Anti-DLL3 antibody, antibody-drug conjugate, and use thereof

Abstract

Disclosed in the present invention are an anti-DLL3 antibody, an antibody-drug conjugate, and the use thereof. The anti-DLL3 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3. The antibody-drug conjugate comprises the antibody or the antigen-binding fragment thereof, a linker unit L and a cytotoxic drug. The antibody of the present invention has good affinity for DLL3, and has significant internalization capacity for cells expressing DLL3. The antibody-drug conjugate has strong inhibitory activity against the proliferation of tumor cells both in vivo and in vitro.

Description

An anti-DLL3 antibody, an antibody-drug conjugate and its applications

[0001] This application claims priority to Chinese patent application 2024106746688, filed on May 28, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to an anti-DLL3 antibody, an antibody-drug conjugate, and their applications. Background Technology

[0003] Delta-like ligand 3 (DLL3) is a member of the Notch ligand family and is a transmembrane protein that attaches to the cell surface. The human DLL3 protein consists of 619 amino acids and is characterized by a DSL domain, six EGF-like repeat sequences, and a transmembrane domain.

[0004] DLL3 can bind to Notch receptors (Notch 1-4) to activate the Notch pathway, a highly conserved cell signaling pathway involved in malignant transformation, cell proliferation, cell cycle arrest and apoptosis, epithelial-mesenchymal transition, and inhibition of neuroendocrine differentiation. During DLL activation of Notch, the membrane DLL binds trans-to the EGF repeat sequence of the Notch receptor, subsequently triggering Notch signaling via endocytosis. After endocytosis, the internalized DLL can be degraded via the proteasome / lysosome or recycled back to the plasma membrane. The endocytosis / recycling event of DLLs depends on ubiquitination (β) of the DLL's intracellular domain (DICD).

[0005] Studies have shown that DLL3 is rarely expressed in normal tissues, but multiple studies suggest that DLL3 is specifically highly expressed in small cell lung cancer (SCLC), thus DLL3 is a promising target for the treatment of lung cancer.

[0006] Current solutions mainly involve preparing corresponding antibodies, which competitively bind to target proteins to block and interfere with signaling pathways to prevent and treat diseases, or utilizing antibody-specific ADCC and CDC effects to induce therapeutic effects.

[0007] Existing DLL3 antibodies have low affinity for antigens and cells expressing antigens. Furthermore, existing antibodies or their drug conjugates do not have strong enough inhibitory activity against tumors. Summary of the Invention

[0008] To address the technical problems of low affinity of DLL3 antibodies and low inhibitory activity of antibody-drug conjugates against tumors in existing technologies, this invention discloses an anti-DLL3 antibody, antibody-drug conjugates, and their applications. The antibodies of this invention, especially the B6 and B275 antibodies, exhibit good affinity for DLL3 and significant internalization ability in cells expressing DLL3; the antibody-drug conjugates derived from B6 and B275 demonstrate extremely strong inhibitory activity against tumor cell proliferation both in vitro and in vivo.

[0009] The first aspect of this invention provides an anti-DLL3 antibody or its antigen-binding fragment, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region VL comprises LCDR1, LCDR2, and LCDR3.

[0010] The HCDR1 contains an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:9;

[0011] The HCDR2 contains an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:10;

[0012] The HCDR3 contains an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:11;

[0013] The LCDR1 contains the amino acid sequence shown in SEQ ID NO:23;

[0014] The LCDR2 contains an amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:14;

[0015] The LCDR3 contains an amino acid sequence as shown in SEQ ID NO:24.

[0016] In some preferred embodiments, the anti-DLL3 antibody or its antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH as shown in SEQ ID NO:4, and LCDR1, LCDR2, and LCDR3 of the light chain variable region VL as shown in SEQ ID NO:8; or comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH as shown in SEQ ID NO:12, and LCDR1, LCDR2, and LCDR3 of the light chain variable region VL as shown in SEQ ID NO:16.

[0017] In some specific embodiments, the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:7.

[0018] Alternatively, the amino acid sequence of HCDR1 is shown in SEQ ID NO:9, the amino acid sequence of HCDR2 is shown in SEQ ID NO:10, the amino acid sequence of HCDR3 is shown in SEQ ID NO:11; the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:15.

[0019] In some preferred embodiments, the heavy chain variable region VH comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with, for example, SEQ ID NO:4; and / or, the light chain variable region VL comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with, for example, SEQ ID NO:8; or,

[0020] The heavy chain variable region VH contains an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with a sequence such as SEQ ID NO:12; and / or, the light chain variable region VL contains an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with a sequence such as SEQ ID NO:16;

[0021] The amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same antigen-binding function as the original sequence; this identity is based on the aforementioned CDR combination, i.e., the site of sequence change is the FR region.

[0022] In some preferred embodiments, the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:4; and / or, the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO:8; or,

[0023] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:12; and / or, the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:16.

[0024] In some specific embodiments, the anti-DLL3 antibody or its antigen-binding fragment includes one or more of the following:

[0025] (1) Fully human antibodies, humanized antibodies, chimeric antibodies, proantibodies, bispecific antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies; and / or,

[0026] (2) Fab, Fab', F(ab')2, Fv, ScFv, biantibody, Fd, sdAb, VHH and complementarity-determining region (CDR); and / or,

[0027] (3) The DLL3 is a human DLL3.

[0028] In some preferred embodiments, the anti-DLL3 antibody or its antigen-binding fragment is a full-length antibody, comprising a heavy chain constant region of the heavy chain of a human antibody, preferably a heavy chain constant region of human antibody IgG1; and / or, comprising a light chain constant region of the light chain of a human antibody, preferably a light chain constant region of the κ chain of a human antibody.

[0029] In some preferred embodiments, the amino acid sequence of the heavy chain constant region of the human antibody IgG1 is as shown in SEQ ID NO:17, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO:17; and / or, the amino acid sequence of the light chain constant region of the human antibody κ chain is as shown in SEQ ID NO:18, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO:18; the amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same antigen-binding function as the original sequence; this identity is based on the aforementioned CDR combination being determined, i.e., the site of sequence change is the FR region.

[0030] In some specific embodiments, the amino acid sequence of the heavy chain of the anti-DLL3 antibody or its antigen-binding fragment is as shown in SEQ ID NO:19; and / or, the amino acid sequence of the light chain is as shown in SEQ ID NO:20; or, the amino acid sequence of the heavy chain of the anti-DLL3 antibody or its antigen-binding fragment is as shown in SEQ ID NO:21; and / or, the amino acid sequence of the light chain is as shown in SEQ ID NO:22.

[0031] A second aspect of the present invention provides an isolated nucleic acid that encodes the anti-DLL3 antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0032] A third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid described in the second aspect of the present invention.

[0033] A fourth aspect of the present invention provides a transformant comprising, in a host cell, the recombinant expression vector described in the third aspect of the present invention.

[0034] In some specific implementations, the host cell is a prokaryotic cell, such as Escherichia coli.

[0035] The fifth aspect of the present invention provides a method for preparing an anti-DLL3 antibody or an antigen-binding fragment thereof, the method comprising culturing the transformant described in the fourth aspect of the present invention on a culture medium suitable for its growth, thereby obtaining the anti-DLL3 antibody or the antigen-binding fragment thereof.

[0036] The sixth aspect of the present invention provides a method for detecting DLL3, which includes the step of contacting a sample to be tested with an anti-DLL3 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0037] In some preferred embodiments, the detection is for non-diagnostic and / or therapeutic purposes.

[0038] The seventh aspect of the present invention provides an antibody-drug conjugate comprising the anti-DLL3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, a linker unit L, and a cytotoxic drug.

[0039] In some preferred embodiments, the cytotoxic drug has the structure shown in formula (A-1), its stereoisomers, pharmaceutically usable salts, solvates, or solvates of salts thereof.

[0040] in,

[0041] M is -L 2 -L 1 -C(O)-;

[0042] L 2 It is -O- or -S-, and L 2 Connect to the aforementioned connector unit L;

[0043] L 1 -(C(R) 1a (R) 1b )) m -CH2-, C3-C6 saturated cycloalkyl or 3-6 saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and the 3-6 saturated heterocyclic group are each independently and optionally converted by one or more R 2a replace;

[0044] m is selected from 1, 2, 3 or 4; the heteroatoms in the 3-6 saturated heterocyclic groups are each independently N, O and S, and the number of heteroatoms is 1, 2 or 3;

[0045] Each R 1a R 1b and R 2a Independently hydrogen, halogen, hydroxyl, amino, or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more R;

[0046] Each R is independently either hydrogen or halogen.

[0047] In some preferred embodiments, L in the antibody-drug conjugate 1 -(C(R) 1a (R) 1b )) m -CH2-; each R 1a Independently hydrogen, halogen, or C1-C6 alkyl; each R 1b Independently hydrogen, halogen, or C1-C6 alkyl; or,

[0048] Where L 1 It is a C3-C6 saturated cycloalkyl group, wherein the C3-C6 saturated cycloalkyl group is optionally surrounded by one or more R 2a Replace, each R 2a It can be hydrogen, halogen, or C1-C6 alkyl independently.

[0049] In some specific implementations, L in the antibody-drug conjugate 1 for

[0050] In some preferred embodiments, the cytotoxic drug has any of the following structures:

[0051] In some preferred embodiments, the connector unit L is -L a -L b -L c -; and the L c Connected to the cytotoxic drug described above;

[0052] -L a -for Preferred End a is connected to Ab, and end b is connected to L. b Connected; wherein Ab is the anti-DLL3 antibody or its antigen-binding fragment as described in the first aspect of the present invention;

[0053] And / or, -L b - For any of the following structures:

[0054] Preferred More Among them, C-end and L a Connected, d end and L c Connected; -L c -for

[0055] In some implementations, the connector unit L is Preferred

[0056] In some preferred embodiments, the antibody-drug conjugate structure is shown in formula (A-2):

[0057] Wherein, p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 3 to 9; more preferably an integer or decimal from 7 to 8, such as 7.8 or 7.9;

[0058] Ab refers to the anti-DLL3 antibody or its antigen-binding fragment as described in the first aspect of this invention; M and L are as defined above.

[0059] In some specific implementations, the antibody-drug conjugate is selected from the following structural formulas:

[0060] in,

[0061] p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9;

[0062] Ab is the anti-DLL3 antibody or its antigen-binding fragment as described in the first aspect of this invention.

[0063] In some embodiments, the antibody-drug conjugate is any of the following conjugates:

[0064] p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably any integer or decimal from 6 to 8; for example, p is 8;

[0065] p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably any integer or decimal from 6 to 8; for example, p is 7.8;

[0066] B6 and B275 are anti-DLL3 antibodies. The heavy chain amino acid sequence of B6 is shown in SEQ ID NO:19, and the light chain amino acid sequence is shown in SEQ ID NO:20. The heavy chain amino acid sequence of B275 is shown in SEQ ID NO:21, and the light chain amino acid sequence is shown in SEQ ID NO:22.

