio) propionate,

[0249] SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,

[0250] IT = iminothiolane.

[0251] "LD" is the linker-drug moiety resulting from the attachment of a drug (D) to a linker (L).

[0252] "Drug loading", also known as the drug-to-antibody ratio (DAR), is the average number of drugs conjugated to each antibody in the ADC. It can be, for example, in the range of about 1 to about 10 drugs conjugated to each antibody, and in certain embodiments, in the range of about 1 to about 8 drugs conjugated to each antibody, preferably in the range of 2-8, 2-7, 2-6, 2-5, 2-4, 1-3, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8 and 6-8. The ADC formula disclosed herein includes a collection of antibody-drug conjugates within the aforementioned ranges. In the embodiments disclosed herein, the drug loading can be expressed as n, which is a decimal or an integer. The drug loading can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, HIC and RP-HPLC.

[0253] In one embodiment of the present disclosure, the drug is conjugated to a reactive group (such as a sulfhydryl group) of the antibody via a linker.

[0254] The drug loading of ADCs can be controlled by the following non-limiting methods, including:

[0255] (1) Control the molar ratio of the linker and the monoclonal antibody,

[0256] (2) Control reaction time and temperature,

[0257] (3) Select different reaction reagents.

[0258] The term "alkyl" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., C 1-20 The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms (i.e., C 1-12 alkyl), more preferably an alkyl group having 1 to 6 carbon atoms (i.e., C 1-6Non-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, 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. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl 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.

[0259] The term "alkylene" refers to a divalent alkyl group, wherein alkyl is as defined above, having from 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 preferably an alkylene group having 1 to 12 carbon atoms (i.e., C 1-12 alkylene), more preferably an alkylene group having 1 to 6 carbon atoms (i.e., C 1-6Alkylene). Non-limiting examples include: -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH(CH2CH3)-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc. Alkylene can be substituted or unsubstituted. When substituted, it can be substituted at any available point of attachment. The substituent is preferably selected from one or more of a deuterium atom, a halogen, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl 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.

[0260] The term "alkenyl" refers to an alkyl group containing at least one carbon-carbon double bond in the molecule, wherein alkyl is as defined above and has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) carbon atoms (i.e., C 2-12 The alkenyl group is preferably an alkenyl group having 2 to 6 carbon atoms (i.e., C 2-6 Alkenyl). Non-limiting examples include: ethenyl, propenyl, isopropenyl, butenyl, etc. Alkenyl can be substituted or unsubstituted. When substituted, it can be substituted at any available point of attachment, and the substituent is preferably selected from one or more of a deuterium atom, an alkoxy group, a halogen, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl 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.

[0261] The term "alkynyl" refers to an alkyl group containing at least one carbon-carbon triple bond in the molecule, wherein alkyl is as defined above and has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) carbon atoms (i.e., C 2-12 The alkynyl group is preferably an alkynyl group having 2 to 6 carbon atoms (i.e., C 2-6 Alkynyl). Non-limiting examples include: ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Alkynyl can be substituted or unsubstituted. When substituted, it can be substituted at any available point of attachment. The substituent is preferably selected from one or more of a deuterium atom, an alkoxy group, a halogen, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl 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.

[0262] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy. Alkoxy groups may be substituted or unsubstituted. When substituted, they may be substituted at any available point of attachment, with the substituent preferably being selected from one or more of a deuterium atom, a halogen, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl 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.

[0263] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic all-carbon ring (i.e., monocyclic cycloalkyl) or polycyclic ring system (i.e., polycyclic cycloalkyl) 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., 3 to 20-membered cycloalkyl). The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 ring atoms (i.e., 3 to 12-membered cycloalkyl), more preferably a cycloalkyl group having 3 to 8 ring atoms (i.e., 3 to 8-membered cycloalkyl), and most preferably a cycloalkyl group having 3 to 6 ring atoms (i.e., 3 to 6-membered cycloalkyl, C 3-6 cycloalkyl).

[0264] Non-limiting examples of the monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl and cyclooctyl.

[0265] The polycyclic cycloalkyl group includes: spirocycloalkyl group, fused cycloalkyl group and bridged cycloalkyl group.

[0266] The term "spiroalkyl" refers to a polycyclic ring system having a common carbon atom (called a spiro atom) between the rings, which may contain one or more double bonds within the ring, or one or more heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized to form nitrogen oxides; the sulfur may be optionally oxoed to form sulfoxides or sulfones, but does not include -OO-, -OS- or -SS-), provided that it contains at least one all-carbon ring and the point of attachment is on the all-carbon 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) ring atoms (i.e., a 5- to 20-membered spiroalkyl). The spiroalkyl preferably has 6 to 14 ring atoms (i.e., a 6- to 14-membered spiroalkyl), and more preferably has 7 to 10 ring atoms (i.e., a 7- to 10-membered spiroalkyl). The spirocycloalkyl group includes a monospirocycloalkyl group and a polyspirocycloalkyl group (such as a bispirocycloalkyl group, etc.), preferably a monospirocycloalkyl group or a bispirocycloalkyl group, more preferably a 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered or 7-membered / 6-membered monospirocycloalkyl group. Non-limiting examples include:

[0267] Its connection point can be at any position;

[0268] wait.

[0269] The term "fused cycloalkyl" refers to a polycyclic ring system in which two adjacent carbon atoms are shared between the rings, which is a monocyclic cycloalkyl fused to one or more monocyclic cycloalkyls, or a monocyclic cycloalkyl fused to one or more heterocyclyls, aryls, or heteroaryls, wherein the point of attachment is on the monocyclic cycloalkyl, which 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) ring atoms (i.e., a 5- to 20-membered fused cycloalkyl). The fused cycloalkyl is preferably a fused cycloalkyl having 6 to 14 ring atoms (i.e., a 6- to 14-membered fused cycloalkyl), more preferably a fused cycloalkyl having 7 to 10 ring atoms (i.e., a 7- to 10-membered fused cycloalkyl). The fused cycloalkyl group includes bicyclic fused cycloalkyl groups and polycyclic fused cycloalkyl groups (such as 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 include:

[0270] , whose connection points can be at any position;

[0271] wait.

[0272] The term "bridged cycloalkyl" refers to a full carbon polycyclic ring system that shares two carbon atoms that are not directly connected between the rings, which 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). The bridged cycloalkyl preferably has a bridged cycloalkyl of 6 to 14 carbon atoms (i.e., a 6 to 14-membered bridged cycloalkyl), more preferably a bridged cycloalkyl of 7 to 10 carbon atoms (i.e., a 7 to 10-membered bridged cycloalkyl). The bridged cycloalkyl includes bicyclic bridged cycloalkyl and polycyclic bridged cycloalkyl (e.g., tricyclic bridged cycloalkyl, tetracyclic bridged cycloalkyl, etc.), preferably bicyclic bridged cycloalkyl or tricyclic bridged cycloalkyl. Non-limiting examples include:

[0273] Its connection point can be at any position.

[0274] The cycloalkyl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0275] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., a monocyclic heterocyclyl) or a polycyclic heterocyclic ring system (i.e., a polycyclic heterocyclyl) containing at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxoed, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-) in the ring, 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). The heterocyclic group is preferably a heterocyclic group having 3 to 12 ring atoms (i.e., a 3- to 12-membered heterocyclic group); further preferably a heterocyclic group having 3 to 8 ring atoms (i.e., a 3- to 8-membered heterocyclic group); more preferably a heterocyclic group having 3 to 6 ring atoms (i.e., a 3- to 6-membered heterocyclic group); and most preferably a heterocyclic group having 5 or 6 ring atoms (i.e., a 5- or 6-membered heterocyclic group).

[0276] Non-limiting examples of the monocyclic heterocyclic group include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl and homopiperazinyl.

[0277] The polycyclic heterocyclic group includes a spiro heterocyclic group, a fused heterocyclic group and a bridged heterocyclic group.

[0278] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic ring system in which the rings share one atom (called a spiro atom), which may contain one or more double bonds in the ring and at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS- or -SS-), provided that it contains at least one monocyclic heterocyclic group and the point of attachment is on the monocyclic heterocyclic 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). The spiro heterocyclic radical preferably has a spiro heterocyclic radical (i.e., a 6 to 14 yuan spiro heterocyclic radical) of 6 to 14 ring atoms, more preferably a spiro heterocyclic radical (i.e., a 7 to 10 yuan spiro heterocyclic radical) with 7 to 10 ring atoms. The spiro heterocyclic radical includes monospiro heterocyclic radical and polyspiro heterocyclic radical (such as dispiro heterocyclic radical etc.), preferably monospiro heterocyclic radical or dispiro heterocyclic radical, more preferably 3 yuan / 4 yuan, 3 yuan / 5 yuan, 3 yuan / 6 yuan, 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 3 yuan, 5 yuan / 4 yuan, 5 yuan / 5 yuan, 5 yuan / 6 yuan, 5 yuan / 7 yuan, 6 yuan / 3 yuan, 6 yuan / 4 yuan, 6 yuan / 5 yuan, 6 yuan / 6 yuan, 6 yuan / 7 yuan, 7 yuan / 5 yuan or 7 yuan / 6 yuan monospiro heterocyclic radical. Non-limiting examples include:

[0279] wait.

[0280] The term "fused heterocyclyl" refers to a polycyclic heterocyclic ring system that shares two adjacent atoms between the rings, which may contain one or more double bonds within the ring and at least one (e.g., 1, 2, 3, or 4) heteroatom selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxoed, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS-, or -SS-), which is a monocyclic heterocyclyl fused to one or more monocyclic heterocyclyls, or a monocyclic heterocyclyl fused to one or more cycloalkyl, aryl, or heteroaryl groups, wherein the point of attachment is on the monocyclic heterocyclyl, 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). The fused heterocyclic radical preferably has a fused heterocyclic radical of 6 to 14 ring atoms (i.e., a 6 to 14-membered fused heterocyclic radical), more preferably a fused heterocyclic radical of 7 to 10 ring atoms (i.e., a 7 to 10-membered fused heterocyclic radical). The fused heterocyclic radical includes bicyclic and polycyclic fused heterocyclic radicals (such as tricyclic fused heterocyclic radicals, tetracyclic fused heterocyclic radicals, etc.), preferably bicyclic fused heterocyclic radicals or tricyclic fused heterocyclic radicals, more preferably 3 yuan / 4 yuan, 3 yuan / 5 yuan, 3 yuan / 6 yuan, 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 3 yuan, 5 yuan / 4 yuan, 5 yuan / 5 yuan, 5 yuan / 6 yuan, 5 yuan / 7 yuan, 6 yuan / 3 yuan, 6 yuan / 4 yuan, 6 yuan / 5 yuan, 6 yuan / 6 yuan, 6 yuan / 7 yuan, 7 yuan / 5 yuan or 7 yuan / 6 yuan bicyclic fused heterocyclic radicals. Non-limiting examples include:

[0281] wait.

