Anti-DLL3 antibody and use

Abstract

Provided are an anti-DLL3 antibody, an antibody-drug conjugate comprising the antibody, and a diagnosis and treatment method for a disease related to expression of DLL3, such as cancer.

Description

Anti-DLL3 antibodies and their applications

[0001] This application claims priority to a prior application, patent application number 202410125989.2, filed with the State Intellectual Property Office of China on January 30, 2024, entitled “Anti-DLL3 Antibodies and Applications thereof.” The entirety of this prior application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to an anti-DLL3 antibody and uses thereof. Background Art

[0003] Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine malignancy, accounting for approximately 15% of all lung cancers. Many patients have already entered the extensive-stage stage of the disease upon diagnosis. Although SCLC is sensitive to chemoradiation and chemotherapy, it is highly recurrent. Etoposide combined with carboplatin or cisplatin has long been the standard first-line treatment for patients with extensive-stage SCLC, with response rates of 70%-90% for limited-stage SCLC and 50%-60% for extensive-stage SCLC. Within one year of first-line treatment, approximately 80% of patients with limited-stage SCLC and nearly all patients with extensive-stage SCLC relapse or progress, leaving a significant unmet clinical need. Studies have found that DLL3 is highly expressed in over 80% of patients with SCLC, and elevated DLL3 expression in SCLC is negatively correlated with patient survival; higher DLL3 expression is associated with lower patient survival.

[0004] DLL3 (Delta-Like Ligand 3, DLL3) is a single-pass transmembrane protein composed of 619 amino acids. Its complete structure includes one DSL domain, one intracellular domain, and six epidermal growth factor-like domains. This target is rarely expressed in normal tissues; however, it is specifically expressed in high concentrations in neuroendocrine tumors. Therefore, DLL3 is expected to become a potential target for the treatment of lung cancer. Currently, multiple drugs targeting DLL3 are under development, mainly bispecific antibodies, cell therapies, and ADC drugs. For example, Amgen's AMG757 (Patent No.: US2017037130A1) bispecific antibody is currently in Phase 3 clinical trials and has shown certain advantages in improving median DOR, median OS, and safety. Rovalpituzumab tesirine (Rova-T, patent number: US9770518B1) is the first ADC drug developed by AbbVie that targets the DLL3 target and is also the first targeted treatment drug for SCLC in clinical research. However, the drug's Phase 3 clinical study was terminated due to the excessive toxic side effects of the cytotoxic active drug PBD, which was intolerable to patients and resulted in insufficient effectiveness.

[0005] In order to provide patients with more diversified treatment options and meet their more diverse treatment needs, the present invention has obtained antibodies with higher affinity for DLL3. Compared with Rova-T, the antibody-drug conjugate of the present invention has the ability to kill tumor cells in vitro and in vivo to a greater extent. Summary of the Invention

[0006] In a first aspect, the present invention provides an anti-DLL3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein:

[0007] (1) the sequence of HCDR1 is the sequence shown in SEQ ID NO. 25 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 25, the sequence of HCDR2 is the sequence shown in SEQ ID NO. 26 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 26, and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 27 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 27;

[0008] (2) the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.1, the sequence of HCDR2 is the sequence shown in SEQ ID NO.2 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.2, and the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.3;

[0009] (3) the sequence of HCDR1 is the sequence set forth in SEQ ID NO. 9 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto, the sequence of HCDR2 is the sequence set forth in SEQ ID NO. 10 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto, and the sequence of HCDR3 is the sequence set forth in SEQ ID NO. 11 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto;

[0010] (4) the sequence of HCDR1 is the sequence shown in SEQ ID NO. 17 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 17, the sequence of HCDR2 is the sequence shown in SEQ ID NO. 18 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 18, and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 19 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 19; or

[0011] (5) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 33 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 33, the sequence of HCDR2 is the sequence shown in SEQ ID NO. 34 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 34, and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 35 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 35.

[0012] In a second aspect, the present invention provides an anti-DLL3 antibody or antigen-binding fragment thereof, comprising a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0013] (1) the sequence of LCDR1 is the sequence shown in SEQ ID NO. 28 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 28, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 29 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 29, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 30 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 30;

[0014] (2) the sequence of LCDR1 is the sequence shown in SEQ ID NO. 4 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 4, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 5 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 5, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 6 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 6;

[0015] (3) the sequence of LCDR1 is the sequence shown in SEQ ID NO. 12 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 12, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 13 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 13, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 14 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 14;

[0016] (4) the sequence of LCDR1 is the sequence shown in SEQ ID NO. 20 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 20, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 21 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 21, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 22 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 22; or

[0017] (5) The sequence of LCDR1 is the sequence shown in SEQ ID NO. 36 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 36, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 37 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 37, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 38 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 38.