[0067] In some embodiments, the antibody-drug conjugate is any of the following conjugates or a pharmaceutically acceptable salt thereof:

[0068] Where p represents the number of connections, and p is any integer from 1 to 10, preferably any integer from 3 to 9; more preferably any integer from 4 to 8; for example, p is 4, 5, 6, 7 or 8;

[0069] B6 is an anti-DLL3 antibody. The heavy chain amino acid sequence of B6 is shown in SEQ ID NO:19, and the light chain amino acid sequence is shown in SEQ ID NO:20.

[0070] In some embodiments, the antibody-drug conjugate is any of the following conjugates or a pharmaceutically acceptable salt thereof:

[0071] Where p represents the number of connections, and p is any integer from 1 to 10, preferably any integer from 3 to 9; more preferably any integer from 4 to 8; for example, p is 4, 5, 6, 7 or 8;

[0072] B275 is an anti-DLL3 antibody. The heavy chain amino acid sequence of B275 is shown in SEQ ID NO:21, and the light chain amino acid sequence is shown in SEQ ID NO:22.

[0073] In some embodiments, the antibody-drug conjugate comprises antibody-drug conjugates as defined above, wherein the number of links p is the same or different, and the average number of links p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably any integer or decimal from 6 to 8; for example, the average number of links p is 7.8 or 8.

[0074] An eighth aspect of the present invention provides a method for preparing an antibody-drug conjugate as described in the seventh aspect of the present invention, the method comprising reacting an anti-DLL3 antibody or its antigen-binding fragment as described in the first aspect of the present invention with a compound represented by Formula II to obtain the antibody-drug conjugate.

[0075] L'-cytotoxic drugs

[0076] II;

[0077] L' is a linker unit L formed with the anti-DLL3 antibody or its antigen-binding fragment as defined above;

[0078] The cytotoxic drugs are as defined above.

[0079] In some embodiments, the antibody-drug conjugate satisfies one or more of the following conditions:

[0080] (1) The compound shown in Formula II is

[0081] or

[0082] (2) The anti-DLL3 antibody or its antigen-binding fragment is B6 or B275;

[0083] The amino acid sequence of the heavy chain of B6 is preferably as shown in SEQ ID NO:19, and the amino acid sequence of the light chain is preferably as shown in SEQ ID NO:20.

[0084] The preferred amino acid sequence of the heavy chain of B275 is shown in SEQ ID NO:21, and the preferred amino acid sequence of the light chain is shown in SEQ ID NO:22.

[0085] The ninth aspect of the present invention provides a pharmaceutical composition comprising the anti-DLL3 antibody or its antigen-binding fragment as described in the first aspect of the present invention and / or the antibody-drug conjugate as described in the seventh aspect of the present invention, and a pharmaceutically acceptable carrier.

[0086] The tenth aspect of this invention provides the use of the anti-DLL3 antibody or its antigen-binding fragment described in the first aspect of this invention, the drug conjugate described in the seventh aspect of this invention, and / or the pharmaceutical composition described in the ninth aspect of this invention in the preparation of medicaments for the diagnosis, prevention, and / or treatment of cancer.

[0087] In some specific embodiments, the cancer is a DLL3-expressing cancer, such as small cell lung cancer. The eleventh aspect of the present invention provides the use of the anti-DLL3 antibody or its antigen-binding fragment described in the first aspect of the present invention, the drug conjugate described in the seventh aspect of the present invention, and / or the pharmaceutical composition described in the ninth aspect of the present invention in the preparation of medicaments for diagnosing, preventing, and / or treating cancers with high DLL3 expression.

[0088] In some specific implementations, the cancer with high DLL3 expression is small cell lung cancer.

[0089] The twelfth aspect of the present invention provides a method for diagnosing, preventing and / or treating cancer, the method comprising administering to a patient in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in the first aspect, an antibody-drug conjugate as described in the seventh aspect, and / or a pharmaceutical composition as described in the eighth aspect.

[0090] In some specific implementations, the cancer is a DLL3-expressing cancer, such as small cell lung cancer.

[0091] The thirteenth aspect of the present invention provides a method for diagnosing, preventing and / or treating cancer, the method comprising administering to a patient in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in the first aspect, an antibody-drug conjugate as described in the seventh aspect, and / or a pharmaceutical composition as described in the eighth aspect.

[0092] In some specific implementations, the cancer is a cancer with high DLL3 expression, such as small cell lung cancer.

[0093] The fourteenth aspect of the present invention provides an anti-DLL3 antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, an antibody-drug conjugate as described in the seventh aspect of the present invention, and / or a pharmaceutical composition as described in the ninth aspect of the present invention for the diagnosis, prevention, and / or treatment of cancer.

[0094] In some specific implementations, the cancer is a DLL3-expressing cancer, such as small cell lung cancer.

[0095] The fifteenth aspect of the present invention provides an anti-DLL3 antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, an antibody-drug conjugate as described in the seventh aspect of the present invention, and / or a pharmaceutical composition as described in the ninth aspect of the present invention for the diagnosis, prevention, and / or treatment of cancer.

[0096] In some specific implementations, the cancer is a cancer with high DLL3 expression, such as small cell lung cancer.

[0097] The sixteenth aspect of the present invention provides a combination therapy comprising administering, to a subject in need, the anti-DLL3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the antibody-drug conjugate as described in the seventh aspect of the present invention, and / or the pharmaceutical composition as described in the ninth aspect of the present invention, and a second therapeutic agent.

[0098] In some preferred embodiments, the second therapeutic agent comprises other anti-DLL3 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates or pharmaceutical compositions comprising said other anti-DLL3 antibodies or antigen-binding fragments thereof, and / or other drugs for treating cancer.

[0099] In some specific implementations, the cancer is a DLL3-expressing cancer, such as small cell lung cancer.

[0100] The seventeenth aspect of the present invention provides a combination therapy comprising administering, to a subject in need, the anti-DLL3 antibody or its antigen-binding fragment as described in the first aspect of the present invention, the antibody-drug conjugate as described in the seventh aspect of the present invention, and / or the pharmaceutical composition as described in the ninth aspect of the present invention, and a second therapeutic agent.

[0101] In some preferred embodiments, the second therapeutic agent comprises other anti-DLL3 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates or pharmaceutical compositions comprising said other anti-DLL3 antibodies or antigen-binding fragments thereof, and / or other drugs for treating cancer.

[0102] In some specific implementations, the cancer is a cancer with high DLL3 expression, such as small cell lung cancer.

[0103] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0104] The reagents and raw materials used in this invention are all commercially available.

[0105] The positive and progressive effects of this invention are as follows:

[0106] The anti-DLL3 antibody of this invention has good affinity for human DLL3 antigen;

[0107] The anti-DLL3 antibody of this invention has good internalization ability in SHP77 cells expressing DLL3, and is superior to the control antibody Rovalpituzumab.

[0108] The anti-DLL3 antibody-drug conjugate of the present invention has good inhibitory activity against the in vitro proliferation of HEK293 cells, SHP77 cells, and LU2514 PDC cells containing human DLL3 protein.

[0109] The anti-DLL3 antibody-drug conjugate of this invention exhibits significant antitumor activity against SHP77 tumor-bearing mice, NCI-H82 tumor-bearing mice, and DMS53 tumor-bearing mice with small cell lung cancer.

[0110] The anti-DLL3 antibody-drug conjugate of this invention exhibits good stability in human, rat, and monkey plasma in vitro.

[0111] The anti-DLL3 antibody-drug conjugate of this invention exhibits good p-value (exposure level and half-life) in rats and monkeys.

[0112] Therefore, this invention has promising applications in DLL3 expression diseases (such as cancer). Attached Figure Description

[0113] Figure 1 is a schematic diagram showing the internalization ability of B6 and B275 antibodies in H_DLL3 HEK-293 cells.

[0114] Figure 2 is a schematic diagram of the internalization ability of B6 and B275 antibodies in SHP77 cells.

[0115] Figure 3 is a schematic diagram of the in vivo antitumor efficacy of the anti-DLL3 antibody-drug conjugate DLL3-ADC on the DMS53 xenograft model.

[0116] Figure 4 is a schematic diagram of the in vivo antitumor efficacy of the anti-DLL3 antibody-drug conjugate DLL3-ADC on the SHP77 xenograft model. Detailed Implementation

[0117] definition

[0118] In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968):

[0119] Alanine: Ala-A, Arginine: Arg-R, Aspartic acid: Asp-D, Cysteine: Cys-C, Glutamine: Gln-Q, Glutamic acid: Glu-E, Histidine: His-H, Glycine: Gly-G, Asparagine: Asn-N, Tyrosine: Tyr-Y, Proline: Pro-P, Serine: Ser-S, Methionine: Met-M, Lysine: Lys-K, Valine: Val-V, Isoleucine: Ile-I, Phenylalanine: Phe-F, Leucine: Leu-L, Tryptophan: Trp-W, Threonine: Thr-T.

[0120] In this invention, the term "and / or" should be understood to mean any one of the options or any combination of two or more of the options.

[0121] In this invention, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as "only one" or "exactly one" or when used in the claims as "consisting of...", will it refer to only one number or one element of the list.

[0122] In this invention, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker unit. In this application, "antibody-drug conjugate" can refer to an antibody or its antigen-binding fragment linked to a biologically active cytotoxic drug fragment via a stable linker unit.

[0123] In this invention, the term "cytotoxic drug" generally refers to a toxic drug that possesses chemical molecules within tumor cells that strongly disrupt their normal growth. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 (or radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics or ribolysins, or their derivatives, for example, can be toxic drugs, including but not limited to camptothecin derivatives, such as camptothecin derivative essanotecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione).

[0124] In this invention, the term "antibody" generally refers to an immunoglobulin that reacts to a specified protein or peptide or fragment thereof. Antibodies can be from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Antibodies may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies may also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of this application can be derived from any species. The term "antibody" can include complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antibodies, and any other modified immunoglobulin molecules, provided that these antibodies exhibit the desired biological activity.

[0125] In this invention, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule containing amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an "epitope." As described above, the antigen-binding domain typically includes a variable region (VL) and a variable region (VH) of the antibody light chain; however, it is not necessary to include both. Fd fragments, for example, have two VH regions and typically retain some of the antigen-binding functionality of the complete antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light chain (CL) and CH1 domains; (2) F(ab′)2 fragments, bivalent fragments having two Fab fragments connected by disulfide bridges of hinge regions; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains of antibody single arms; (5) dAb fragments (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli”, Nature 341:544-546 (1989), which are incorporated herein by reference in their entirety) having a VH domain; (6) separate complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv), for example derived from scFV libraries.Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined together using a recombination method via a synthetic linker. The synthetic linker allows it to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv)) (see, for example, Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-ChainFv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)); (8) “VHH” refers to the variable antigen-binding domain of heavy chain antibodies from camelids (camels, dromedaries, llamas, alpacas, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and a review by Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH can also be called nanobody (Nb).