[0282] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic ring system that shares two atoms that are not directly connected between the rings, which may contain one or more double bonds within the ring and at least one (e.g., 1, 2, 3, or 4) heteroatom selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS-, or -SS-), 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 bridged heterocyclic group). The bridged heterocyclic group is preferably a bridged heterocyclic group having 6 to 14 ring atoms (i.e., a 6- to 14-membered bridged heterocyclic group), and more preferably a bridged heterocyclic group having 7 to 10 ring atoms (i.e., a 7- to 10-membered bridged heterocyclic group). According to the number of constituent rings, heterocyclic groups can be divided into bicyclic bridged heterocyclic groups and polycyclic bridged heterocyclic groups (such as tricyclic bridged heterocyclic groups, tetracyclic bridged heterocyclic groups, etc.), preferably bicyclic bridged heterocyclic groups or tricyclic bridged heterocyclic groups. Non-limiting examples include:

[0283] wait.

[0284] The heterocyclic group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment. The substituents are preferably selected from one or more of a deuterium atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclic groupoxy group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group.

[0285] The term "aryl" refers to a monocyclic all-carbon aromatic ring (i.e., a monocyclic aromatic group) or a polycyclic aromatic ring system (i.e., a polycyclic aromatic group) having a conjugated π electron system, which has 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., a 6- to 14-membered aromatic group). The aryl group is preferably an aromatic group having 6 to 10 ring atoms (i.e., a 6- to 10-membered aromatic group). The monocyclic aromatic group is, for example, a phenyl group. Non-limiting examples of the polycyclic aromatic group include: naphthyl, anthracenyl, phenanthrenyl, etc. The polycyclic aromatic group also includes a phenyl group fused with one or more heterocyclic groups or cycloalkyl groups, or a naphthyl group fused with one or more heterocyclic groups or cycloalkyl groups, wherein the connection point is on the phenyl group or naphthyl group, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic aromatic ring system, non-limiting examples include:

[0286] wait.

[0287] The aryl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0288] The term "heteroaryl" refers to a monocyclic heteroaromatic ring (i.e., a monocyclic heteroaryl) or a polycyclic heteroaromatic ring system (i.e., a polycyclic heteroaryl) having a conjugated π electron system, which contains at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS-, or -SS-), and has 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). The heteroaryl is preferably a heteroaryl having 5 to 10 ring atoms (i.e., a 5- to 10-membered heteroaryl), more preferably a heteroaryl having 5 or 6 ring atoms (i.e., a 5- or 6-membered heteroaryl).

[0289] The monocyclic heteroaryl groups include, but are not limited to, furyl, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furazanyl, pyrrolyl, N-alkylpyrrolyl, pyridyl, pyrimidinyl, pyridonyl, N-alkylpyridone (e.g. etc.), pyrazinyl, pyridazinyl, etc.

[0290] The polycyclic heteroaryl groups include, but are not limited to, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, phthalazinyl, benzimidazolyl, benzothiophenyl, quinazolinyl, benzothiazolyl, carbazolyl, and the like. The polycyclic heteroaryl groups also include monocyclic heteroaryl groups fused to one or more aromatic groups, wherein the point of attachment is on the aromatic ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaromatic ring system. The polycyclic heteroaryl groups also include monocyclic heteroaryl groups fused to one or more cycloalkyl or heterocyclic groups, wherein the point of attachment is on the monocyclic heteroaromatic ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaromatic ring system. Non-limiting examples include:

[0291] wait.

[0292] The heteroaryl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a deuterium atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl 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.

[0293] The term "amino protecting group" refers to a group that is easily removed and introduced onto an amino group in order to keep the amino group unchanged while reacting other parts of the molecule. Non-limiting examples include: (trimethylsilyl)ethoxymethyl, tetrahydropyranyl, tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), methyloxycarbonyl (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.

[0294] The term "hydroxy protecting group" refers to a group that is introduced on a hydroxy group and is easily removed, and is used to block or protect the hydroxy group while reacting on other functional groups of the compound. 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, etc.

[0295] The term "cycloalkyloxy" refers to a cycloalkyl-O- group in which cycloalkyl is as defined above.

[0296] The term "heterocyclyloxy" refers to a heterocyclyl-O- group in which heterocyclyl is as defined above.

[0297] The term "aryloxy" refers to an aryl-O- group in which the aryl group is as defined above.

[0298] The term "heteroaryloxy" refers to a heteroaryl-O- group in which heteroaryl is as defined above.

[0299] The term "alkylthio" refers to an alkyl-S- group in which alkyl is as defined above.

[0300] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0301] The term "haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.

[0302] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0303] The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups, wherein alkyl is as defined above.

[0304] The term "methylidene" refers to =CH2.

[0305] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0306] The term "hydroxy" refers to -OH.

[0307] The term "mercapto" refers to -SH.

[0308] The term "amino" refers to -NH2.

[0309] The term "cyano" refers to -CN.

[0310] The term "nitro" refers to -NO2.

[0311] The term "oxo" or "oxo" refers to "=0".

[0312] The term "carbonyl" refers to C=O.

[0313] The term "carboxy" refers to -C(O)OH.

[0314] The term "carboxylate" refers to -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.

[0315] The abbreviation "Me" in the chemical formula is methyl.

[0316] The abbreviation "Ph" in the chemical formula is phenyl.

[0317] The term "THF" refers to tetrahydrofuran.

[0318] The term "EtOAc" refers to ethyl acetate.

[0319] The term "MeOH" refers to methanol.

[0320] The term "DMF" refers to N,N-dimethylformamide.

[0321] The term "DIPEA" refers to diisopropylethylamine.

[0322] The term "TFA" refers to trifluoroacetic acid.

[0323] The term "MeCN" refers to acetonitrile.

[0324] The term "DMA" refers to N,N-dimethylacetamide.

[0325] The term "Et2O" refers to diethyl ether.

[0326] The term "DCE" refers to 1,2-dichloroethane.

[0327] The term "DIPEA" refers to N,N-diisopropylethylamine.

[0328] The term "NBS" refers to N-bromosuccinimide.

[0329] The term "NIS" refers to N-iodosuccinimide.

[0330] The term "Cbz-Cl" refers to benzyl chloroformate.

[0331] The term "Pd2(dba)3" refers to tris(dibenzylideneacetone)dipalladium.

[0332] The term "Dppf" refers to 1,1'-bisdiphenylphosphinoferrocene.

[0333] The term "HATU" refers to 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0334] The term "KHMDS" refers to potassium hexamethyldisilazide.

[0335] The term "LiHMDS" refers to lithium bistrimethylsilylamide.

[0336] The term "MeLi" refers to methyllithium.

[0337] The term "n-BuLi" refers to n-butyllithium.

[0338] The term "NaBH(OAc)3" refers to sodium triacetoxyborohydride.

[0339] The term "DCM" refers to dichloromethane.

[0340] The term "DMAP" refers to 4-dimethylaminopyridine.

[0341] The term "DMBOH" refers to 2,4-dimethoxybenzyl alcohol.

[0342] The term "EDCI" refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0343] The term "MTBE" refers to methyl tert-butyl ether.

[0344] The term "DMF" refers to N,N-dimethylformamide.

[0345] The term "DMTMM" refers to 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride.

[0346] The term "EtOAc" refers to ethyl acetate.

[0347] The compounds disclosed herein may exist in specific stereoisomeric forms. The term "stereoisomer" refers to isomers having identical structures but different arrangements of atoms in space. It includes cis and trans (or Z and E) isomers, (-)- and (+)-isomers, (R)- and (S)-enantiomers, diastereomers, (D)- and (L)-isomers, tautomers, atropisomers, conformers and mixtures thereof (such as racemates, mixtures of diastereomers). The substituents in the compounds disclosed herein may have additional asymmetric atoms. All of these stereoisomers and their mixtures are included within the scope of the disclosure. Optically active (-)- and (+)-isomers, (R)- and (S)-enantiomers and (D)- and (L)-isomers may be prepared by chiral synthesis, chiral reagents or other conventional techniques. An isomer of a compound disclosed herein can be prepared by asymmetric synthesis or chiral auxiliary, or, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), by forming a diastereomeric salt with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art to obtain the pure isomer. Furthermore, separation of enantiomers and diastereoisomers is typically accomplished by chromatography.

[0348] In the chemical structure of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Two configurations.

[0349] 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 is easily converted from one isomeric form to another isomeric form. It includes all possible tautomers, i.e., in the form of a single isomer or in the form of a mixture of any proportions of the tautomers. Non-limiting examples include: keto-enol, imine-enamine, lactam-lactim, etc. The equilibrium of lactam-lactim is shown below:

[0350] For example, when referring to pyrazolyl, it is understood to include either of the following two structures or a mixture of two tautomers:

[0351] All tautomeric forms are within the scope of the present disclosure, and the naming of compounds does not exclude any tautomer.

[0352] The compounds of the present disclosure include all suitable isotopic derivatives of the compounds thereof. The term "isotopic derivative" refers to a compound in which at least one atom is replaced by 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, etc., preferably deuterium.

[0353] Compared to non-deuterated drugs, deuterated drugs offer advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged biological half-life. All isotopic variations of the compounds disclosed herein, whether radioactive or not, are encompassed by this disclosure. Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom, where the deuterium replacement can be partial or complete. Partial deuterium replacement refers to the replacement of at least one hydrogen atom with at least one deuterium atom.