[0018] In a third aspect, the present invention provides an anti-DLL3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0019] (1) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 25 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 25; the sequence of HCDR2 is the sequence shown in SEQ ID NO. 26 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 26; the sequence of HCDR3 is the sequence shown in SEQ ID NO. 27 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 27; the sequence of LCDR1 is the sequence shown in SEQ ID NO. 28 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 28; the sequence of LCDR2 is the sequence shown in SEQ ID NO. 29 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. The sequence of SEQ ID NO. 29 is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 29, and the sequence of LCDR3 is the sequence of SEQ ID NO. 30 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 30;

[0020] (2) The sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.1, the sequence of HCDR2 is the sequence shown in SEQ ID NO.2 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.2, and the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.3, the sequence of LCDR1 is the sequence shown in SEQ ID NO.4 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.4, and the sequence of LCDR2 is the sequence shown in SEQ ID NO.5 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. The sequence of LCDR3 is the sequence of SEQ ID NO. 6 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence of SEQ ID NO. 6;

[0021] (3) the sequence of HCDR1 is the sequence set forth in SEQ ID NO. 9 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 9, the sequence of HCDR2 is the sequence set forth in SEQ ID NO. 10 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 10, and the sequence of HCDR3 is the sequence set forth in SEQ ID NO. 11 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 11, the sequence of LCDR1 is the sequence set forth in SEQ ID NO. 12 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 12, and the sequence of LCDR2 is the sequence set forth in SEQ ID NO. 13 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. The sequence of SEQ ID NO. 13 is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 13, and the sequence of LCDR3 is the sequence of SEQ ID NO. 14 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 14;

[0022] (4) the sequence of HCDR1 is the sequence set forth in SEQ ID NO. 17 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 17, the sequence of HCDR2 is the sequence set forth in SEQ ID NO. 18 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 18, and the sequence of HCDR3 is the sequence set forth in SEQ ID NO. 19 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 19, the sequence of LCDR1 is the sequence set forth in SEQ ID NO. 20 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 20, and the sequence of LCDR2 is the sequence set forth in SEQ ID NO. 21 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. The sequence of LCDR3 is the sequence of SEQ ID NO. 22 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 21; or

[0023] (5) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 33 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 33, the sequence of HCDR2 is the sequence shown in SEQ ID NO. 34 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 34, and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 35 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 35, the sequence of LCDR1 is the sequence shown in SEQ ID NO. 36 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 36, and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 37 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. The sequence shown in NO.37 has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.38 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.38.

[0024] In the present invention, the CDR sequences are defined according to Kabat.

[0025] The anti-DLL3 antibody or antigen-binding fragment thereof described in the present invention is a murine, chimeric, humanized or fully human antibody or antigen-binding fragment. Preferably, the anti-DLL3 antibody or antigen-binding fragment thereof described in the present invention is a humanized antibody or antigen-binding fragment.

[0026] In a fourth aspect, the present invention provides an anti-DLL3 antibody or antigen-binding fragment thereof, wherein the anti-DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein:

[0027] (1) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 59 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 59;

[0028] (2) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 7 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 7;

[0029] (3) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 15 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 15;

[0030] (4) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 23 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 23;

[0031] (5) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 31 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 31;

[0032] (6) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 39 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 39;

[0033] (7) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 54 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 54; or

[0034] (8) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 56 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 56.

[0035] In a fifth aspect, the present invention provides an anti-DLL3 antibody or an antigen-binding fragment thereof, wherein the anti-DLL3 antibody or antigen-binding fragment thereof comprises a light chain variable region, wherein:

[0036] (1) The light chain variable region sequence is the sequence shown in SEQ ID NO. 59 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 59;

[0037] (2) The light chain variable region sequence is the sequence shown in SEQ ID NO. 8 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 8;

[0038] (3) The light chain variable region sequence is the sequence shown in SEQ ID NO. 16 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 16;

[0039] (4) the light chain variable region sequence is the sequence shown in SEQ ID NO. 24 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 24;

[0040] (5) The light chain variable region sequence is the sequence shown in SEQ ID NO. 32 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 32;

[0041] (6) The light chain variable region sequence is the sequence shown in SEQ ID NO. 40 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 40;

[0042] (7) the light chain variable region sequence is the sequence shown in SEQ ID NO. 55 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 55; or

[0043] (8) The light chain variable region sequence is the sequence shown in SEQ ID NO. 57 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 57.