[0126] In this invention, the term "variable region" or "variable domain" generally refers to the structural domain of the antibody heavy or light chain involved in the binding of the antibody to the antigen. In this application, the term "variable" generally means that certain portions of the sequence of the variable domain of the antibody vary strongly, resulting in various specific antibodies binding to and specificizing their antigens. This variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs), namely LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), mostly in a β-sheet configuration, connected by three CDR structural loop regions. The CDRs in each chain are closely packed together through the FR region and together with the CDRs from the other chain, they form the antigen-binding site of the antibody.

[0127] In this invention, the amino acid sequences of the listed CDRs are all given according to the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia et al. (Chothia et al., (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined by any of the known schemes described above. While the scope of protection claimed by this invention is based on the sequence shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention. Therefore, when referring to antibodies defined by a specific CDR sequence as defined by this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0128] The sequence identity between sequences is calculated as follows. To determine the percentage of identity between two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in the first and second amino acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being compared is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, the molecules are identical at that position. Sequence comparison and the calculation of the percentage of identity between two sequences can be performed using mathematical algorithms. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program, which is integrated into the GCG software package. The Blossum 62 matrix or PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6 are used to determine the percentage of identity between two amino acid sequences. A particularly preferred set of parameters (and one set of parameters that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. Alternatively, the PAM120 weighted remainder table, gap length penalty of 12, and gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17), which has been incorporated into the ALIGN program (version 2.0). Additionally or alternatively, the protein sequence described in this invention can be further used as a "query sequence" to perform a search against a public database to, for example, identify other family member sequences or related sequences.

[0129] In this invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of Y consists of the second and third constant regions (and a fourth constant region for IgE and IgM) of two heavy chains linked together, with disulfide bonds (interchain) forming hinges. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains, and hinge regions for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y includes a variable region of a single heavy chain and a first constant region that binds to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0130] In this invention, "Fab" consists of a light chain and a heavy chain, CH1, and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain segments containing the CH2 and CH3 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. "Fab'" contains a portion of a light chain and a heavy chain containing the VH and CH1 domains, as well as the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab's to form the F(ab')2 molecule. "F(ab')2" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0131] In this invention, the scFv (single chain antibody fragment) can be a conventional single chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids. The VL and VH domains enable them to pair as linkers to form monovalent molecules as single polypeptide chains [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating G4S amino acid sequences or variants thereof. For example, linkers with the amino acid sequences (G4S)4 or (G4S)3 can be used, but variants thereof can also be used.

[0132] In this invention, the term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies by any particular method.

[0133] The term "multispecific antibody," used in its broadest sense, encompasses antibodies that exhibit multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and triabodies, antibody fragments covalently or non-covalently linked, etc.

[0134] In this invention, the term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, human antibodies comprise at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Human antibodies may optionally include at least a portion of the constant region (Fc) of human immunoglobulins.

[0135] In this invention, "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K). D The equilibrium dissociation constant represents the dissociation rate constant and the binding rate constant (k, k, k) respectively. dis and k on The ratio of affinity to antigen. Affinity can be measured by common methods known in the art. In some embodiments of the invention, affinity is measured using surface plasmon resonance (SPR) technology, such as the affinity between the antibody and antigen of the present invention. In some preferred embodiments of the invention, a specific method for measuring affinity is the BIAcore method described herein.

[0136] In this invention, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine; for example, it can be fluorine or chlorine.

[0137] In this invention, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. Alkyl groups can be substituted or unsubstituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having residues derived from the removal of hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane. It can be a straight-chain or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, such as a chain alkyl containing 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc.

[0138] In this invention, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring comprises 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropane, cyclobutane, cyclopentane, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cyclohepttrienyl, cyclooctane, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0139] In this invention, the term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic light substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or sulfur, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, wherein 1 to 4 are heteroatoms; more preferably, it comprises 3 to 8 ring atoms, wherein 1 to 3 are heteroatoms; even more preferably, it comprises 3 to 6 ring atoms, wherein 1 to 3 are heteroatoms; most preferably, it comprises 5 or 6 ring atoms, wherein 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl groups. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups. The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, and the ring connected to the parent structure is the heterocyclic group.

[0140] In this invention, the term "independently" generally means that the variable applies to any situation, regardless of whether the variable has the same or different definitions in the same compound. For example, the variable may refer to the type or number of substituents in the compound, or the type of atoms in the compound. For example, when R appears twice in a compound and R is defined as "independently carbon or nitrogen", both Rs can be carbon, both Rs can be nitrogen, or one R can be carbon and the other R can be nitrogen.

[0141] In this invention, the terms "optional" or "optionally" generally mean that the event or environment described below may but does not have to occur, and the description includes situations in which the event or environment occurs or does not occur. For example, "optionally alkyl-substituted heterocyclic group" means that an alkyl group may but does not have to be present, and the description can include cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0142] In this invention, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, independently substituted by the corresponding number of substituents. Substituents are only considered in their possible chemical positions, and those skilled in the art can determine (experimentally or theoretically) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (e.g., alkene) bond.

[0143] In this invention, as those skilled in the art will know, terms such as "alkyl," "alkenyl," and "cycloalkyl" can be preceded by an identifier to indicate the number of atoms present in the group under specific conditions, for example, C1-C4 alkyl, C3-C7 cycloalkoxy, C1-C4 alkylcarbonylamino, etc., where the subscript number following "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl); C...1-10 In this context, the members of the group can have any number of carbon atoms falling within the range of 1-10.

[0144] In this invention, the compounds or antibody-drug conjugates of this invention comprise their tautomers, meso compounds, racemates, enantiomers, and / or diastereomers. In this application, the term "diastereomer" generally refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. In this application, the terms "tautomer" or "tautomer form" are used interchangeably and generally refer to structural isomers with different energies that can be interconverted through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via the rearrangement of some bonding electrons. In this application, the term "meta-polymorph" generally refers to a molecule containing asymmetric atoms but possessing symmetry factors that result in zero total optical rotation within the molecule. The term "racemic mixture" or "racemic mixture" refers to a composition consisting of two enantiomers in equimolar amounts.

[0145] In this invention, the terms "connector unit" or "connector structure" generally refer to a chemical structural fragment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end. Alternatively, it may be connected to other connectors before being linked to a cytotoxic drug. The direct or indirect connection to the ligand can refer to the group directly connecting to the ligand via a covalent bond, or it can refer to the connection of the ligand via a connector structure. For example, chemical structural fragments or bonds containing acid-labile connector structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) connector structures, light-labile connector structures, dimethyl connector structures, or disulfide-containing connector structures can be used as connector structures.

[0146] In some embodiments of the present invention, antibody-drug conjugates refer to compositions containing different DAR distributions. In this invention, the term "drug loading" generally refers to the average number of cytotoxic drugs loaded onto each ligand, which may be called the average number of links, or expressed as the ratio of cytotoxic drug to antibody amount. The range of cytotoxic drug loading can be 0-12 links per ligand (Ab), for example, 1-10 cytotoxic drugs. The drug loading of each ADC molecule after the conjugation reaction can be identified using conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization. The average number of links p can be an integer or decimal from 1 to 10. For example, the average number of links p can be an integer or decimal from 2 to 8. For example, the average number of links p can be an integer or decimal from 3 to 8. For example, the average number of links p can be an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0147] In some embodiments of the present invention, antibody-drug conjugates refer to compounds containing the same DAR distribution. The term "drug loading" refers to the number of cytotoxic drugs loaded on each ligand, which can be called the number of links or expressed as the ratio of cytotoxic drug to antibody. The range of cytotoxic drug loading can be 0-12 links per ligand (Ab), for example, 1-10 cytotoxic drugs. The number of links p can be any integer from 1 to 10. For example, the number of links p can be any integer from 3 to 9. For example, the number of links p can be any integer from 6 to 8. For example, the number of links p can be 4, 5, 6, 7, or 8.

[0148] In this invention, certain atoms of the compounds or antibody-drug conjugates of this invention may appear in the form of more than one isotope. For example, hydrogen may be in the form of protium (…). 1 H), deuterium ( 2 H) and tritium ( 3 Carbon exists in the form of H, and it may exist in three different isotopes (H). 12 C 13 C and 14 C) Naturally occurring. Examples of isotopes that may be incorporated into the compounds of this application include, but are not limited to, those that exist naturally. 15 N、 18 O、 17 O、 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125I, or similar isotopes. Therefore, the compounds or antibody-drug conjugates of the present invention can be enriched in one or more of these isotopes relative to their natural abundance. As those skilled in the art will know, such isotope-enriched compounds can be used for a variety of purposes. For example, with heavy isotopes such as deuterium (I, or similar isotopes). 2 H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability. For example, deuterium (H) 2 The natural abundance of deuterium (H) is approximately 0.015%. Therefore, there is approximately one deuterium atom for every 6500 hydrogen atoms in nature. Thus, the deuterium-containing compounds or antibody-drug conjugates of the present invention have a deuterium abundance greater than 0.015% at one or more positions (as the case may be). Unless otherwise specified, the structures described in the present invention may also include compounds or antibody-drug conjugates that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds or antibody-drug conjugates that are otherwise identical to the structure of the present invention, except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon-13 or carbon-14, are within the scope of the present invention.

[0149] As is known in the art, in this invention, "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate capable of being incorporated into the chain by a DNA or RNA polymerase.

[0150] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors.

[0151] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In an exemplary aspect, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacterium or protozoan. In an exemplary aspect, as described herein, the host cell is a cell originating from or obtained from an individual. In an exemplary aspect, the host cell is derived from or obtained from a mammal.

[0152] In this invention, the methods and conditions for culturing the resulting transformants and for recovering the resulting antibody molecules are known to those skilled in the art and can be modified or optimized based on methods known in this specification and the prior art, depending on the specific expression vector and mammalian host cell used.

[0153] In this invention, applications of detection for non-diagnostic and / or therapeutic purposes include: detecting the presence or absence of DLL3 protein in the laboratory; screening other antibodies targeting DLL3 as positive antibodies; or competing with other anti-DLL3 antibodies for binding to detect whether there is competition between antibodies, i.e., whether the antigenic epitopes are the same or similar.