[0354] When a position is specifically designated as "deuterium" or "D," the position is understood to have an abundance of deuterium that is at least 1000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 15% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 1000 times greater than the natural abundance of deuterium (i.e., at least 15% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 2000 times greater than the natural abundance of deuterium (i.e., at least 30% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3000 times greater than the natural abundance of deuterium (i.e., at least 45% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3340 times greater than the natural abundance of deuterium (i.e., at least 50.1% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3500 times greater than the natural abundance of deuterium (i.e., at least 52.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 4000 times greater than the natural abundance of deuterium (i.e., at least 60% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 4500 times greater than the natural abundance of deuterium (i.e., at least 67.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 5000 times greater than the natural abundance of deuterium (i.e., at least 75% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 5500 times greater than the natural abundance of deuterium (i.e., at least 82.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6000 times greater than the natural abundance of deuterium (i.e., at least 90% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6333.3 times greater than the natural abundance of deuterium (i.e., at least 95% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6466.7 times greater than the natural abundance of deuterium (i.e., at least 97% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6600 times greater than the natural abundance of deuterium (i.e., at least 99% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6633.3 times greater than the natural abundance of deuterium (ie, at least 99.5% deuterium incorporation).

[0355] "Substitution" or "substituted" means that one or more hydrogen atoms, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms, in a group are independently replaced by a corresponding number of substituents. Those skilled in the art can determine (by experiment or theory) whether substitution is possible or not without undue effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0356] The present disclosure also includes various deuterated forms of antibody-drug conjugates of formula (Pc-LYD). Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of antibody-drug conjugates of formula (Pc-LYD) with reference to relevant literature. When preparing deuterated forms of antibody-drug conjugates of formula (Pc-LYD), commercially available deuterated starting materials can be used, or they can be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane.

[0357] "Optional" or "optionally" means that the event or circumstances described subsequently may but need not occur, and includes both situations in which the event or circumstances occur and do not occur. For example, "alkyl optionally (optionally) substituted with halogen or cyano" includes both situations in which the alkyl is substituted with halogen or cyano and situations in which the alkyl is not substituted with halogen and cyano.

[0358] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0359] The pharmaceutical composition can be in the form of a sterile injectable aqueous solution. Acceptable vehicles and solvents include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable formulation can be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion can be injected into the subject's bloodstream via local bolus injection. Alternatively, the solution or microemulsion can be administered in a manner that maintains a constant circulating concentration of the disclosed compound. To maintain this constant concentration, a continuous intravenous drug delivery device can be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous pump.

[0360] Pharmaceutical compositions can be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions prepared in a nontoxic, parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, sterile fixed oils can conveniently be used as solvents or suspending media. For this purpose, any blended fixed oil, including synthetic mono- or diglycerides, can be used. Furthermore, fatty acids, such as oleic acid, can also be used to prepare injectable formulations.

[0361] The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the antibody-drug conjugates of the present disclosure that is safe and effective when administered to a subject and exhibits the desired biological activity. As an example, the antibody-drug conjugates of the present disclosure contain at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0362] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, stabilizers, or preservatives.

[0363] The term "excipient" refers to any additive in a pharmaceutical preparation other than the active ingredient, also known as an adjuvant. Examples include binders, fillers, disintegrants, and lubricants in tablets; the base component of semisolid ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0364] The term "diluent," also known as filler, primarily increases the weight and volume of a tablet. The addition of a diluent not only maintains a certain volume but also reduces dosage variations of the main ingredient and improves the drug's compressibility. When the tablet contains an oily component, an absorbent is added to absorb the oil and maintain a "dry" state, facilitating tablet production. Examples include starch, lactose, inorganic calcium salts, and microcrystalline cellulose.

[0365] 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" are used interchangeably herein. As used herein, the term "cynomolgus monkey" or "cynomolgus monkey" refers to cynomolgus monkeys (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

[0366] "Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

[0367] The term "sample" refers to a collection of fluid, cells, or tissue isolated from a subject, as well as fluid, cells, or tissue present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.

[0368] "Treatment" and "treatment" (and grammatical variations thereof) refer to clinical interventions that attempt to alter the pathological process of the individual being treated and can be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the development of the disease or slow the progression of the disease.

[0369] "Effective amount" is generally enough to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring and / or improve or ameliorate the amount of damage caused by or associated with the disease state. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactic effective amount. A "therapeutically effective amount" is enough to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "prophylactic effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, 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 related symptoms. A complete treatment or prophylactic effect may not occur after administering one dose, but may occur after administering a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more doses. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on factors such as 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.

[0370] 2. Description of Specific Implementation Methods

[0371] A. Antibody Structure in Antibody-Drug Conjugates

[0372] In certain embodiments, the antibodies provided herein are full-length antibodies.

[0373] In certain embodiments, the antibodies provided herein are antibody fragments.

[0374] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH or F(ab')2 fragment, in particular a Fab fragment. "Fab" is a monovalent fragment consisting of the VL, VH, CL and CH1 domains. A "Fab fragment" can be produced by papain cleavage of an antibody. "Fab'" contains VL, CL, VH and CH1, and also contains a region between the CH1 and CH2 domains so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule. "Fab'-SH" is a Fab' fragment in which the cysteine ​​residues in the constant region have a free sulfhydryl group. "F(ab')2" is a bivalent fragment comprising two Fab fragments connected by a disulfide bond at the hinge region.

[0375] In another embodiment, the antibody fragment is a diabody, triabody, or tetrabody. A diabody is an antibody fragment with two antigen-binding sites, which contains a linked VH and VL in the same polypeptide chain (VH-VL). By using a linker that is too short to prevent pairing between the two domains on the same chain, these domains are forced to pair with complementary domains from another chain, thereby generating two antigen-binding sites. The two antigens can be the same or different.

[0376] 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 the linker have one of the following orders in the N-terminal to C-terminal direction: 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 between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via the native disulfide bond between CL and CH1. In addition, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., at 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.

[0377] In another embodiment, the antibody fragment is a single-chain variable fragment (scFv). "scFv" is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously connected by a short flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFv herein can have the VL and VH variable regions in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0378] In another embodiment, the antibody fragment is an Fd fragment consisting of the VH and CH1 domains.

[0379] In another embodiment, the antibody fragment is an Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0380] In another embodiment, the antibody fragment is a dsFv 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 to be substituted with the cysteine ​​residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method (Protein Engineering. 7: 697 (1994)).

[0381] In another embodiment, the antibody fragment is a single domain antibody, which is an antibody fragment comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.

[0382] In another embodiment, the antibody fragment is a domain antibody (dAb); see, e.g., U.S. Patent No. 6,248,516. Domain antibodies (dAbs) are functional binding domains of antibodies that correspond to the variable regions of the heavy (VH) or light (VL) chains of human antibodies. dAbs have a molecular weight of approximately 13 kDa, or less than one-tenth the size of a full-length antibody. dAbs express well in a variety of hosts, including bacteria, yeast, and mammalian cell systems. Furthermore, dAbs are highly stable and retain activity even under harsh conditions, such as lyophilization or heat denaturation. See, for example, U.S. Patents 6,291,158; 6,582,915; 6,593,081; 6,172,197; U.S. Serial No. 2004 / 0110941; European Patent 0368684; U.S. Patent 6,696,245; WO04 / 058821; WO04 / 003019 and WO03 / 002609.

[0383] 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.

[0384] In certain embodiments, antibody is a humanized antibody. Generally, non-human antibodies are passed through humanization to reduce immunogenicity to people, while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies comprise one or more variable regions, wherein CDR or its portion are derived from non-human antibodies, and FR or its portion are derived from human antibodies. Optionally, humanized antibodies also may comprise a part for human constant region. In some embodiments, some FR residues in humanized antibodies may be substituted with corresponding residues from non-human antibodies (for example, antibodies providing CDR sequences).

[0385] Humanized antibodies and methods for their generation are reviewed in, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, 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 specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfuacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the "guided selection" method of FR shuffling).

[0386] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light chain variable regions or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol., 151: 2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0387] B. Modification of Antibodies in Antibody-Drug Conjugates

[0388] In certain embodiments, the amino acid sequence variants of the antibody in the antibody conjugates provided herein are encompassed. For example, it is desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of the residues in the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution can be performed to obtain the final construct, as long as the final construct has the desired characteristics, such as antigen binding.

[0389] a) Substitution, insertion, and deletion variants

[0390] In certain embodiments, antibody variants with one or more amino acid replacements are provided. Interested sites of substitution mutagenesis include CDRs and FRs. Conservative replacements are shown under the heading of "preferred replacements" in Table 2. More substantial changes are provided under the heading of "exemplary replacements" in Table 2, and are further described below with reference to amino acid side chain classes. Amino acid replacements can be introduced into the antibody of interest, and the product is screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0391] Table 2

[0392] Based on common side chain properties, amino acids can be grouped as follows:

[0393] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0394] (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0395] (3) Acidic: Asp, Glu;

[0396] (4) Basic: His, Lys, Arg;

[0397] (5) Residues that affect chain orientation: Gly, Pro;

[0398] (6) Aromatic: Trp, Tyr, Phe.

[0399] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0400] In certain embodiments, substitution, insertion or deletion can occur in one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) can be made to the CDRs, which do not substantially reduce binding affinity. Such changes can be, for example, outside the antigen contact residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged, or contains no more than 1, 2 or 3 amino acid substitutions.

[0401] A method for identifying residues or regions that can be used as mutagenesis targets in antibodies is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or residue group (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that initially show functional sensitivity. In addition, the contact points between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as alternative candidates. Variants can be screened to determine whether they contain desired properties.

[0402] Amino acid sequence insertions include amino and / or carboxyl terminus fusions ranging in length from 1 residue to 100 or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of an antibody with an enzyme or a polypeptide that extends the serum half-life of the antibody.

[0403] b) Fc region modification

[0404] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein.

[0405] In some embodiments, the one or more amino acid modifications can reduce Fc binding to an Fc receptor, such as its binding to an Fcγ receptor, and reduce or eliminate effector function. In some embodiments, the engineered Fc region has a binding affinity for an Fc receptor decreased by 50%, 80%, 90%, or 95% or more compared to a native Fc region. In some embodiments, the Fc receptor is a human Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIB, or FcγRIIIa. In some embodiments, the engineered Fc region also has a reduced binding affinity for complement, such as C1q, compared to a native Fc region. In some embodiments, the engineered Fc region has an enhanced binding affinity for the neonatal Fc receptor (FcRn) compared to a native Fc region; for example, by introducing the M252Y / S254T / T256E mutations into the Fc region. In some embodiments, the transformed Fc region has reduced effector function, which can include but is not limited to one or more of the following: 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 uptake of antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation or reduced T cell priming. For IgG1 Fc region, amino acid residues at positions 238, 265, 269, 270, 297, 327 and 329 replace the effector function that can be reduced. In some embodiments, the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index. For the IgG4 Fc region, substitution of amino acid residues at positions such as 228 can reduce effector function.