[0044] In a sixth aspect, the present invention provides an anti-DLL3 antibody or an antigen-binding fragment thereof, wherein the anti-DLL3 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0045] (1) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 58 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 58, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 59 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 59;

[0046] (2) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 7 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 7, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 8 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 8;

[0047] (3) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 15 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 15, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 16 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 16;

[0048] (4) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 23 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 23, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 24 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 24;

[0049] (5) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 31 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 31, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 32 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 32;

[0050] (6) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 39 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 39, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 40 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 40;

[0051] (7) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 54 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 54, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 55 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 55; or

[0052] (8) The heavy chain variable region sequence is the sequence shown in SEQ ID NO.56 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO.56, and the light chain variable region sequence is the sequence shown in SEQ ID NO.57 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO.57.

[0053] In some embodiments of aspects 1 to 6, the anti-DLL3 antibody or antigen-binding fragment thereof specifically binds to human DLL3. In some specific embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof binds to a DLL3 protein epitope that is conserved between DLL3s from different species (e.g., between human and cynomolgus monkey DLL3).

[0054] In the present invention, the antigen-binding fragment is Fv, VHH, scFv, Fab, Fab' or (Fab')2.

[0055] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody or antigen-binding fragment thereof has ADCC activity.

[0056] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody or antigen-binding fragment thereof has CDC activity.

[0057] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody or antigen-binding fragment thereof has ADCP activity.

[0058] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody is a full-length antibody.

[0059] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody is a monoclonal antibody.

[0060] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody is an IgM, IgD, IgG, IgA, or IgE antibody. In some specific embodiments, the anti-DLL3 antibody is an IgG antibody. In some more specific embodiments, the anti-DLL3 antibody is an IgG1 antibody.

[0061] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody is of the IgG1, IgG2, or IgG4 isotype.

[0062] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody comprises a light chain constant region of the kappa subtype or the lambda subtype.

[0063] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody comprises a human IgG1 heavy chain constant region and a human kappa light chain constant region.

[0064] In some embodiments of the first to sixth aspects, the anti-DLL3 antibody comprises a wild-type Fc region.

[0065] In some embodiments of aspects 1 to 6, the anti-DLL3 antibody or antigen-binding fragment thereof is engineered to have enhanced ADCC, CDC, and / or ADCP activity. In some specific embodiments, the anti-DLL3 antibody comprises an engineered Fc region, wherein the engineered Fc region confers enhanced ADCC, CDC, and / or ADCP effects to the antibody.

[0066] In some embodiments of aspects 1 to 6, the anti-DLL3 antibody or antigen-binding fragment thereof is engineered to have reduced ADCC, CDC, and / or ADCP activity. In some specific embodiments, the anti-DLL3 antibody comprises an engineered Fc region that imparts reduced ADCC, CDC, and / or ADCP activity. For the in vivo use of the DLL3 target and antibodies of the present invention, antibodies with reduced or even eliminated ADCC, CDC, and / or ADCP activity may be advantageous, as an excessively strong Fc effect may affect or even diminish the therapeutic efficacy of the antibody. Methods for reducing or eliminating Fc effects are known in the art, including, for example, 1) amino acid residue mutations (primarily to reduce binding to relevant receptors), 2) glycosylation modifications, and 3) IgG4 antibody modifications.

[0067] In the present invention, as a non-limiting example, the Fc region of the anti-DLL3 antibody may comprise amino acid mutations that reduce ADCC, CDC and / or ADCP effects.

[0068] In a seventh aspect, the present invention provides an isolated nucleic acid molecule encoding the anti-DLL3 antibody or antigen-binding fragment thereof according to aspects 1 to 6.

[0069] In some embodiments, the present invention provides a combination of isolated polynucleotides comprising a polynucleotide encoding a light chain of an antibody or antigen-binding fragment thereof of the present invention and a polynucleotide encoding a heavy chain of an antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the polynucleotides are operably linked to a regulatory sequence that can be recognized by a host cell transformed with the vector.

[0070] In an eighth aspect, the present invention provides a vector comprising the nucleic acid molecule described in the seventh aspect.

[0071] In some embodiments, the expression vectors of the present invention comprise a nucleic acid molecule or a combination of polynucleotides of the present invention operably linked to regulatory sequences that allow expression of the encoded polypeptide in a host cell or a cell-free expression system. The choice of expression vector depends on the choice of host cell and can be selected so as to have the desired expression and regulatory characteristics in the selected host cell.

[0072] The nucleic acid in the vector can be operably connected to one or more expression control sequences. As used herein, "operably connected" means incorporated into a genetic construct so that the expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence composed of a region of a DNA molecule generally within 100 nucleotides upstream of the transcription start point (generally near the start site of RNA polymerase II). In order to place the coding sequence under the control of the promoter, the translation start site of the polypeptide translation reading frame must be positioned between 1 and about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, position, and level. Unlike promoters, enhancers can work when located at different distances from the transcription site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe the coding sequence into mRNA, and then the mRNA can be translated into the protein encoded by the coding sequence, the coding sequence is "operably connected" to the expression control sequence in the cell and is "under the control" of the expression control sequence.