[0154] In this invention, the term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds described in this application or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The pharmaceutical composition may facilitate administration to the organism, promote the absorption of the active ingredient, and thus exert its biological activity. Conventional preparation methods for pharmaceutical compositions can be found in the Chinese Pharmacopoeia. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. Sterile injectable formulations may also be sterile injectable solutions or suspensions prepared in non-toxic, parenteral-acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Furthermore, sterile fixative oils can be conveniently used as solvents or suspension media. For example, any blended fixative oil, including synthetic mono- or diglycerides, may be used. Additionally, fatty acids such as oleic acid can also be used to prepare injectable formulations.

[0155] In this invention, the terms "pharmaceutically acceptable salt" or "pharmaceutically usable salt" generally refer to salts of compounds or antibody-drug conjugates of this invention, or salts of compounds or antibody-drug conjugates described in this invention. Such salts are safe and / or effective when used in mammals and can have the desired biological activity. The compounds or antibody-drug conjugates of this invention can form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0156] In this invention, pharmaceutically acceptable carriers are any of those conventionally used carriers, limited only by physicochemical considerations (such as solubility and lack of reactivity with antibodies targeting DLL3) and by route of administration. Pharmaceutically acceptable carriers described herein, such as mediators, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of a pharmaceutical composition and does not have adverse side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce adverse, allergic, or other inappropriate reactions when administered to animals or humans. In some aspects, the pharmaceutical composition is free of pyrogens and other impurities that would be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption delay agents, etc.; their uses are well known in the art.

[0157] As used herein, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "therapeuticly effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0158] In this invention, the term "cancer" refers to a malignant tumor, a disease caused by the dysregulation of mechanisms controlling cell growth and proliferation. The term "DLL3-expressing cancer" refers to a malignant tumor in which DLL3 is abnormally expressed on the cell surface.

[0159] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0160] Example 1: Obtaining anti-DLL3 antibody

[0161] Using a fully human antibody library with hundreds of billions of entries, a combination of solid-phase screening (human hu-DLL3-His / Fc purchased from ACRO, catalog number DL3-H52H4 / DL3-H5255, and DLL3-His purchased from Kaika, catalog number DLL-CM103, used as antigens) and liquid-phase screening (biotinylated hu-DLL3 and biotinylated Cyno-DLL3 used as antigens, and biotinylated reagent purchased from Thermo Fisher, catalog number 21335) was employed for cross-screening. After multiple rounds of screening, positive clones that specifically bind to human and monkey DLL3 were obtained. The specific steps are as follows:

[0162] Liquid-phase antibody library screening: The natural library phage suspension was diluted and blocked with 2.5% BSA, then co-incubated with streptavidin-conjugated Dynabeads™. Phages after negative screening were collected. The phage suspension collected after negative screening was incubated with Dynabeads cells coated and blocked with biotin-labeled antigen (hu-DLL3 or cyno-DLL3), and bound and washed according to the Kingfisher magnetic bead screening system method. Phages were eluted with Typsin to infect the library plates; the eluted phage solution was thoroughly mixed with logarithmic-phase SS320 cells (purchased from Sanyou Biopharmaceutical Co., Ltd.), incubated at 37°C for 30 min, and then plated on 2YT-Car+-Tet+ plates and incubated overnight at 37°C. Input phages were prepared by scraping and screened four times, with each round using a 3-fold gradient of antigen concentration decreasing.

[0163] Solid-phase antibody library screening: Diluted antigen (hu-DLL3-His / Fc or cyno-DLL3-His) was added to immunotubes, coated overnight at 4°C, washed, and blocked with 5% PBSM. The phage suspension blocked with 5% PBSM was incubated with the antigen-coated and blocked immunotubes, followed by binding and washing according to the immunotube screening system. Phage was eluted with Typsin to infect the library plates; the eluted phage solution was thoroughly mixed with logarithmic-phase SS320 cells and incubated at 37°C for 30 min. The mixture was then plated on 2YT-Car+-Tet+ plates (Car is carbenicillin, Tet is tetracycline, both purchased from Shanghai Sangon Biotech Co., Ltd., catalog numbers A600469-0005 and A100422-0010, respectively) and incubated overnight at 37°C. The input phages were prepared by scraping and screened in four rounds, with each round using a 3-fold gradient of antigen concentration.

[0164] The monoclonal antibodies obtained from the washing process were sequenced and full-length antibodies were constructed. Further cellular-level FACS binding verification (using engineered HEK293 cells expressing human DLL3 protein, H_DLL3 HEK-293, purchased from Jiman Biotechnology, catalog number GM-C12896, experimental method as in Example 2) and ELISA protein-level non-binding verification of DLL1 (purchased from ACRO catalog number DL1-H52H8) and DLL4 (purchased from ACRO catalog number DL4-H5227) (experimental method as in Example 2) were performed. Finally, candidate antibodies that can specifically bind to human DLL3 and monkey DLL3 at the cellular level were obtained.

[0165] The names and sequence numbers of some of the antibodies constructed in this embodiment are shown in Table 1.

[0166] Table 1. Heavy and light chain variable region sequences (KABAT) of the antibody.

[0167] The underlined areas below the amino acid sequences in the table represent CDR regions defined according to the Kabat numbering system.

[0168] The heavy chain and light chain variable region sequences are linked to the heavy chain constant region and light chain constant region sequences of the human antibody, respectively. For example, the antibody heavy chain constant region is selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO:17; the antibody light chain constant region is selected from the human κ chain constant region as shown in SEQ ID NO:18, and the antibody constant region sequences are shown in Table 2.

[0169] Table 2. Sequence Numbering of Constant Regions

[0170] The heavy chain amino acid sequence of B6 is (SEQ ID NO:19):

[0171] The light chain amino acid sequence of B6 is (SEQ ID NO:20):

[0172] The heavy chain amino acid sequence of B275 is (SEQ ID NO:21):

[0173] The light chain amino acid sequence of B275 is (SEQ ID NO:22):

[0174] Example 2: Detection of in vitro binding activity of anti-DLL3 antibody

[0175] 1. In vitro indirect ELISA binding assay

[0176] Human DLL3-His protein (AcroBiosystems, Cat#DL3-H52H4) was diluted to a concentration of 2 μg / mL with PBS at pH 7.4. 100 μL of this solution was added to each well of a 96-well high-affinity ELISA plate and incubated overnight (12-16 hours) at 4°C. After washing three times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 200 μL of 1% bovine serum albumin (BSA) blocking buffer diluted with PBST was added to each well, and the plate was incubated at 37°C for 1 hour. After blocking, the blocking buffer was discarded, and the plate was washed once with PBST buffer. The antibody to be tested was diluted with PBST containing 1% BSA, starting at 3 μg / mL, and serially diluted 3-fold to obtain 8 concentrations. 100 μL of each solution was added to each well of the plate, and the plate was incubated at 37°C for 1 hour. After incubation, wash the plate three times with PBST, add 100 μL / well of HRP-labeled goat anti-human secondary antibody (abcam, ab97225) diluted with PBST containing 1% BSA, and incubate at room temperature for 50 minutes. After washing the plate six times with PBST, add 100 μL / well of TMB chromogenic substrate (Suzhou Yake Chemical Reagent Co., Ltd., cat#S0025), and incubate at room temperature in the dark for 10 minutes. Stop the reaction by adding 50 μL / well of 1M HCl. Read the absorbance at 450 nm using a microplate reader (Thermo, Ascent) and analyze the data. The concentration-signal curve analysis results are shown in Table 3 below. The results show that the antibody of this invention has good affinity for human DLL3 antigen.

[0177] Table 3. Affinity of antibodies to human DLL3 antigen detected by ELISA (ECG) 50 value)

[0178] 2. FACS detection of the binding activity of anti-DLL3 antibody on H_DLL3 HEK-293 cells.

[0179] HEK293 cells (H_DLL3 HEK-293, purchased from Jimon Biotechnology, catalog number GM-C12896) engineered to express human DLL3 protein were prepared into a cell suspension and the cell density was adjusted to 1×10⁶. 6Cells / mL. Take a 96-well round-bottom plate and add 100 μL of cell suspension to each well. Dilute the antibody to be tested with FACS buffer to eight concentration gradients: 5.0000, 1.6667, 0.5556, 0.1852, 0.0617, 0.0206, 0.0069, and 0.0007 μg / mL. Add 100 μL of each concentration of antibody to the corresponding well, 100 μL per well, and incubate at 4°C for 60 min. Wash the plate twice with FACS buffer. Dilute the secondary antibody PE labelled anti-human IgG Fc (Abcam) 1:300 with FACS buffer, 100 μL / well, and incubate at 4°C for 30 min. After washing the plate twice with FACS buffer, use flow cytometry to detect the mean fluorescence intensity (MFI) of cells incubated with each antibody concentration, and perform concentration-curve analysis to determine the antibody binding EC50. 50 The concentrations and results are shown in Table 4. FACS combined with experimental results showed that B6 and B275 antibodies have good affinity for human DLL3 antigen.

[0180] Table 4. Affinity of antibodies to human DLL3 antigen as detected by FACS (EC50) 50 value)

[0181] Example 3: Evaluation of the internalization capacity of anti-DLL3 antibody based on live-cell imaging

[0182] Anti-DLL3 target cells were resuscitated and expanded. H_DLL3 HEK-293 cells (purchased from Jiman Biotechnology, catalog number GM-C12896) and SHP77 cells (purchased from ATCC, catalog number CRL-2195) were used to adjust the target cell density with culture medium and then seeded into experimental plates (Corning 3599 96-well cell culture plates, H_DLL3 HEK-293 cells: 10,000 / well; SHP77 cells: 10,000 / well). Each test antibody (B6 and B275), positive control antibody (Rovalpituzumab, expressed from the heavy and light chain sequences disclosed in patent US20190225685), and negative control antibody (GenScript Human IgG1) were diluted to the corresponding working solutions (H_DLL3 HEK-293 cells: maximum incubation concentration 30 nM, 2-fold dilution, 8 concentration points, minimum incubation concentration 0.234 nM; SHP77 cells: two incubation concentrations of 60 nM and 30 nM). After cell adhesion, the test antibodies were mixed with... Fabfluor-pH antibody labeling dye (Sartorius, The Fabfluor-pH Antibody Labeling Dyes kit (Cat. No: 4722) was thoroughly mixed (molar ratio 1:3) and incubated at 37°C for 15 min. Samples of each concentration were transferred to the corresponding wells of the experimental plate. The plate was then transferred to the Incucyte live-cell analysis system, and the appropriate scanning and imaging program was set up. Images were acquired over 48 hours using the Incucyte live-cell analysis system. The analytical results are expressed as total red object integrated intensity (RCU × μm² / Image). For antibodies at multiple concentrations, fitted curves were exported using Incucyte software and analyzed using GraphPad Prism software. The results are shown in Table 5 and Figures 1 and 2.