[0406] In certain embodiments, the antibodies comprise one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (using the EU numbering system) of the Fc region.

[0407] In certain embodiments, the Fc domain of the antibodies herein comprises a "knob-in-hole" mutation. A "knob-in-hole" is a design strategy for engineering antibody heavy chain homodimers to undergo heterodimerization (e.g., to efficiently generate bispecific antibodies, multispecific antibodies, or one-armed antibodies). Generally, such techniques involve introducing a protuberance ("knob") at the interface of a first polypeptide (such as the first CH3 domain in the first antibody heavy chain) and a cavity ("hole") at the corresponding interface of a second polypeptide (such as the second CH3 domain in the second antibody heavy chain), such that the protuberance can be positioned in the cavity to promote heterodimer formation and hinder homodimer formation. The protuberance is constructed by replacing smaller amino acid side chains from the interface of the first polypeptide (such as the first CH3 domain in the first antibody heavy chain) with larger side chains (e.g., arginine, phenylalanine, tyrosine, or tryptophan). A compensatory cavity of the same or similar size as the protuberance is created in the interface of a second polypeptide (such as the second CH3 domain in a second antibody heavy chain) by replacing larger amino acid side chains with smaller side chains (e.g., alanine, serine, valine, or threonine). The protuberance and cavity can be generated by altering the nucleic acid encoding the polypeptide (e.g., by site-specific 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 comprising the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge 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-to-hole mutations include, but are not limited to, those described in Table 3.

[0408] Table 3

[0409] Details regarding the pestle-and-mortar technique are described in, for example, 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).

[0410] The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a truncated C-terminus ending with PG. Therefore, in some embodiments, a composition of intact antibodies can include a population of antibodies in which all K447 residues and / or G446+K447 residues have been removed. In some embodiments, a composition of intact antibodies can include a population of antibodies in which the K447 residue and / or G446+K447 residues have not been removed. In some embodiments, a composition of intact antibodies comprises a mixture of antibodies with and without the K447 residue and / or G446+K447 residues.

[0411] C. Recombination Methods

[0412] Anti-DLL3 antibodies can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0413] In one embodiment, the present disclosure provides isolated nucleic acids encoding antibodies as described above. Such nucleic acids can independently encode any of the aforementioned 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, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0414] In order to recombinantly produce an antibody, the nucleic acid encoding the antibody is separated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily separated and sequenced using conventional procedures, or produced by recombinant methods or obtained by chemical synthesis.

[0415] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector functions are not desired. Following expression, the soluble fraction can be isolated from the bacterial cell paste and further purified.

[0416] D. Determination

[0417] The antibody drug conjugates provided herein can be identified, screened or characterized for their physical / chemical characteristics and / or biological activity by a variety of assays known in the art. In one aspect, the antibody drug conjugate activity of the present disclosure can be tested, for example, by known methods such as ELISA, Western blotting, etc.

[0418] E. Treatment Methods and Administration Routes

[0419] Any of the aforementioned antibody-drug conjugates provided herein, or pharmaceutically acceptable salts thereof, can be used to treat a disease.

[0420] In one aspect, the present disclosure provides use of an anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof in the preparation of a medicament. In some embodiments, the present disclosure provides 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 includes but is not limited to lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, large cell lung cancer), head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer (e.g., medullary thyroid cancer), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer (e.g., colon cancer and rectal cancer), kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer, glioblastoma, skin cancer and melanoma; preferably, the lung cancer is small cell lung cancer.

[0421] In some embodiments, the present disclosure provides use of an anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a DLL3-related disease.

[0422] In one such embodiment, the use further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent (eg, one, two, three, four, five, or six additional therapeutic agents).

[0423] In yet another aspect, a pharmaceutical composition comprising the anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof is provided, e.g., for use in any of the above pharmaceutical uses or therapeutic methods. In one embodiment, the pharmaceutical composition comprises any polypeptide or drug conjugate provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0424] The anti-DLL3 antibody-drug conjugates or pharmaceutically acceptable salts thereof of the present disclosure can be used alone or in combination with other agents for treatment. For example, the anti-DLL3 antibody-drug conjugates or pharmaceutically acceptable salts thereof of the present disclosure can be co-administered with at least one additional therapeutic agent.

[0425] The anti-DLL3 antibody-drug conjugates of the present disclosure, or pharmaceutically acceptable salts thereof, can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusions.

[0426] The anti-DLL3 antibody-drug conjugates disclosed herein, or pharmaceutically acceptable salts thereof, should be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered 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 site of delivery of the agent, the method of administration, the timing of administration, and other factors known to medical practitioners. The anti-DLL3 antibody or drug conjugate thereof may be formulated with one or more additional agents. The effective amount of such additional agents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used in the same dosages and by administration routes as described herein, or at approximately 1 to 99% of the dosages described herein, or at other dosages and by any route determined empirically / clinically appropriate.

[0427] For the prevention or treatment of disease, the appropriate dosage of an anti-DLL3 antibody-drug conjugate of the present disclosure, or a pharmaceutically acceptable salt thereof (when used alone or in combination with one or more other additional therapeutic agents), 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 for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient at one time or over a series of treatments.

[0428] F. Products

[0429] In another aspect of the present disclosure, an article (e.g., a kit) is provided that includes materials useful for treating, preventing, and / or diagnosing the aforementioned conditions. The article 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 can be formed from a variety of materials, such as glass or plastic.

[0430] The container contains an anti-DLL3 antibody-drug conjugate of the present disclosure, or a pharmaceutically acceptable salt thereof, alone or in combination with another composition. The container can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper). At least one active agent in the composition is an 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 to treat the condition of choice.

[0431] Additionally, an article of manufacture may comprise: (a) a first container having an anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof contained therein; and (b) a second container having a composition contained therein, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent.

[0432] Alternatively, or additionally, the product may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer. From a commercial and user perspective, it may further comprise other materials required, including other buffers, diluents, filters, needles, and syringes.

[0433] Example

[0434] The present disclosure is further described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0435] Experimental methods in the disclosed embodiments or test examples where specific conditions are not specified are generally performed under conventional conditions or those recommended by the raw material or product manufacturers. Reagents where the specific sources are not specified are conventional reagents purchased from the market.

[0436] 1. Antibody Preparation

[0437] Example 1: Preparation of DLL3 antigen, detection protein, and stably transfected cell lines

[0438] DLL3 genes from different species, along with human DLL1 and DLL4 genes, were transfected into Chinese hamster ovary (CHO) cells (Invitrogen, R80007) to construct CHO-S cell lines expressing DLL3 proteins from different species for subsequent antibody screening and identification. The amino acid sequences of the relevant proteins are as follows:

[0439] Human DLL3 full-length protein (Uniprot, Q9NYJ7):

[0440] Cynomolgus monkey DLL3 full-length protein (Uniprot, A0A2K5WSR4):

[0441] Rat DLL3 full-length protein (Uniprot, O88671):

[0442] Mouse DLL3 full-length protein (Uniprot, O88516):

[0443] Human DLL1 full-length protein (Uniprot, O00548):

[0444] Human DLL4 full-length protein (Uniprot, Q9NR61):

[0445] 1.1 Construction of cell lines with high expression of DLL3, DLL1, and DLL4

[0446] The pCDH lentiviral expression vector plasmid containing SEQ ID NOs: 1-6 (synthesized by GENEWIZ) was transfected into 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17) using Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent with pVSVG and pCMV lentiviral packaging vectors. The supernatant containing the virus was collected, filtered, and subjected to ultracentrifugation. The supernatant was discarded and resuspended in 0.2 mL of sterile PBS. The concentrated virus was used to infect Chinese hamster ovary cells (CHO-S, Invitrogen, R80007), DMS53 (ATCC, CRL-2062), and H82 (ATCC, HTB-175). The cells were selected with puromycin for two to three weeks and then single-cell sorted by FACS. The selected monoclonal cell lines were expanded and frozen.

[0447] 1.2 Preparation of Antigens

[0448] Using human DLL3 (Uniprot, Q9NYJ7), cynomolgus macaque DLL3 (Uniprot, A0A2K5WSR4), and mouse DLL3 (Uniprot, O88516) sequences as templates, DLL3 ECD fusion proteins containing different tags were designed and cloned into the pTT5 vector. After expression in 293E cells, the antigens were obtained. The amino acid sequences of the relevant proteins are as follows:

[0449] 1) His-hDLL3 (ECD):

[0450] Note: The dot-dashed line represents part of the signal peptide sequence, the single-dash line represents the his tag and linker, and the double-dash line represents the DLL3 extracellular region.

[0451] 2) Fc-hDLL3 (ECD):

[0452] Note: The dot-dashed part is the signal peptide sequence, the single-dash part is the Fc tag and linker, and the double-dash part is the DLL3 extracellular region.

[0453] 3)hDLL3(ECD)-strep twin:

[0454] Note: The dot-dashed part is the signal peptide sequence, the double-dashed part is the DLL3 extracellular region, and the single-dashed part is the strep twin tag.

[0455] 4)cynoDLL3(ECD)-strep twin:

[0456] Note: The dot-dashed part is the signal peptide sequence, the double-dashed part is the DLL3 extracellular region, and the single-dashed part is the strep twin tag.

[0457] 5) mouDLL3(ECD)-strep twin:

[0458] Note: The dot-dashed part is the signal peptide sequence, the double-dashed part is the DLL3 extracellular region, and the single-dashed part is the strep twin tag.

[0459] Example 2: Preparation of mouse anti-human DLL3 monoclonal antibodies

[0460] 1. Immunity

[0461] Anti-human DLL3 monoclonal antibodies were produced by immunizing mice. Female SJL mice, 6-8 weeks old (Shanghai Slake Laboratory Animal Co., Ltd., Animal Production License No. SCXK(Shanghai)2017-0005) were used in this experiment. Mice were housed in an SPF-grade laboratory environment for one week under a 12 / 12 hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. Acclimated mice were immunized according to the following protocol.