[0183] Table 5 Antibody internalization ability

[0184] The results showed that B6 and B275 had a high internalization rate in cells expressing DLL3, which was higher than that of the positive control antibody Rovalpituzumab.

[0185] Example 4: Synthesis of Connector-Cytoxin

[0186] (1) Connector-cytotoxin X1:

[0187] Synthesis route:

[0188] first step:

[0189] Under nitrogen protection, benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a DMF (50 mL) solution of 27a (5.00 g, 43.0 mmol) and NaHCO3 (10.9 g, 129 mmol), and the reaction was carried out at 25 °C for 17 hours. TLC (PE / EA = 2 / 1) showed that the reaction was complete. The reaction solution was added to 500 mL of water and extracted twice with EA (250 mL). After separation, the solution was washed with saturated sodium chloride aqueous solution (500 mL), dried over anhydrous Na2SO4, concentrated, and column filtered (PE:EA = 3:2) to give 5.1 g of colorless liquid, yield: 57.1%.

[0190] Step Two:

[0191] Under nitrogen protection, a solution of 27b (4.50 g, 21.8 mmol) in THF (30 mL) was added dropwise to a solution of KI2 (4.00 g, 10.9 mmol), TsOH (800 mg, 4.65 mmol), and reacted at 0 °C for 2 hours at 25 °C. TLC (PE / EA = 1 / 2) showed the reaction was complete. The reaction solution was added to 200 mL of water, extracted twice with EA (200 mL), dried over anhydrous Na2SO4, and concentrated by column chromatography (PE / EA = 3 / 2) to give 1.56 g of a white solid, yield: 26%.

[0192] Step 3:

[0193] Under hydrogen atmosphere and at 0°C, Pd / C (80 mg) was added to a mixed solution of EtOH (8 mL) and EA (8 mL) at 27°C (800 mg, 1.55 mmol), and the mixture was stirred at 0°C for 2.5 hours. LC-MS showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with EA (200 mL), concentrated, dissolved in THF (20 mL), and evaporated to dryness to obtain 600 mg of white solid, yield: 91%.

[0194] Step 4:

[0195] Under nitrogen protection, at 0°C, DIEA (152 mg, 1.18 mmol) was added to a DMF (6 mL) solution of 27 d (220 mg, 0.515 mmol), KI4 (250 mg, 0.47 mmol), and HATU (214 mg, 0.56 mmol), and the reaction was carried out at 0°C for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was added to an aqueous citric acid solution (pH = 4) (150 mL), filtered, and the filter cake was washed with 175 mL of water. After drying, the solution was pulled dry with an oil pump to obtain 260 mg of brown solid, yield: 66%.

[0196] Step 5:

[0197] Under nitrogen protection, diethylamine (8 mL) was added dropwise to a 30 mL solution of DCM (27°C, 260 mg, 0.309 mmol) at 0 °C, and the reaction was carried out at 0 °C for 3 hours. LC-MS showed that the reaction was complete. The reaction solution was added to a 600 mL solution of petroleum ether at 0 °C, and a solid precipitated out. After standing for the solid to be adsorbed to the bottom of the flask, the solution was poured out and dried using an oil pump to obtain 90 mg of brown solid, yield: 47.1%.

[0198] Step 6:

[0199] Under nitrogen protection, at 0°C, HATU (74 mg, 0.19 mmol) was added to a DMF (2.5 mL) solution containing 27f (90 mg, 0.13 mmol), KI-1 (92 mg, 0.19 mmol), and DIEA (50 mg, 0.39 mmol), and the reaction was carried out at 0°C for 2 hours. LC-MS showed that the basic reaction was complete. At 0°C, the reaction solution was added to an aqueous solution of citric acid (pH 4) (30 mL), and a flocculent solid precipitated. After filtration, the solid was analyzed by preparative agar (DCM / MecOH = 10 / 1) to give 9.2 mg of a pale yellow solid (X1), yield: 6%.

[0200] MS m / z (ESI): 1074 [M+1].

[0201] H-NMR (400MHz, MeOD): 7.65(d,1H),7.62(s,1H),7.30-7.21(m,5H),6.79(s,2H),5.69-5.65(m, 1H),5.57(d,1H),5.43-5.10(m,3H),4.70(d,2H),4.48-4.39(m,2H),4.10-4.05(m,1H),4.01-3 .75(m,5H),3.46(t,2H),3.22-3.15(m,2H),3.07-3.00(m,1H),2.75(m,1H),2.62(m,1H),2.45( s,3H),2.37-2.20(m,6H),2.10-2.02(m,2H),2.00-1.92(m,2H)1.68-1.57(m,6H),1.01(t,3H).

[0202] (2) Connector-Cytoxin X2:

[0203] Synthesis route:

[0204] first step

[0205] 34a (5 g, 48.0 mmol) and K₂CO₃ (19.9 g, 144.0 mmol) were dissolved in DMF (20 mL), and benzyl bromide (12.3 g, 72.0 mmol) was added dropwise. The reaction was carried out at 25 °C for 17 hours. TLC (PE / EA = 3 / 1) was used to determine if the reaction was complete. The reaction solution was added to water (200 mL), extracted with EA (250 mL), washed with saturated NaCl, dried over anhydrous Na₂SO₄, and concentrated and column filtered (PE:EA = 2:1) to give 8.7 g of colorless liquid 34b, yield 93%. MS-ESI: m / z 195.1 [M+H]⁺.

[0206] Step 2

[0207] Dissolve 7.3 g (19.8 mmol) of 43c and 1.46 g (8.5 mmol) of TsOH in 20 mL of THF. Under nitrogen protection and cooling to 0 °C, add 10 mL of THF solution containing 7.7 g (39.6 mmol) of 34b. After the addition is complete, react at 0 °C for 2 hours. TLC (PE / EA = 2 / 1) shows that most of the starting material has reacted. Pour the reaction solution into 100 mL of water, extract with 100 mL of DCM, separate the layers, wash with saturated NaCl, dry with anhydrous Na₂SO₄, and pass through a column (PE / EA = 1 / 1) to obtain 3.9 g of colorless viscous 34d, yield: 39%. MS-ESI: m / z 503.3 [M+H]+.

[0208] Step 3

[0209] Under hydrogen atmosphere and at 0°C, Pd / C (1 g, 10 wt.%) was added to a mixed solution of 1.9 g (3.78 mmol) of EtOH (100 mL) and EA (100 mL), and the reaction was carried out at 0°C for 3 hours. TLC (PE / EA = 2 / 1) showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with EA / EtOH (1:1, 100 mL × 3). The filtrate was concentrated, dissolved in THF (50 mL × 3), and evaporated to dryness. This process was repeated three times to obtain 1 g of gray solid 34e, yield: 64%. MS-ESI: m / z 435.2 [M+Na]+.

[0210] Step 4

[0211] Under nitrogen protection, DIEA (303 mg, 2.35 mmol) was added dropwise to a DMF (20 mL) solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol), and HATU (429 mg, 1.13 mmol) at 0 °C. After the addition was complete, the reaction was allowed to proceed at 0 °C for 2 hours. LC-MS showed the reaction was complete. The reaction solution was added dropwise to 300 mL of water, stirred, and allowed to stand for 5 minutes. After filtration, the filter cake was dissolved in a DCM / MeOH (10:1, 100 mL) solution, dried, and stirred. Column chromatography (EA:MeOH = 30:1) yielded 600 mg of yellow solid 34f, yield: 77%. MS-ESI: m / z 830.3 [M+H]+.

[0212] Step 5

[0213] Under nitrogen protection, diethylamine (5 mL) was added dropwise to a 34f (150 mg, 0.18 mmol) DCM (5 mL) solution at 0 °C, and the reaction was carried out at 0 °C for 2 hours. LCMS showed that the reaction was complete. Petroleum ether solution (100 mL × 6) was added to the reaction solution, and a solid precipitated. After standing to allow the solid to settle, the solution was poured off and then dried using an oil pump to obtain 34 g of 120 mg white powder. LCMS showed that the product content was 70%, yield: 76%. MS-ESI: m / z 608.3 [M+H]+.

[0214] Step 6

[0215] Under nitrogen protection, HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) solutions at 0 °C, and the reaction was carried out at 0 °C for 2 h. LC-MS showed that the starting material reacted completely. The reaction solution was directly passed through a reversed-phase column with eluent (MeCN / MeOH = 1 / 1):H2O = 60%:40%) to purify 14.8 mg of yellow solid x 2, yield 14%.

[0216] MS-ESI: m / z 1062.4[M+H]+.

[0217] 1H NMR(400MHz, Methanol-d4)δ7.69–7.61(m,2H),7.22–7.16(m,2H),7.16–7.09(m,3H),6.76(s,2H),5.70–5.64(m,1H),5.6 0(d,J=16.4Hz,1H),5.40–5.31(m,2H),5.26(d,J=19.0Hz,1H),4.65–4.50(m,7H),4.25–4.16(m,1H),3.87(d,J=16.7Hz,1 H),3.83–3.76(m,3H),3.72(d,J=17.0Hz,2H),3.44(t,J=7.1Hz,2H),3.25–3.17(m,2H),3.10–3.02(m,1H),2.92–2.83(m, 1H),2.45–2.39(m,5H),2.32–2.20(m,5H),1.97–1.89(m,2H),1.63–1.50(m,4H),1.34–1.20(m,6H),0.99(t,J=7.3Hz,3H).

[0218] (3) Connector-Cytoxin X3:

[0219] Synthesis route:

[0220] first step:

[0221] To a mixture of 32a (2.00 g, 6.6 mmol), K₂CO₃ (1.82 g, 13.2 mmol), and MeCN (20 mL), bromopropene (960 mg, 7.92 mmol) was added, and the mixture was stirred at 20 °C for 5 hours. TLC (PE / EA = 1 / 2) showed the reaction was complete. The reaction mixture was poured into 100 mL of water, the pH was adjusted to 5, and the mixture was extracted three times with EA (100 mL). The extract was dried over anhydrous sodium sulfate, evaporated to dryness, and purified by column chromatography (PE / EA = 2 / 1) to give 1.83 g of white solid 32b, yield: 81%.

[0222] Step Two:

[0223] TFA (10 mL) was added to 10 mL of DCM containing 32b (1.38 g, 4.02 mmol), and the mixture was stirred at 25 °C for 17 hours. TLC (PE / EA = 1 / 3) showed that the reaction was complete. The reaction solution was evaporated to dryness to obtain 0.91 g of yellow viscous substance 32c, yield negligible.