[0462] Immunization regimen:

[0463] The first group of mice was immunized with His-hDLL3 (ECD) (SEQ ID NO: 7). Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No. 77161) adjuvant cross immunization. Antigen and adjuvant The ratio of Gold Adjuvant is 1:1, antigen and adjuvant Thermo The alum ratio was 3:1, 50 μg / mouse / time (primary immunization) and 25 μg / mouse / time (boost immunization). Antigen was emulsified and inoculated, with immunization times of 0, 7, 14, and 21 days. Blood was collected on days 7 and 21, and the antibody titer in the mouse serum was determined by ELISA. After the fourth immunization, mice with high antibody titers in serum and titers that reached a plateau were selected for spleen cell fusion. Three days before spleen cell fusion, booster immunization was performed, with an intraperitoneal (ip) injection of 25 μg / mouse of an antigen solution prepared in saline.

[0464] The second group of mice were immunized with DLL3CHO-s and Fc-hDLL3 (ECD) (SEQ ID NO: 8) in alternating immunization with cell and protein antigens. Gold Adjuvant (Sigma Cat No. T2684) 0.1 mL / mouse was injected intraperitoneally. Half an hour later, each mouse was injected intraperitoneally with 0.1 mL diluted to 10% with normal saline. 8 / mL concentration of cell solution. After the cells are evenly dispersed, they are inoculated on days 0, 14, 28, and 42. Fc-hDLL3 (ECD) antigen is used Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No. 77161) adjuvant cross immunization. Antigen and adjuvant The ratio of Gold Adjuvant is 1:1, antigen and adjuvant Thermo The alum ratio was 3:1, with 50 μg / mouse / dose for the primary immunization and 25 μg / mouse / dose for the booster immunization. hDLL3-Fc immunizations were administered on days 7, 21, 35, and 49. Blood samples were collected on days 14, 35, and 49, and serum antibody titers were determined by ELISA. After the eighth immunization, mice with high serum antibody titers that plateaued were selected for spleen cell fusion. Three days prior to spleen cell fusion, a booster immunization was performed with an intraperitoneal (ip) injection of 50 μg / mouse of hDLL3-Fc protein antigen in saline.

[0465] 2. Spleen Cell Fusion

[0466] The optimized electrofusion method was used to combine spleen lymphocytes with myeloma cells Sp2 / 0 cells ( CRL-8287 TM ) were fused to obtain hybridoma cells.

[0467] The fused hybridoma cells were 3-4×10 5 Resuspend the cells at a density of 100 μL / well in complete culture medium (IMDM medium containing 20% ​​FBS, 1× HAT, and 1× OPI) at a density of 100 μL / well and seed 150 μL / well in a 96-well plate. Incubate at 37°C, 5% CO2 for 3-4 days, remove the supernatant, add 200 μL / well of HT complete culture medium (IMDM medium containing 20% ​​FBS, 1× HAT, and 1× OPI), and incubate at 37°C, 5% CO2 for 3 days before screening.

[0468] 3. Hybridoma cell screening and antibody sequence determination

[0469] Based on hybridoma cell growth density, hybridoma culture supernatants were assayed for binding to DLL3 protein using ELISA and FACS for binding to DLL3 CHO-S cells. Clones that bound to human DLL3 protein, monkey DLL3 protein, and DLL3 CHO-S cells, while not binding to wild-type CHO-S cells, were promptly frozen, expanded, seeded, and subcloned once or twice until single-cell clones were obtained. These screening experiments yielded hybridoma clones mAb100 and mAb6.

[0470] The hybridoma clones were expanded and cultured, RNA was extracted, and reverse transcription amplification (RT-PCR) was performed using degenerate primers of mouse-Ig to finally obtain the variable region sequence of the antibody.

[0471] mAb6 Heavy Chain Variable Region:

[0472] mAb6 light chain variable region:

[0473] mAb100 heavy chain variable region:

[0474] mAb100 light chain variable region:

[0475] Table 4. CDR sequences of mouse antibodies

[0476] Note: The amino acid residues of the VH / VL CDRs are determined and annotated according to the Kabat numbering system.

[0477] The heavy chain variable region and light chain variable region of the mouse antibody were cloned into the pTT 5 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.

[0478] Human IgG1 heavy chain constant region:

[0479] Human kappa light chain constant region:

[0480] Example 3: Humanization of murine anti-DLL3 monoclonal antibody

[0481] By comparing the Kabat human antibody heavy and light chain variable region germline gene database, the heavy and light chain variable region germline genes with high homology were selected as templates, and the CDRs of the mouse antibody were grafted onto the corresponding human templates, forming a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acids in the variable region were backmutated and then recombined with the constant region (exemplarily, with the human IgG1 heavy chain constant region set forth in SEQ ID NO: 28 and the human kappa light chain constant region set forth in SEQ ID NO: 29) to obtain a full-length antibody.

[0482] The human germline light chain variable region FR1, FR2, and FR3 templates for mAb6 are IGKV1-16*01, and the light chain FR4 region template is IGKJ4*01. The human germline heavy chain variable region FR1, FR2, and FR3 templates are IGHV1-3*01, IGHV7-4-1*02, or IGHV3-73*01, and the heavy chain FR4 region template is IGHJ6*01. The human germline light chain variable region FR1, FR2, and FR3 templates for mAb100 are IGKV1-27*01, and the light chain FR4 region template is IGKJ4*01. The human germline heavy chain variable region FR1, FR2, and FR3 templates are IGHV3-11*01, and the heavy chain FR4 region template is IGHJ6*01.

[0483] In addition, the amino acid residue at position 4 in the HCDR3: PLYYYGRSYNAVAY (SEQ ID NO: 24) of the heavy chain variable region of mAb100 was mutated from Y to H, and the amino acid residue at position 11 was mutated from A to G to obtain new HCDR3: PLYHYGRSYNAVAY (SEQ ID NO: 30) and PLYYYGRSYNGVAY (SEQ ID NO: 31).

[0484] Table 5. Humanized templates and corresponding point mutations of mAb6 and mAb100 antibodies

[0485] Note: Taking A100EG in the table as an example, it means that the A at position 100E under Kabat numbering has mutated to G.

[0486] The sequences of the obtained humanized antibody variable regions are as follows:

[0487] hAb6 VH1(Q1E,R71V,T73K)

[0488] hAb6 VH2(Q1E,I69L,R71V,T73K,S76N)

[0489] hAb6 VH3(Q1E,M48I,V67A,I69L,R71V,T73K,S76N)

[0490] hAb6 VH4(Q1E,Q43K,I69L,R71V,T73K,S76N)

[0491] hAb6 VH5(Q1E,F69L,L71V,T73K)

[0492] hAb6 VH6(Q1E,F69L,L71V,T73K,V75S,S76N)

[0493] hAb6 VH7(F27Y,S30T,I69L,R71V,D73K,T93A)

[0494] hAb6VH8(GraftIGHV1-3*01)

[0495] hAb6VH9(GraftIGHV7-4-1*02)

[0496] hAb6VH10(GraftIGHV3-73*01)

[0497] hAb6VH11(Q1E,R38K,F69L,L71V,T73K,V75S,S76N)

[0498] hAb6VH12(Q1E,V68A,F69L,L71V,T73K,V75S,S76N)

[0499] hAb6VH13(Q1E,R38K,V68A,F69L,L71V,T73K,V75S,S76N)

[0500] hAb6 VL1(F36L,S46G)

[0501] hAb6 VL2(F36L,S46G,T69A,F71Y)

[0502] hAb6 VL3(F36L,A43S,P44F,S46G,T69A,F71Y)

[0503] hAb6 VL4(F36L,S46G,T69A,F71Y,T85D)

[0504] hAb6VL5(GraftIGKV1-16*01)

[0505] hAb100 VH1(Q1E,R94S)

[0506] hAb100 VH2(Q1E,S49A,R94S)

[0507] hAb100 VH3(Q1E,S49A,R94S,Y98H)

[0508] hAb100 VH4(Q1E,S49A,R94S,A100EG)

[0509] hAb100VH5(Graft IGHV3-11*01)

[0510] hAb100 VL1(Graft IGKV1-27*01)

[0511] hAb100 VL2(V43I)

[0512] Note: The single-line part is the CDR region, and the double-line part is the mutation site.

[0513] Table 6. General formula of the CDR region of hAb100 antibody

[0514] Wherein, X1 is Y or H; X2 is A or G.

[0515] Exemplary combinations of heavy and light chain variable regions of humanized antibodies are as follows:

[0516] Table 7. Humanized antibodies of mAb6

[0517] Note: hAb6L1H1 indicates that the antibody comprises a heavy chain variable region hAb6VH1 and a light chain variable region hAb6VL1, and the sequence of its heavy chain constant region is SEQ ID NO: 28, and the sequence of its light chain constant region is SEQ ID NO: 29, and so on.

[0518] Table 8. Humanized antibodies of mAb100

[0519] Note: hAb100L1H1 indicates that the antibody comprises a heavy chain variable region hAb100VH1 and a light chain variable region hAb100VL1, and the sequence of its heavy chain constant region is SEQ ID NO: 28, and the sequence of its light chain constant region is SEQ ID NO: 29, and so on.

[0520] The above antibodies were cloned, expressed, and purified, and then humanized antibodies with better activity were selected through protein binding experiments (Test Example 1), cell binding experiments (Test Example 2), and Biacore (Test Example 4). The amino acid sequences of the heavy and light chains of exemplary humanized antibodies are as follows:

[0521] Hu6 (also known as hAb6L4H12) heavy chain:

[0522] Note: The underlined part in the sequence is the variable region, and the italic part is the constant region.

[0523] SEQ ID NO: 58

[0524] Hu6 (also known as hAb6L4H12) light chain:

[0525] Note: The underlined part in the sequence is the variable region, and the italic part is the constant region.

[0526] SEQ ID NO: 59

[0527] Hu100 (also known as hAb100L1H1) heavy chain:

[0528] Note: The underlined part in the sequence is the variable region, and the italic part is the constant region.

[0529] SEQ ID NO: 60

[0530] Hu100 (also known as hAb100L1H1) light chain:

[0531] Note: The underlined part in the sequence is the variable region, and the italic part is the constant region.

[0532] SEQ ID NO: 61.

[0533] The positive control antibody used in this disclosure is BI-764532 (constructed with reference to WO2019234220A1), and the negative control is C25 (wherein the VH / VL sequence is from patent US6114143A), and their sequences are as follows:

[0534] BI-764532 heavy chain:

[0535] Note: The underlined part in the sequence is the variable region, and the italic part is the constant region.

[0536] SEQ ID NO: 62

[0537] BI-764532 light chain:

[0538] Note: The underlined part in the sequence is the variable region, and the italic part is the constant region.

[0539] SEQ ID NO: 63

[0540] C25 heavy chain:

[0541] C25 light chain:

[0542] Note: The underlined part in the sequence is the variable region, and the italic part is the constant region.

[0543] 2. Preparation of ADC

[0544] Example 4. Preparation of ADC-1

[0545] To a solution of Hu100 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 3 mL, 203 nmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 50.7 μL, 507 nmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0546] Compound 9-A (2.18 mg, 2030 nmol, prepared with reference to Example 9 in patent application WO2020063676A1) was dissolved in 150 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 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 stored frozen at 4°C.

[0547] RP-HPLC calculated average value: n=4.43.

[0548] Example 5. Preparation of ADC-2

[0549] To a solution of Hu100 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 3 mL, 203 nmol) at 37°C was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 121.7 μL, 1217 nmol). The mixture was shaken in a water bath at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0550] Compound 9-A (3.27 mg, 3042 nmol) was dissolved in 150 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at 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), which was stored frozen at 4°C.

[0551] RP-HPLC calculated average value: n=7.32.

[0552] Example 6. Preparation of ADC-3

[0553] To a solution of Hu100 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 6.5 mL, 440 nmol) at 37°C was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 159.4 μL, 1594 nmol). The mixture was shaken in a water bath at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0554] Compound 9-A (5.66 mg, 5265 nmol) was dissolved in 330 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at 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) and stored frozen at 4°C.

[0555] RP-HPLC calculated average value: n=6.12.

[0556] Example 7. Preparation of ADC-4

[0557] To a solution of Hu6 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 76 mL, 5140 nmol) at 37°C was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 1.14 mL, 11.4 μmol). The solution was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0558] Compound 9-A (49.0 mg, 45.6 μmol) was dissolved in 3.8 mL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-4 in PBS buffer (4.07 mg / mL, 167 mL), which was stored frozen at 4°C.

[0559] RP-HPLC calculated average value: n=3.53.

[0560] Example 8. Preparation of ADC-5

[0561] To a solution of Hu6 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 68 mL, 4595 nmol) at 37°C was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 2.74 mL, 27.4 μmol). The solution was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0562] Compound 9-A (82.75 mg, 77.04 μmol) was dissolved in 3.4 mL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-5 (3.12 mg / mL in PBS buffer, 174.1 mL) and stored frozen at 4°C.

[0563] RP-HPLC calculated average value: n=7.43.

[0564] Example 9. Preparation of ADC-6

[0565] To a solution of antibody C25 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.6 mL, 108 nmol) at 37°C was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 35.6 μL, 356 nmol). The mixture was shaken in a water bath at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0566] Compound 9-A (1.39 mg, 1.294 μmol) was dissolved in 80 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at 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), which was stored frozen at 4°C.

[0567] RP-HPLC calculated average value: n=6.76.

[0568] Example 10. Preparation of ADC-7

[0569] To a solution of antibody C25 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.6 mL, 108 nmol) at 37°C was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (10 mM, 64.8 μL, 648 nmol). The mixture was shaken in a water bath at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0570] Compound 9-A (1.74 mg, 1.62 μmol) was dissolved in 80 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at 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), which was stored frozen at 4°C.

[0571] RP-HPLC calculated average value: n=7.75.

[0572] Analysis of drug loading in ADC stock solution

[0573] ADCs are antibody-drug conjugates that treat diseases by relying on the antibody's targeted properties to deliver the drug into cells, thereby killing or inhibiting cell growth. The drug loading plays a decisive role in its efficacy.

[0574] This disclosure uses RP-HPLC to analyze drug loading, and the process is basically as follows:

[0575] Reagents and instruments:

[0576] Trifluoroacetic acid (TFA): produced by Sigma, 100 mL / bottle; acetonitrile: LC grade, 4 L / bottle, produced by Thermo Fisher; DTT: produced by Sigma, 1 g / bottle.

[0577] High performance liquid chromatograph: Agilent 1200.

[0578] Solution preparation:

[0579] 1) 0.25M DTT solution:

[0580] Preparation example: Take 5.78 mg of DTT and add 150 μL of purified water to fully dissolve it to prepare 0.25 M DTT solution. Store at -20°C.

[0581] 2) Mobile phase A (0.1% TFA aqueous solution):

[0582] Preparation example: Measure 1000 mL of purified water into a graduated cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.

[0583] 3) Mobile phase B (0.1% TFA acetonitrile solution):

[0584] Preparation example: Measure 1000 mL of acetonitrile into a measuring cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.

[0585] Naked antibody and test sample (concentration 1 mg / mL, about 200 μL) were reduced by adding 4 μL DTT and incubated in a 37°C water bath for 1 hour. After reduction, the samples were removed from the tube and used for injection.

[0586] Chromatographic conditions:

[0587] Chromatographic column: Agilent PLRP-S 1000A 8μm 4.6*250mm; column temperature: 80℃;

[0588] DAD detector: detection wavelength 280 nm; sample chamber temperature: 4°C; flow rate: 1 mL / min;

[0589] Injection volume: 40 μL;

[0590] The chromatographic gradient is shown in Table 9-1:

[0591] Table 9-1

[0592] Data Analysis:

[0593] By comparing the spectra of the sample and naked antibody, the positions of the light and heavy chains are distinguished, and then the spectrum of the test sample is integrated to calculate the DAR value. The calculation formula is as follows:

[0594] Table 9-2

[0595] Sum of LC peak areas = LC peak area + LC+1 peak area

[0596] Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area

[0597] LC DAR = Σ(number of connected drugs * peak area percentage) / total LC peak area

[0598] HC DAR = Σ(number of connected drugs * peak area percentage) / total HC peak area

[0599] DAR=LC DAR+HC DAR

[0600] The following biochemical assays were used to verify the activity of the antibodies and ADCs disclosed herein.

[0601] Test Example 1: ELISA detection of protein binding of antibodies

[0602] Coat the plate with streptavidin (abcam, ab136200, 1 μg / mL) at 100 μL / well at 4°C overnight. Wash the plate three times with PBST solution (PBS containing 0.1% Tween 20) at 250 μL / well. Block the plate with 5% milk at 250 μL / well at 37°C for 2 hours. Wash the plate three times with PBST solution at 250 μL / well. Add biotinylated DLL3 antigen (1 μg / mL) (SEQ ID NO: 9) and incubate at 37°C for 1 hour. Wash the plate three times with PBST solution at 250 μL / well. Prepare antibodies Hu6 and Hu100 (maximum concentration 100 nM, 4-fold serial dilutions) and incubate at 37°C for 1 hour. Wash the plate six times with PBST solution at 250 μL / well. Add 100 μL / well of human IgG (H+L)-HRP (Jackson, 109-035-003, 1:4000 dilution) at a working concentration and incubate at 37°C for 1 hour. Wash the plate six times with 250 μL / well of PBST solution. Add 100 μL / well of TMB (KPL, 5120-0077) colorimetric solution and develop at room temperature for 5-10 minutes. Stop color development by adding 100 μL / well of 1 M H₂SO₄. Read the plate at 450 nm using a microplate reader (Molecular Devices, VERSA max).

[0603] Table 10. Antibody binding activity to proteins

[0604] The results showed that both antibodies Hu6 and Hu100 had excellent binding ability to DLL3.

[0605] Test Example 2: FACS detection of antibody binding at the cellular level

[0606] The DLL3-expressing small cell lung cancer cell lines H1184 (ATCC, catalog number CRL-5858), DLL3 / H82, cynoDLL3 / CHO-s, and RatDLL3 / CHO-s cells were prepared with FACS buffer (1% BSA + pH 7.4 PBS) to a concentration of 1×10 6 / mL cell suspension, 100μL / well was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add different concentrations of the test antibody, 100μL / well. Incubate in a 4°C refrigerator in the dark for 1 hour. After washing three times by centrifugation at 300g, add working concentrations of APC anti-human IgG Fc (BioLegend, 410712) or PE F(ab')2-goat anti-human IgG (invitrogen, H10104) and incubate in a 4°C refrigerator in the dark for 40 minutes. After washing three times by centrifugation at 300g, the geometric mean fluorescence intensity was detected on an Invitrogen flow cytometer to calculate the binding EC value of the antibody to cells expressing DLL3. 50 The results are shown in Table 11-1, Table 11-2, Table 11-3 and Figures 1A to 1C.

[0607] Table 11-1. Binding activity of antibodies to cell-expressed DLL3

[0608] Table 11-2. Antibody binding activity to DLL3 / H82 cells expressing human DLL3

[0609] Table 11-3. Antibody binding activity to H1184 cells

[0610] The results showed that all antibodies disclosed herein specifically bind to cells expressing DLL3. Hu6 showed strong binding to cells expressing DLL3 from various species. Hu100 also showed excellent binding to cells expressing both human and cynomolgus macaque DLL3, but did not bind to cells expressing rat DLL3. BI-764532 only bound to cells expressing human and cynomolgus macaque DLL3, and its binding activity was weaker than that of Hu6 and Hu100.

[0611] Test Example 3: FACS detection of antibody binding to DLL1 and DLL4 cells

[0612] Stably transfected human DLL1 / CHO-s and human DLL4 / CHO-s cells were prepared into 1×10 6100 μL / well of a 100 μL / mL cell suspension was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300 g for 5 minutes and remove the supernatant. 100 μL / well of the antibody to be tested was added and incubated in a 4°C refrigerator protected from light for 1 hour. After washing three times by centrifugation at 300 g, a working concentration of PE F(ab')2-goat anti-human IgG Fc secondary antibody (Invitrogen, H10104) was added and incubated in a 4°C refrigerator protected from light for 40 minutes. After washing three times by centrifugation at 300 g, the geometric mean fluorescence intensity was measured on an Invitrogen flow cytometer.