[0224] Step 3:

[0225] 41d (1.92 g, 4.87 mmol) was added to a mixture of 32c (910 mg, 4.87 mmol) and NaHCO3 (613 mg, 7.3 mmol) in DME / H2O (20 mL / 10 mL), and the mixture was stirred at 25 °C for 3 hours. TLC (DCM / MeOH = 1 / 1) showed the reaction was complete. The reaction mixture was poured into 100 mL of water, the pH was adjusted to 5 with aq.HCl (1 N), and the mixture was extracted twice with EA (150 mL). The extract was dried over anhydrous sodium sulfate, evaporated to dryness, and purified by column chromatography (DCM / MeOH = 20 / 1) to give 1.53 g of white solid 32e, yield: 67%. MS-ESI: m / z 467.4 [M+H]+.

[0226] Step 4:

[0227] Pd / C (600 mg) was added to 50 mL of MeOH at 32°C (3 g, 5.83 mmol), and the mixture was stirred for 5 hours under a hydrogen balloon at 25°C. TLC (EA) showed the reaction was complete. The reaction solution was filtered and evaporated to dryness to give 32 g of a white solid (1.9 g), yield: 77%.

[0228] Step 5:

[0229] HATU (707 mg, 1.86 mmol) was added to 10 mL of DMF containing 32 g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol), and triethylamine (342 mg, 3.38 mmol). The mixture was stirred at 0 °C for 3.5 h. TLC (EA) showed that the reaction was complete. The reaction mixture was poured into 80 mL of H2O, extracted twice with EA (100 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (EA) to give 1.186 g of white solid after 32 h (yield: 83%). MS-ESI: m / z 842.3 [M+H]+.

[0230] Step 6:

[0231] The DCM / diethylamine (20 mL, 20 / 1) solution (1.186 g, 1.41 mmol) was stirred at 25 °C for 17 hours. TLC (DCM / MeOH = 10 / 1) showed the reaction was complete. The reaction mixture was poured into petroleum ether (200 mL) and filtered to give 768 mg of white solid 32i, yield: 88%. MS-ESI: m / z 620.3 [M+H]+.

[0232] Step 7:

[0233] HATU (414 mg, 1.09 mmol) was added to 10 mL of DMF containing 32i (676 mg, 1.09 mmol), 32e (508 mg, 1.09 mmol), and DIEA (423 mg, 3.27 mmol), and the mixture was stirred at 20 °C for 17 hours. TLC (PE / EA = 1 / 5) showed the reaction was complete. The reaction mixture was poured into water (30 mL), filtered, and the filter cake was purified by column chromatography (DCM / MeOH = 50 / 1) to obtain 511 mg of white solid 32j, yield: 44%. MS-ESI: m / z 1068.3 [M+H]+.

[0234] Step 8:

[0235] A solution of 32 J (482 mg, 0.451 mmol) of diethylamine / DCM (10 mL, 1 / 5) was stirred at 10 °C for 17 hours. TLC (EA) showed that the reaction was complete. The reaction solution was poured into PE (300 mL) and filtered to give 301 mg of white solid 32 K, yield negligible.

[0236] Step 9:

[0237] Morpholine (93 mg, 1.07 mmol) was added to 5 mL of THF containing 32 kJ (301 mg, 0.356 mmol) and Pd(PPh3)4 (82 mg, 0.071 mmol), and the mixture was stirred at 25 °C for 5 hours. LC-MS showed that the reaction was complete. 108 mg of a white solid was prepared in 32 mL of the reaction solution, yield: 38%. MS-ESI: m / z 806.3 [M+H]+.

[0238] Step 10:

[0239] Add 27 mg of acetyl bromide (0.134 mmol) to 32 mL of THF (2 mL) and DMF (2 mL) containing 108 mg (0.134 mmol) of triethylamine (41 mg (0.402 mmol) and 41 mg (0.402 mmol), and stir at 0 °C for 1 hour. TLC (DCM / MeOH = 10 / 1) showed the reaction was complete. The reaction solution was directly used to prepare 15 mg of white solid x 3, yield: 12%.

[0240] MS-ESI: m / z 926.3[M+H]+.

[0241] 1H NMR (400MHz, DMSO-d6) δ12.11(s,1H),8.54–8.42(m,3H),8.27–8.16(m,2H),7.78(d,J=11.0Hz,1H),7.3 0(s,1H),6.53(s,1H),5.61–5.51(m,1H),5.42(s,2H),5.20–5.05(m,2H),4.56–4.42(m,2H),4.32–4.22( m,1H),3.96–3.87(m,3H),3.79(d,J=5.6Hz,2H),3.70(d,J=5.9Hz,2H),3.25–3.08(m,2H),2.61–2.53(m ,2H),2.45–2.36(m,4H),2.36–2.22(m,3H),2.20–2.03(m,4H),1.99–1.68(m,4H),0.87(t,J=7.3Hz,3H).

[0242] (4) Connector-Cytoxin X4:

[0243] Synthesis route:

[0244] first step:

[0245] Pd / C (400 mg, 10 wt.%) was added to 20 mL of MeOH containing 2.00 g (2.58 mmol) of 33a, and the mixture was stirred at 20 °C for 5 hours. TLC (EA) showed that the reaction was complete. The reaction solution was filtered and evaporated to dryness to give 1.3 g of white solid 33b, yield: 74%.

[0246] Step Two:

[0247] HATU (305 mg, 0.802 mmol) was added to 5 mL of DMF containing 33b (0.55 g, 0.802 mmol), KI4 (427 mg, 0.802 mmol), and DIPEA (310 mg, 2.40 mmol), and the mixture was stirred at 0 °C for 2 hours. TLC (DCM / MeOH = 1 / 10) showed the reaction was complete. The reaction mixture was poured into 40 mL of water, filtered to obtain a crude product, and purified by column chromatography (DCM / MeOH = 20 / 1) to give 360 ​​mg of a yellow solid 33c, in 41% yield.

[0248] Step 3:

[0249] Diethylamine (2 mL) was added to 10 mL of DCM (33°C, 360 mg, 0.326 mmol). The mixture was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 5 / 1) showed that the reaction was complete. The reaction mixture was poured into PE (100 mL) and filtered to give 205 mg of white solid 33°C, yield: 71%. MS-ESI: m / z 881.3 [M+H]+.

[0250] Step 4:

[0251] A solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL) was added to a mixture of 33 d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) and water (1 mL). The mixture was stirred at 0 °C for 1 hour. The reaction solution directly yielded 15 mg of white solid X4, yield: 6%.

[0252] MS-ESI: m / z 1001.2[M+H]+.

[0253] 1H NMR(400MHz,DMSO-d6)δ8.57–8.50(m,1H),8.50–8.43(m,2H),8.35–8.29(m,1H),8.19–8.12(m,2H),7 .80(d,J=10.8Hz,1H),7.27–7.14(m,7H),6.53(s,1H),5.59–5.51(m,1H),5.44–5.39(m,2H),5.20–5.0 7(m,2H),4.56–4.44(m,3H),3.92(s,3H),3.80–3.68(m,5H),3.41(s,1H),3.21–3.12(m,2H),2.83–2.7 4(m,1H),2.58–2.55(m,3H),2.39(s,4H),2.18–2.03(m,4H),1.93–1.78(m,2H),0.87(t,J=7.3Hz,3H).

[0254] Example 5: Preparation of anti-DLL3 drug conjugates

[0255] 1. Preparation of anti-DLL3 drug conjugate B6-X2

[0256] B6-X2 (DAR8)

[0257] Add 1 / 9 volume ratio of 200 mM histidine-acetic acid pH 5.5 solution to the antibody B6 buffer (PB pH 7.4; 47 mg, 9.4 mg / mL), then add the prepared tris(2-carbonylethyl) phosphate hydrochloride solution (7.0 mM, 0.454 mL, 3.18 μmol), place in a constant temperature stirrer, stir at 60 rpm, react at 37 °C for 3 hours, and then stop the reaction.

[0258] The adapter-cytotoxin X2 (10.0 mM, 3.50 μmol) was added to the above solution, and the mixture was placed in a constant temperature stirrer at 60 rpm and reacted at 22 °C for 2 hours. The reaction was then stopped. The reaction solution was desalted and purified on an AKTA using a G-25 gel column (desalting column: HiPrep 26 / 10 desalting column; eluent: 20 mM histidine-hydrochloric acid, pH 5.5), and concentrated by ultrafiltration using a 30 KD ultrafiltration tube to obtain a solution of the exemplary product B6-X2 (20 mM histidine-hydrochloric acid, pH 5.5; 31 mg, 2.06 mg / mL, yield: 66%), which was stored at -80 °C.

[0259] MSDAR analysis and calculation yielded a DAR value of p = 8.00.

[0260] 2. Preparation of anti-DLL3 drug conjugate B275-X2

[0261] B275-X2 (DAR 7.8)

[0262] Add 1 / 9 volume ratio of 200 mM histidine-acetic acid pH 5.5 solution to the antibody B275 buffer (PB pH 7.4; 60 mg, 9.72 mg / mL, 0.405 μmol), then add the prepared tris(2-carbonylethyl) phosphate hydrochloride solution (7.0 mM, 0.376 mL, 2.63 μmol), place in a constant temperature stirrer, stir at 60 rpm, react at 22 °C for 3 hours, and then stop the reaction.

[0263] The adapter-cytotoxin X2 (3.87 mg, 3.65 μmol) was dissolved in 0.365 mL of DMA and added to the above solution. The mixture was placed in a constant temperature stirrer at 60 rpm and reacted at 22 °C for 2 hours with shaking. The reaction was then stopped. The reaction solution was desalted and purified on an AKTA using a G-25 gel column (desalting column: HiPrep 26 / 10 desalting column; eluent: 20 mM histidine-hydrochloric acid, pH 5.5). The solution was then concentrated by ultrafiltration using a 30 KD ultrafiltration tube to obtain a solution of the exemplary product B275-X2 (20 mM histidine-hydrochloric acid, pH 5.5; 47.8 mg, 3.79 mg / mL, yield: 80%), which was stored at -80 °C.

[0264] HIC DAR analysis and calculation yielded a DAR value of p = 7.8.

[0265] Example 6: In vitro cell proliferation inhibitory activity test of anti-DLL3 antibody-drug conjugate (DLL3-ADC) Example 6.1: In vitro cell proliferation inhibitory activity against HEK293 cells containing human DLL3 protein

[0266] The inhibitory effect of various anti-DLL3 antibody-drug conjugates (ADCs) on cell proliferation was evaluated by incubating the cells with engineered HEK293 cells (H_DLL3 HEK-293, purchased from Jimon Biotechnology, catalog number GM-C12896) expressing human DLL3 protein for 3 days using the MTS assay (Promega, CellTiter 96 AQueous One Solution Cell Proliferation Assay System kit).

[0267] Cells in the logarithmic growth phase were collected and seeded at a density of 6000 cells / well (50 μL / well). The cell culture plates were incubated overnight at 37°C with 5% CO2. On the second day of the experiment, each ADC drug was diluted 3-fold with complete culture medium to obtain nine concentration gradients: 800, 200, 50, 12.5, 3.125, 0.781, 0.195, 0.049, and 0.012 nM. 50 μL / well of each drug was added to the cell culture plate, with complete culture medium used as a blank control. Three replicates were set up. The cells were incubated at 37°C with 5% CO2 for another 3 days. Before detection at the end of incubation, the cell culture plates were removed from the incubator and allowed to equilibrate to room temperature for 10 min. Add 7.5 μL of Triton X-100 solution to each well of a Cell+Triton culture plate equilibrated to room temperature, gently tap to mix, and incubate the cell culture plate at 37°C for 30 min. Then add 20 μL of MTS to each well and incubate at 37°C for 1-4 h until the color deepens. Read the data using a 492 nm wavelength microplate reader. Use GraphPad Prism software to plot an S-shaped dose-response curve using a nonlinear regression model and calculate the IC50 value. Cell viability is calculated as follows: (Lum of test drug - Lum of blank control) / (Lum of solvent blank control - Lum of blank control) × 100%. Proliferation inhibition rate % = 100% - cell viability %.

[0268] The experimental results are shown in Table 6 below. The results show that B6-X2 and B275-X2 have good inhibitory activity on the proliferation of H_DLL3 HEK-293 cells.

[0269] Table 6. Inhibitory activity of DLL3-ADC on the proliferation of H_DLL3 HEK-293 cells.

[0270] Example 6.2: Inhibitory activity against human DLL3 protein in SHP77 cells during in vitro cell proliferation

[0271] The inhibitory effect of various anti-DLL3 antibody-drug conjugate (ADC) drugs on cell proliferation after incubation for 3 days in SHP77 cells (purchased from ATCC, catalog number CRL-2195) was evaluated using the MTS assay (Promega, CellTiter 96 AQueous One Solution Cell Proliferation Assay System kit).

[0272] Cells in the logarithmic growth phase were collected and seeded at a density of 6000 cells / well (50 μL / well). The cell culture plates were incubated overnight at 37°C with 5% CO2. On the second day of the experiment, each ADC drug was diluted 3-fold with complete culture medium to obtain nine concentration gradients: 800, 200, 50, 12.5, 3.125, 0.781, 0.195, 0.049, and 0.012 nM. 50 μL / well of each drug was added to the cell culture plate, with complete culture medium used as a blank control. Three replicates were set up. The cells were incubated at 37°C with 5% CO2 for another 3 days. Before detection at the end of incubation, the cell culture plates were removed from the incubator and allowed to equilibrate to room temperature for 10 min. Add 7.5 μL of Triton X-100 solution to each well of a Cell+Triton culture plate equilibrated to room temperature, gently tap to mix, and incubate the cell culture plate at 37°C for 30 min. Then add 20 μL of MTS to each well and incubate at 37°C for 1-4 h until the color deepens. Read the data using a 492 nm wavelength microplate reader. Use GraphPad Prism software to plot an S-shaped dose-response curve using a nonlinear regression model and calculate the IC50 value. Cell viability is calculated as follows: (Lum of test drug - Lum of blank control) / (Lum of solvent blank control - Lum of blank control) × 100%. Proliferation inhibition rate % = 100% - cell viability %.

[0273] The experimental results are shown in Table 7 below. The results show that B6-X2 and B275-X2 have good inhibitory activity on the proliferation of SHP77 cells.

[0274] Table 7. Inhibitory activity of DLL3-ADC on the proliferation of human DLL3 SHP77 cells.

[0275] Example 7: In vitro cell proliferation inhibitory activity test of anti-DLL3 antibody-drug conjugate against patient-derived tumor sample-derived stable passaged cell line (PDC) LU2514.

[0276] LU2514 is a stable cell line prepared from a patient-derived tumor sample. It was derived from a small cell lung cancer patient. LU2514 belongs to Crown Bioscience, Inc., and this experiment was commissioned to Crown Bioscience.

[0277] use Chemiluminescent cell viability assay (CTG method, Promega G7572) was used to evaluate the inhibitory effect of various anti-DLL3 antibody-conjugated camptothecin toxoid ADC drugs (B6-X2, B275-X2) on cell proliferation after incubation for 6 days in DLL3-positive LU2514 cells.

[0278] The specific experimental steps were as follows: Logarithmic growth phase cells were collected and seeded at a density of 2000 cells / well. The cell culture plates were incubated overnight at 37°C in a 5% CO2 incubator. On the second day, each ADC drug was diluted 4-fold with complete culture medium to obtain 9 concentration gradients. 20 μL / well was added to each well of the cell culture plate, with a maximum final concentration of 2000 nM. The complete culture medium served as a blank control, and 3 replicates were set up. The plates were incubated at 37°C in a 5% CO2 incubator for 6 days. After incubation, the cell culture plates were removed and allowed to equilibrate to room temperature. 75 μL of CTG detection reagent (Promega, Cat#: G7573) was added to each well, and the mixture was shaken and incubated in the dark for 10 minutes. The signal values ​​were then read using an EnVision multi-functional plate reader (PerkinElmer, EquipID: TAREA0011). GraphPadPrism software was used to plot an S-shaped dose-response curve using a nonlinear regression model and to calculate the IC50. 50 Cell viability is calculated using the formula: (Lum test drug - Lum blank control) / (Lum solvent blank control - Lum blank control) × 100%. Proliferation inhibition rate % = 100% - cell viability %.

[0279] The experimental results are shown in Table 8 below. The results show that B6-X2 and B275-X2 have good inhibitory activity against the in vitro proliferation of LU2514 PDC cells.

[0280] Table 8. Inhibitory activity of DLL3-ADC on the proliferation of LU2514 PDC cells

[0281] Example 8: In vivo tumor-suppressive activity of anti-DLL3 antibody-drug conjugate against human small cell lung cancer cells DMS53 in tumor-bearing mice

[0282] To evaluate the inhibitory effect of DLL3-ADCs on tumor formation in vivo, the antitumor efficacy of each DLL3-ADC was evaluated after xenografts were formed in mice using DLL3-positive human small cell lung cancer cells DMS53 (purchased from ATCC, catalog number CRL-2062).

[0283] 1. Test drugs and materials

[0284] Normal saline (control group): Normal saline was administered via tail vein injection as a single dose.

[0285] B6-X2 (treatment group): 6 mg / kg, administered via tail vein injection, once in total;

[0286] B275-X2 (treatment group): 6 mg / kg, administered via tail vein injection, once in total.

[0287] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0288] 3. Experimental animals: 8-week-old female NCG mice, purchased from Jicui Pharmaceutical Co., Ltd.

[0289] 4. Test methods:

[0290] 5×10 6 One DMS53 cell was subcutaneously instilled into the right anterior scapula of an 8-week-old female NCG mouse. When the tumor grew to approximately 150 mm... 3 Tumor-bearing mice were randomly assigned to groups using StudyDirector™ and began receiving intravenous (iv) injections of ADC drug on day 0, with a total of one injection at a dose of 6 mg / kg. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0291] Five mice were included in each of the solvent control group and the treatment group. The tumor inhibition rate was calculated by measuring tumor volume. Tumor inhibition rate (TGI%) = 100% - (tumor volume of the treatment group on the day of measurement - tumor volume of the treatment group on day 0) / (tumor volume of the control group on the day of measurement - tumor volume of the control group on day 0).

[0292] The experimental results are shown in Figure 3 and Table 9. The results indicate that antibody-drug conjugates B6-X2 and B275-X2 exhibited significant antitumor activity after a single dose.

[0293] Table 9. In vivo antitumor efficacy of anti-DLL3 antibody-drug conjugates in DMS53 xenograft model.

[0294] Example 9: In vivo tumor-suppressive activity of anti-DLL3 antibody-drug conjugate on human small cell lung cancer cells SHP77 tumor-bearing mice

[0295] To evaluate the inhibitory effects of B6-X2 and B275-X2 on tumor formation in vivo, the antitumor effects were evaluated after xenografts were formed in mice using SHP77 human small cell lung cancer cells (purchased from ATCC, catalog number CRL-2195) that are positive for DLL3.

[0296] 1. Test drugs and materials

[0297] Normal saline (control group): Normal saline was injected via tail vein once every 7 days for a total of 2 injections;

[0298] B6-X2 (treatment group): 6 mg / kg, administered via tail vein injection, once every 7 days, for a total of 2 injections;

[0299] B275-X2 (treatment group): 6 mg / kg, administered via tail vein injection, once every 7 days, for a total of 2 injections.

[0300] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0301] 3. Experimental animals: 8-week-old female BALBc-Nude mice, purchased from Jicui Pharmaceutical Co., Ltd.

[0302] 4. Test methods:

[0303] 5×10 6 One SHP77 cell was subcutaneously instilled into the right anterior scapula of an 8-week-old female BALBc-Nude mouse. When the tumor grew to approximately 100 mm... 3 Mice bearing tumors were randomly assigned to StudyDirector™ groups and began receiving intravenous (iv) injections of the ADC drug on day 0 (day 0), once every 7 days for a total of 2 injections, at a dose of 6 mg / kg. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0304] Five mice were used in each of the solvent control and treatment groups. Tumor inhibition rate was calculated by measuring tumor volume. Tumor inhibition rate (TGI%) = 100% - (Tumor volume of the treatment group on the day of measurement - Tumor volume of the treatment group on day 0) / (Tumor volume of the control group on the day of measurement - Tumor volume of the control group on day 0). When tumor regression occurred (tumor volume at measurement was less than the tumor volume at the time of initial administration), the tumor inhibition rate = [1 - (Tumor volume of the treatment group on the day of measurement ÷ Tumor volume of the treatment group on day 0) ÷ (Tumor volume of the control group on the day of measurement ÷ Tumor volume of the control group on day 0)] ÷ [1 - (Tumor volume of the control group on day 0 ÷ Tumor volume of the control group on the day of measurement)] × 100%.

[0305] The experimental results are shown in Figure 4 and Table 10. The results indicate that both antibody-drug conjugates B6-X2 and B275-X2 exhibited significant antitumor activity after administration.

[0306] Table 10. In vivo antitumor efficacy of antibody-drug conjugates in SHP77 xenograft model

[0307] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An anti-DLL3 antibody or its antigen-binding fragment, characterized in that, It contains the heavy chain variable region VH and the light chain variable region VL. The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3, and the light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The HCDR1 contains an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:9; The HCDR2 contains an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:10; The HCDR3 contains an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:11; The LCDR1 contains the amino acid sequence shown in SEQ ID NO:23; The LCDR2 contains an amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:14; The LCDR3 contains an amino acid sequence as shown in SEQ ID NO:

24.