[0613] The results showed that antibodies Hu6 and Hu100 did not bind to human DLL1 and DLL4.

[0614] Test Example 4: Biacore antibody affinity test

[0615] The test antibody was affinity-captured for 18 seconds using a Protein A biosensor chip (Cat. #29127556, Cytiva). Antigens (human DLL3 (ACRO, DLL3-H52H4), monkey DLL3 (KACTUS, DLL-RM103), and mouse DLL3 (KACTUS, DLL-MM103)) were then passed over the chip surface for 180 seconds, followed by a 600-second dissociation period. Binding and dissociation curves were obtained using a Biacore 8K (Cytiva) instrument for real-time signal monitoring. After each experimental cycle, the biosensor chip was washed and regenerated 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.

[0616] Table 12-1. Antibody affinity

[0617] Table 12-2. Affinity of Antibodies to Human DLL3

[0618] Table 12-3. Affinity of Antibodies to Human DLL3

[0619] The results showed that the humanized and chimeric antibodies mAb100 and mAb6 specifically bind to human DLL3 with high affinity. Antibody Hu6 has high affinity for human, monkey, and mouse DLL3, while Hu100 has high affinity for human and monkey DLL3 but does not bind to mouse DLL3.

[0620] Test Example 5: Antibody epitope competition binding experiment

[0621] Coat the plate with BI-764532 antibody (1 μg / mL) at 100 μL / well at 4°C overnight. Wash the plate three times with 250 μL / well of PBST solution. Block the plate with 5% milk at 250 μL / well at 37°C for 2 hours. Wash the plate three times with 250 μL / well of PBST solution. Add biotinylated DLL3-Strep (0.1 μg / mL, SEQ ID NO: 9). Prepare competing antibodies: BI-764532, Hu6, and Hu100 (maximum concentration 100 μg / mL, serially diluted 4-fold) and incubate at 37°C for 1 hour. Wash the plate six times with 250 μL / well of PBST solution. Add 100 μL / well of streptavidin-peroxidase (1:2000 dilution) (Jackson Immuno Research, 016-030-084) and incubate at 37°C for 1 hour. Wash the plate six times with 250 μL / well of PBST solution. Add 100 μL / well of TMB (KPL, 5120-0077) colorimetric solution and develop at room temperature for 5-10 min. Add 100 μL / well of 1 M H₂SO₄ to stop color development. Read the plate at 450 nm using a microplate reader. The results are shown in Figure 2.

[0622] The results showed that antibodies Hu6 and Hu100 did not compete with BI-764532, indicating that antibodies Hu6 and Hu100 bound to different epitopes with BI-764532.

[0623] Test Example 6: Endocytic activity detection of anti-DLL3 antibodies

[0624] DT3C is a recombinantly expressed fusion protein composed of the diphtheria toxin fragment A (toxin only) and the group G Streptococcus 3C fragment (IgG-binding portion). This protein has a high affinity for the IgG portion of the antibody and enters the cell together with the antibody during endocytosis. Under the action of intracellular furin, DT is released, which is toxic. DT inhibits EF2-ADP ribosylation, blocks protein translation, and ultimately leads to cell death. DT3C that does not enter the cell has no cytotoxic activity. The endocytic activity of the antibody is evaluated based on the cell killing effect.

[0625] Experimental procedures

[0626] a. Prepare a DMS53 / DLL3 cell suspension in fresh cell culture medium RPMI1640 (GE, SH30809.01) containing 20% ​​FBS and add 2000 cells / 50 μL / well to a 96-well cell culture plate. Do not plate cells in columns 1 and 12, but add only 50 μL of culture medium. Incubate at 37°C with 5% carbon dioxide for 16 hours.

[0627] b. Prepare a 4× concentration of DT3C (9600 nM, expressed and purified by Shanghai Panchao Biotechnology Co., Ltd.) in serum-free medium and filter through a 0.22 μm filter. Prepare a 4× concentration of the antibody (1600 nM) in serum-free medium. Mix 80 μL of the DT3C solution and 80 μL of the antibody solution at a 1:1 ratio and incubate at room temperature for 30 minutes.

[0628] c. Dilute the mixture 5-fold with serum-free medium for a total of 9 concentrations, with the 10th point being pure culture medium.

[0629] d. Add 50 μL of diluted antibody to the cells and incubate in an incubator for three days.

[0630] e. Add 50 μL CTG to each well ( Luminescent Cell Viability Assay, Promega, G7573), incubate at room temperature in the dark for 10 minutes, and read chemiluminescence on Victor3.

[0631] The results are shown in Table 13 and Figure 3 below.

[0632] Table 13. Cell killing caused by antibody internalization

[0633] The results showed that both antibodies Hu6 and Hu100 could be internalized by cells.

[0634] Test Example 7: FACS detection of ADC binding at the cellular level

[0635] hDLL3 / CHO-s and cynoDLL3 / CHO-s cells expressing DLL3 were prepared with FACS buffer (1% BSA + pH 7.4 PBS) to a volume of 1×10 6 / mL cell suspension, 100μL / well was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add different concentrations of ADC to be tested, 100μL / well. Incubate in a 4℃ refrigerator away from light for 1 hour. After washing three times by centrifugation at 300g, add working concentrations of APC anti-human IgG Fc (BioLegend, 410712) or PE F(ab')2-goat anti-human IgG (invitrogen, H10104) and incubate in a 4℃ refrigerator away from light for 40 minutes. After washing three times by centrifugation at 300g, the geometric mean fluorescence intensity was detected on an Invitrogen flow cytometer to calculate the binding EC value of the antibody to cells expressing DLL3. 50 The results are shown in Table 14, Figure 4A and Figure 4B.

[0636] Table 14. Binding activity of antibodies to cell-expressed DLL3

[0637] The results showed that the ADCs disclosed herein were able to specifically bind to human and monkey DLL3 expressed in cells, wherein increasing DAR values ​​had little effect on ADC binding.

[0638] Test Example 8: Biacore ADC affinity test

[0639] The test antibody was affinity-captured for 18 seconds using a Protein A biosensor chip (Cat. #29127556, Cytiva). Antigens, human DLL3 (ACRO, DLL3-H52H4) and monkey DLL3 (KACTUS, DLL-RM103), were then passed over the chip surface for 180 seconds, followed by a 600-second dissociation period. Binding and dissociation curves were obtained using a Biacore 8K (Cytiva) instrument for real-time signal monitoring. After each experimental cycle, the biosensor chip was washed and regenerated 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.

[0640] Table 15. Affinity of ADCs for DLL3 from different species

[0641] The results showed that the ADCs disclosed herein can specifically bind to both human and monkey DLL3 proteins, with increasing DAR values ​​having little effect on ADC binding.

[0642] Test Example 9: Cytotoxicity test of ADC at different DLL3 expression levels

[0643] The experimental steps are as follows:

[0644] a. Prepare a cell suspension using fresh cell culture medium containing 10% FBS and add 135 μL per well to a 96-well cell culture plate (Corning, 3903). Do not plate cells in columns 1 and 12. Add only 135 μL of culture medium and incubate at 37°C in 5% CO2 for 16 hours.

[0645] b. Prepare the ADC sample in PBS to a first-well working solution (10× concentration). Using this as the starting concentration, perform a gradient dilution with PBS at the corresponding multiples. Add 15 μL of the 10× concentration ADC solution to each well and incubate at 37°C in 5% CO2 for 6 days.

[0646] c. Add 70 μL of CTG (Promega, G7573) to each well and incubate at room temperature in the dark for 10 minutes. Read chemiluminescence on Victor3 and process the data using GraphPad Prism5. Graph the antibody or ADC concentration on the X-axis and the light intensity on the Y-axis.

[0647] See Table 16 for the plating density of different cells, the concentration of the working solution in the first well (10× concentration), and the dilution multiple.

[0648] Table 16. Cell plating density and first well working solution concentration

[0649] The cells used in the experiment are as follows:

[0650] H1184 (+++) was purchased from ATCC, CRL-5858;

[0651] DMS53(++) was purchased from ATCC, CRL-2062;

[0652] SK-MEL3(+) was purchased from ATCC, HTB-69;

[0653] CHO-K1(-) was purchased from ATCC, CCL-61.

[0654] “+” indicates the expression level of DLL3, and “-” indicates the absence of DLL3 expression.

[0655] The results are shown in Table 17 and Figures 5A to 5D.

[0656] Table 17. Cytotoxicity of ADCs against cells expressing different DLL3 levels

[0657] The results showed that ADC-1, ADC-2 and ADC-3 had strong target cell killing activity and could kill SK-MEL3, DMS53 and H1184 cells expressing DLL3, but had no killing effect on DLL3-negative CHO-K1 cells.

[0658] Test Example 10: Bystander Killing Activity Experiment

[0659] DMS53 / DLL3high (DMS53 cells stably transfected with DLL3) and U-2OS (ATCC, HTB-96) cells were cultured in RPMI1640 + 20% FBS + 1× Glutamax and McCoy's 5A + 10% FBS, respectively. The cells were trypsinized, neutralized with fresh culture medium, and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded, and the cells were resuspended in RPMI1640 + 20% FBS + 1× Glutamax. After cell counting, the cell density of DMS53 / DLL3high cells was adjusted to 9×104 The U-2OS cell density was adjusted to 3×10 4 Cells / mL. 500 μL of DMS53 / DLL3high cells and 500 μL of U-2OS cells were added to corresponding wells of a 12-well plate. 500 μL of U-2OS cells and 500 μL of RPMI1640 + 20% FBS + 1× Glutamax were added to corresponding wells of a 12-well plate. Incubate at 37°C with 5% CO2 for 24 hours. Prepare the sample to a 40x intermediate concentration (200 nM). 25 μL of each ADC sample was added to the corresponding wells of the 12-well plate. A solvent control group was set up. Incubate at 37°C with 5% CO2 for 6 days. Cells in the 12-well plate were trypsinized and neutralized with fresh culture medium. 20 μL of cells were added to 20 μL of trypan blue and counted. Cells were centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. Cells were washed once with 100 μL of FACS buffer, centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. Resuspend with 100 μL of FACS buffer, add 2 μg / mL of anti-DLL3 positive antibody, and incubate on ice for 60 minutes. Wash once with FACS buffer, centrifuge at 1500 rpm for 3 minutes, add secondary antibody APC anti-human IgG Fc (100x), incubate for 30 minutes, and wash once with FACS buffer. Add 200 μL of FACS buffer to resuspend the cells and detect by FACS. Flow cytometry data were analyzed using FlowJo to obtain the DMS53 / DLL3high ratio and calculate the total number of DMS53 / DLL3high and U-2OS. The data were plotted using GraphPad Prism5, with different samples as the X-axis and the calculated cell number as the Y-axis. The results are shown in Figure 6.

[0660] The results showed that ADC-1, ADC-2 and ADC-3 had obvious bystander cytotoxic effects.

[0661] In vivo biological evaluation

[0662] Test Example 11-1: In vivo efficacy evaluation in the DMS53 cell CDX mouse model

[0663] Human small cell lung cancer DMS53 cells (5×10 6 200 μL of 50% matrigel per mouse (ATCC, CRL-2062) was inoculated subcutaneously on the right flank of Balb / c mice. 3After removing the weight, tumor size, and tumor size, mice were randomized according to tumor volume, with 8 mice in each group, and drug administration began on the same day. ADC was injected intraperitoneally at a dose of 1.5 mg / kg, once a week, for a total of 2 doses. After the second dose, drug administration was stopped and tumor growth was continued to be observed. Tumor volume and body weight were measured twice a week and the data were recorded. Data were recorded using Excel statistical software: mean value was calculated as avg; SD value was calculated as STDEV; SEM value was calculated as STDEV / SQRT (number of animals in each group); GraphPad Prism software was used for graphing, and Two-way ANOVA or One-way ANOVA was used for statistical analysis of data.

[0664] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0665] Relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

[0666] Tumor inhibition rate TGI (%) = 1-T / C (%).

[0667] The results are shown in Table 18 and Figure 7.

[0668] Table 18. Efficacy of ADC on DMS53 xenografts in nude mice

[0669] The results showed that at a dose of 1.5 mpk, both ADC-1 and ADC-4 could significantly inhibit the growth of DMS53 cell subcutaneous transplanted tumors. At doses of 0.5 mpk and 1.5 mpk, both ADC-2 and ADC-5 could significantly inhibit the growth of DMS53 cell subcutaneous transplanted tumors.

[0670] Test Example 11-2: In vivo efficacy evaluation in a DMS53 cell CDX mouse model

[0671] Human small cell lung cancer DMS53 cells (5×10 6 200 μL of 30% matrigel per mouse (ATCC, CRL-2062) was inoculated subcutaneously on the right flank of Balb / c mice. 15 days after inoculation, when the tumor volume was ~180 mm 3After removing the weight, tumor size that was too large or too small, the mice were randomized according to the tumor volume, with 8 mice in each group, and drug administration 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 2 doses. After the second dose, drug administration was stopped and tumor growth was continued to be observed. Tumor volume and body weight were measured twice a week and the data were recorded. Data were recorded using Excel statistical software: the mean value was calculated as avg; the SD value was calculated as STDEV; the SEM value was calculated as STDEV / SQRT (number of animals in each group); GraphPad Prism software was used for graphing, and Two-way ANOVA or One-way ANOVA was used for statistical analysis of the data.

[0672] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0673] Relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

[0674] Tumor inhibition rate TGI (%) = 1-T / C (%).

[0675] The results are shown in Table 19 and Figure 8.

[0676] Table 19. Efficacy of ADC on DMS53 xenografts in nude mice

[0677] The results showed that ADC-3 at doses of 0.5 mpk and 1.5 mpk, and ADC-1 at a dose of 1.5 mpk, could significantly inhibit the growth of subcutaneously transplanted DMS53 cell tumors.

[0678] Test Example 12: In vivo efficacy evaluation in the NCI-H1184 cell CDX mouse model

[0679] NCI-H1184 cells (6×10 6 80 NDG mice were inoculated subcutaneously at the right ribs with 200 μL of 50% Matrix Gel per mouse; 13 days after inoculation, the tumors were kept at an average volume of 185 mm. 3After weight loss and tumor size were eliminated, mice were randomly divided into 7 groups of 9 mice each according to tumor volume. Dosing began on the same day, as shown in Table 19. Antibodies were injected intraperitoneally for 21 days. Tumor volume was measured twice weekly, and body weight was recorded. Data were recorded using Excel statistical software: mean (avg); SD (STDEV); SEM (SEM) (STDEV / SQRT (number of animals in each group)). Graphs were constructed using GraphPad Prism software, and data were statistically analyzed using two-way ANOVA or one-way ANOVA.

[0680] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0681] Relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

[0682] Tumor inhibition rate TGI (%) = 1-T / C (%).

[0683] The results are shown in Table 20 and Figure 9.

[0684] Table 20. Efficacy of ADC on H1184 xenografts in nude mice

[0685] The results showed that both ADC-2 and ADC-3 could significantly inhibit the growth of NCI-H1184 tumors.

[0686] Test Example 13: Rat PK Study

[0687] The single-dose pharmacokinetics of ADC molecules were studied in SD rats (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.).

[0688] The ADC molecule was injected intravenously at 3 mg / kg (n=4 / group). Blood was collected from the fundus vein of rats 5 minutes, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days, and 28 days after administration. The collected blood samples were left at room temperature for half an hour to coagulate, and then centrifuged at 1000g for 15 minutes at 4°C to collect serum. Bioanalytical measurements were performed using the HTRF method to measure the rat serum samples. Biotin-labeled DLL3 was used to capture the monoclonal antibody, and biotin-labeled antitoxin antibody was used to capture the toxin. The content of the test product was quantitatively analyzed using the four-parameter model curve of the standard. The pharmacokinetic parameters were analyzed using the standard non-compartmental model of WinNonlin software (6.4). The experimental results are shown in Table 21 and Figures 10A to 10C.

[0689] Table 21. Pharmacokinetics of ADC molecules in rats

[0690] The results showed that the AUC of the total antibody and the complete ADC of the ADC molecules measured in vivo were similar, indicating that the complete ADC structures of the three ADC molecules were stable.

[0691] Test Example 14: Monkey PK Study

[0692] The single-dose pharmacokinetics of ADC molecules were studied in cynomolgus monkeys (Suzhou Xishan Zhongke Laboratory Animal Co., Ltd.).

[0693] The ADC molecule was injected intravenously at 10 mg / kg (n=3 / group) at 5 minutes, 8 hours, 1 day, 1.5 days, 2 days, 3 days, 4 days, 7 days, 10 days, 14 days, 21 days and 28 days after administration. The collected blood samples were placed at room temperature for half an hour to coagulate, and then centrifuged (4°C, 2600g, 10 minutes). The upper serum was taken and immediately stored at -60°C. Serum was collected for bioanalysis. The DELFIA method was used to measure the serum samples, and the quantitative analysis of the test product content was performed using the four-parameter model curve of the standard. The pharmacokinetic parameters were analyzed using the standard non-compartmental model of WinNonlin software (6.4). The experimental results are shown in Table 22 and Figure 11.

[0694] Table 22. Monkey Pharmacokinetics of ADC Molecules

[0695] Results showed that after intravenous administration of ADC-2 (10 mg / kcal) to cynomolgus monkeys, the half-lives of the total antibody and intact ADC were 9.9±1.1 days and 8.5±0.1 days, respectively. The AUCs for the total antibody and intact ADC were 27,405 μg / mL*h and 25,702 μg / mL*h, respectively. The half-lives of the total antibody and intact ADC in cynomolgus monkeys were essentially identical, with a 94% AUC ratio for the intact ADC to the total antibody, indicating good stability of ADC-2 in cynomolgus monkeys.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, having a structure as shown in the general formula Pc-LYD: in: Pc is an anti-DLL3 antibody comprising a heavy chain variable region and a light chain variable region, wherein: i) the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 57; 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; Among them, SEQ ID NO: 57 is PLYX 1 YGRSYNX 2 VAY, where X 1 Y or H; X 2 is A or G; or ii) the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 16; HCDR2 comprising the amino acid sequence of SEQ ID NO: 17; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; and 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; Preferably, The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and 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; Y is -O-CR 1 R 2 -C(O)-, where R 1 Halogenated C 1-6 Alkyl or C 3-6 Cycloalkyl; R 2 Selected from hydrogen Substance, halogenated C 1-6 Alkyl and C 3-6 Cycloalkyl; or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; n is 1 to 10; L is a connector.

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

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the anti-DLL3 antibody comprises: 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 NOs: 55 or 56; or ii) the heavy chain variable region comprises any one of the amino acid sequences selected from SEQ ID NOs: 43, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 and 44, and / or the light chain variable region comprises any one of the amino acid sequences selected from SEQ ID NOs: 48, 45, 46, 47 and 49; 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.

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

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the anti-DLL3 antibody comprises a heavy chain constant region and a light chain constant region; preferably, 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.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 5, wherein the anti-DLL3 antibody comprises a heavy chain and a light chain, in: 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.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein said Y is selected from: The O-end of Y is connected to L.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein said L is -L 1 -L 2 -L 3 -L 4 -, in: L 1 Selected from -(succinimidyl-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, where W is C 1-6 Alkylene or C 1-6 Alkylene-C 3-6 Cycloalkyl, wherein the C 1-6 Alkylene or C 1-6 Alkylene-C 3-6 The cycloalkyl groups are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -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 by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH 2 ) t - and chemical bonds, 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 halogenated alkyl 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 halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; Preferably, L 1 for 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 comprising glycine-glycine-phenylalanine-glycine; L 4 -NH(CH 2 )t-, t is 1 or 2; The L 1 The end is connected to PC; More preferably, the L is represented by the following structure:

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein the antibody-drug conjugate has the structure shown below: in: Pc is the anti-DLL3 antibody of claim 1; n is 1 to 10; Preferably, the antibody-drug conjugate has the structure shown below: in: Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein: 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.

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

11. Use of the antibody-drug conjugate or a 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 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 tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, medullary thyroid cancer, malignant pleural mesothelioma, breast cancer, triple-negative breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric 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; more preferably, wherein the lung cancer is small cell lung cancer.

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