2. The anti-DLL3 antibody or its antigen-binding fragment as described in claim 1, characterized in that, The anti-DLL3 antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH as shown in SEQ ID NO:4, and LCDR1, LCDR2 and LCDR3 of the light chain variable region VL as shown in SEQ ID NO:8; or comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH as shown in SEQ ID NO:12, and LCDR1, LCDR2 and LCDR3 of the light chain variable region VL as shown in SEQ ID NO:16; Preferably, the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:3; the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

7. Alternatively, the amino acid sequence of HCDR1 is shown in SEQ ID NO:9, the amino acid sequence of HCDR2 is shown in SEQ ID NO:10, the amino acid sequence of HCDR3 is shown in SEQ ID NO:11; the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

15.

3. The anti-DLL3 antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region VH comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with, for example, SEQ ID NO:4; and / or, the light chain variable region VL comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with, for example, SEQ ID NO:8; or, The heavy chain variable region VH contains an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with a sequence such as SEQ ID NO:12; and / or, the light chain variable region VL contains an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with a sequence such as SEQ ID NO:16; The amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same antigen-binding function as the original sequence; Preferably, the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:4; and / or, the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO:8; or, The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:12; and / or, the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:

16.

4. The anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, characterized in that, The anti-DLL3 antibody or its antigen-binding fragment includes one or more of the following: (1) Fully human antibodies, humanized antibodies, chimeric antibodies, proantibodies, bispecific antibodies, multispecific antibodies, monoclonal antibodies, and polyclonal antibodies; and / or, (2) Fab, Fab', F(ab')2, Fv, ScFv, biantibody, Fd, sdAb, VHH and complementarity-determining region; and / or, (3) The DLL3 is a human DLL3.

5. The anti-DLL3 antibody or its antigen-binding fragment as described in claim 4, characterized in that, The anti-DLL3 antibody or its antigen-binding fragment is a full-length antibody, comprising a heavy chain constant region of the heavy chain of a human antibody, preferably a heavy chain constant region of a human antibody IgG1; and / or, comprising a light chain constant region of the light chain of a human antibody, preferably a light chain constant region of the κ chain of a human antibody. Preferably, the amino acid sequence of the heavy chain constant region of the human antibody IgG1 is as shown in SEQ ID NO:17, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO:17; and / or, the amino acid sequence of the light chain constant region of the human antibody κ chain is as shown in SEQ ID NO:18, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO:18; the amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same antigen-binding function as the original sequence; More preferably, the amino acid sequence of the heavy chain of the anti-DLL3 antibody or its antigen-binding fragment is as shown in SEQ ID NO:19; and / or, the amino acid sequence of the light chain is as shown in SEQ ID NO:20; or, the amino acid sequence of the heavy chain of the anti-DLL3 antibody or its antigen-binding fragment is as shown in SEQ ID NO:21; and / or, the amino acid sequence of the light chain is as shown in SEQ ID NO:

22.

6. An isolated nucleic acid, characterized in that, The nucleic acid encodes the anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 6.

8. A transformant, characterized in that, The transformant contains the recombinant expression vector as described in claim 7 in the host cell; Preferably, the host cell is a prokaryotic cell, such as Escherichia coli.

9. A method for preparing an anti-DLL3 antibody or its antigen-binding fragment, characterized in that, The method includes culturing the transformant as described in claim 8 on a culture medium suitable for its growth to obtain the anti-DLL3 antibody or its antigen-binding fragment.

10. A method for detecting DLL3, characterized in that, It includes the step of contacting the sample to be tested with an anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5; preferably, the detection is for non-diagnostic and / or therapeutic purposes.

11. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate comprises an anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5, a linker unit L, and a cytotoxic drug.

12. The antibody-drug conjugate as described in claim 11, characterized in that, The cytotoxic drug has the structure shown in formula (A-1), its stereoisomers, pharmaceutically usable salts, solvates, or solvates of salts thereof. in, M is -L 2 -L 1 -C(O)-; L 2 It is -O- or -S-, and L 2 Connect to the aforementioned connector unit L; L 1 -(C(R) 1a (R) 1b )) m -CH2-, C3-C6 saturated cycloalkyl or 3-6 saturated heterocyclic group, wherein the C3-C6 saturated cycloalkyl and the 3-6 saturated heterocyclic group are each independently and optionally converted by one or more R 2a replace; m is selected from 1, 2, 3 or 4; the heteroatoms in the 3-6 saturated heterocyclic groups are each independently N, O and S, and the number of heteroatoms is 1, 2 or 3; Each R 1a R 1b and R 2a Independently hydrogen, halogen, hydroxyl, amino, or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more R; Each R is independently either hydrogen or halogen.

13. The antibody-drug conjugate as described in claim 12, characterized in that, Where L 1 -(C(R) 1a (R) 1b )) m -CH2-; each R 1a Independently hydrogen, halogen, or C1-C6 alkyl; each R 1b Independently hydrogen, halogen, or C1-C6 alkyl; or, Where L 1 It is a C3-C6 saturated cycloalkyl group, wherein the C3-C6 saturated cycloalkyl group is optionally surrounded by one or more R 2a Replace, each R 2a It can be hydrogen, halogen, or C1-C6 alkyl independently.

14. The antibody-drug conjugate as described in claim 13, characterized in that, Where L 1 for 15. The antibody-drug conjugate as described in claim 12, characterized in that, The cytotoxic drug described herein has any of the following structures:

16. The antibody-drug conjugate according to any one of claims 11 to 15, characterized in that, The connector unit L is -L a -L b -L c -; and the L c Connected to the cytotoxic drug described above; -L a -for Preferred End a is connected to Ab, and end b is connected to L. b Connected; And / or, -L b - For any of the following structures: Preferred More Among them, C-end and L a Connected, d end and L c Connected; -L c -for 17. The antibody-drug conjugate as described in claim 16, characterized in that, The connector unit L is Preferred 18. The antibody-drug conjugate according to any one of claims 11 to 17, characterized in that, in, The structure of the antibody-drug conjugate is shown in formula (A-2): Wherein, p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 3 to 9; more preferably an integer or decimal from 7 to 8, such as 7.8 or 7.9; Ab is the anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5; M is as described in any one of claims 12 to 17; L is as described in any one of claims 11 to 17.

19. The antibody-drug conjugate according to any one of claims 11 to 18, characterized in that, The antibody-drug conjugates mentioned above are selected from the following structural formulas: in, p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; Ab is the anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5.

20. The antibody-drug conjugate according to any one of claims 11 to 19, characterized in that, The antibody-drug conjugate is any one of the following conjugates: p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably any integer or decimal from 6 to 8; for example, p is 8; p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably any integer or decimal from 6 to 8; for example, p is 7.8; B6 and B275 are anti-DLL3 antibodies. The heavy chain amino acid sequence of B6 is shown in SEQ ID NO:19, and the light chain amino acid sequence is shown in SEQ ID NO:

20. The heavy chain amino acid sequence of B275 is shown in SEQ ID NO:21, and the light chain amino acid sequence is shown in SEQ ID NO:

22.

21. The antibody-drug conjugate according to any one of claims 11 to 20, characterized in that, The antibody-drug conjugate is any one of the following conjugates or a pharmaceutically acceptable salt thereof: Where p represents the number of connections, and p is any integer from 1 to 10, preferably any integer from 3 to 9; more preferably any integer from 4 to 8; for example, p is 4, 5, 6, 7 or 8; B6 is an anti-DLL3 antibody. The heavy chain amino acid sequence of B6 is shown in SEQ ID NO:19, and the light chain amino acid sequence is shown in SEQ ID NO:

20.

22. The antibody-drug conjugate according to any one of claims 11 to 20, characterized in that, The antibody-drug conjugate is any one of the following conjugates or a pharmaceutically acceptable salt thereof: Where p represents the number of connections, and p is any integer from 1 to 10, preferably any integer from 3 to 9; more preferably any integer from 4 to 8; for example, p is 4, 5, 6, 7 or 8; B275 is an anti-DLL3 antibody. The heavy chain amino acid sequence of B275 is shown in SEQ ID NO:21, and the light chain amino acid sequence is shown in SEQ ID NO:

22.

23. An antibody-drug conjugate, characterized in that, It comprises the antibody-drug conjugate as described in claim 21 or 22, wherein the number of links p is the same or different, and the average number of links p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably any integer or decimal from 6 to 8; for example, the average number of links p is 7.8 or 8.

24. A method for preparing an antibody-drug conjugate as described in any one of claims 11 to 23, characterized in that, The preparation method includes reacting the anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5 with a compound as shown in formula II to obtain the antibody-drug conjugate, L'-cytotoxic drug ii; L' is formed with the anti-DLL3 antibody or its antigen-binding fragment to form a linker unit L as described in any one of claims 12 to 22; The cytotoxic drug is as described in any one of claims 12 to 22; Preferably, the antibody-drug conjugate satisfies one or more of the following conditions: (1) The compound shown in Formula II is or (2) The anti-DLL3 antibody or its antigen-binding fragment is B6 or B275; The amino acid sequence of the heavy chain of B6 is preferably as shown in SEQ ID NO:19, and the amino acid sequence of the light chain is preferably as shown in SEQ ID NO:

20. The preferred amino acid sequence of the heavy chain of B275 is shown in SEQ ID NO:21, and the preferred amino acid sequence of the light chain is shown in SEQ ID NO:

22.

25. A pharmaceutical composition, characterized in that, It comprises an anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5 and / or an antibody-drug conjugate as described in any one of claims 11 to 23, and a pharmaceutically acceptable carrier.

26. The use of the anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5, the drug conjugate as described in any one of claims 11 to 23, and / or the pharmaceutical composition as described in claim 25 in the preparation of medicaments for the diagnosis, prevention, and / or treatment of cancer; Preferably, the cancer is a DLL3-expressing cancer, such as small cell lung cancer.

27. A method for diagnosing, preventing, and / or treating cancer, characterized in that, The method includes administering to a subject in need an anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5, a drug conjugate as described in any one of claims 11 to 23, and / or a pharmaceutical composition as described in claim 25; Preferably, the cancer is a DLL3-expressing cancer, such as small cell lung cancer.

28. An anti-DLL3 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, an antibody-drug conjugate as described in any one of claims 11 to 23, and / or a pharmaceutical composition as described in claim 25, for the diagnosis, prevention, and / or treatment of cancer; Preferably, the cancer is a DLL3-expressing cancer, such as small cell lung cancer.

29. A combination therapy, characterized in that, It includes administering to a subject in need the anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 5, the antibody-drug conjugate as described in any one of claims 11 to 23, and / or the pharmaceutical composition as described in claim 25, and a second therapeutic agent; Preferably, the second therapeutic agent comprises other anti-DLL3 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates or pharmaceutical compositions comprising said other anti-DLL3 antibodies or antigen-binding fragments thereof, and / or other drugs for treating cancer; More preferably, the cancer is a DLL3-expressing cancer, such as small cell lung cancer.

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