[0073] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0074] The expression vector may include a tag sequence. The tag sequence is typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, an Fc fragment, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein, and protein A. In some embodiments, a nucleic acid molecule encoding a DLL3 fusion polypeptide is present in a vector containing a nucleic acid encoding one or more domains of an Ig heavy chain constant region, e.g., domains corresponding to the amino acid sequences of the hinge, CH2, and CH3 regions of a human immunoglobulin Cγ1 chain (Fc fragment).

[0075] In a ninth aspect, the present invention provides a host cell comprising the nucleic acid molecule of the seventh aspect or the vector of the eighth aspect.

[0076] In some embodiments, the host cell can be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic host cell can be a fungus such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, an insect cell such as S. frugiperda, a plant cell such as tobacco, or a mammalian cell such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the host cell is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell, or COS cell.

[0077] In a tenth aspect, the present invention provides a bispecific antibody having a first antigen-binding site and a second antigen-binding site, wherein one antigen-binding site comprises the anti-DLL3 antibody or antigen-binding fragment thereof described in aspects 1 to 6; preferably, the first antigen-binding site of the bispecific antibody comprises the anti-DLL3 antibody or antigen-binding fragment thereof described in aspects 1 to 6, and the second antigen-binding site comprises another anti-DLL3 antibody or antigen-binding fragment thereof described in aspects 1 to 6; more preferably, the first antigen-binding site and the second antigen-binding site of the bispecific antibody bind to different epitopes of the same antigen.

[0078] Preferably, the bispecific antibody of the present invention is an IgG-like bispecific antibody, wherein the left arm is the first antigen binding site and the right arm is the second antigen binding site.

[0079] Preferably, the first antigen-binding site of the bispecific antibody of the present invention comprises a first heavy chain and a first light chain; and the second antigen-binding site comprises a second heavy chain and a second light chain.

[0080] In an eleventh aspect, the present invention provides a bispecific antibody, wherein:

[0081] (1) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 25 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 25, the HCDR2 sequence is the sequence shown in SEQ ID NO. 26 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 26, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 27 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 27; in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 28 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 28, and the LCDR2 sequence is the sequence shown in SEQ ID NO. 29 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. NO.29, and the LCDR3 sequence is the sequence shown in SEQ ID NO.30 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.30; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.17 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.17, the HCDR2 sequence is the sequence shown in SEQ ID NO.18 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.18, and the HCDR3 sequence is the sequence shown in SEQ ID NO.19 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. No. 19, wherein the LCDR1 sequence is SEQ ID NO. 20 or a sequence having 85%, 90%, 95%, 98% or 99% identity thereto; the LCDR2 sequence is SEQ ID NO. 21 or a sequence having 85%, 90%, 95%, 98% or 99% identity thereto; and the LCDR3 sequence is SEQ ID NO. 22 or a sequence having 85%, 90%, 95%, 98% or 99% identity thereto;

[0082] (2) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 25 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 25, the HCDR2 sequence is the sequence shown in SEQ ID NO. 26 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 26, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 27 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 27; in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 28 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 28, and the LCDR2 sequence is the sequence shown in SEQ ID NO. 29 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. NO.29, and the LCDR3 sequence is the sequence shown in SEQ ID NO.30 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.30; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.1 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.1, the HCDR2 sequence is the sequence shown in SEQ ID NO.2 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.2, and the HCDR3 sequence is the sequence shown in SEQ ID NO.3 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. NO.3, in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.4 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.4, the LCDR2 sequence is the sequence shown in SEQ ID NO.5 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.5, and the LCDR3 sequence is the sequence shown in SEQ ID NO.6 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.6; or

[0083] (3) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.17 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.17, the HCDR2 sequence is the sequence shown in SEQ ID NO.18 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.18, and the HCDR3 sequence is the sequence shown in SEQ ID NO.19 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.19; in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.20 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.20, and the LCDR2 sequence is the sequence shown in SEQ ID NO.21 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. NO.21, and the LCDR3 sequence is the sequence shown in SEQ ID NO.22 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.22; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.1 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.1, the HCDR2 sequence is the sequence shown in SEQ ID NO.2 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.2, and the HCDR3 sequence is the sequence shown in SEQ ID NO.3 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. NO.3 has 85%, 90%, 95%, 98% or 99% identity with the sequence shown in NO.3; in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.4 or a sequence with 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.4, the LCDR2 sequence is the sequence shown in SEQ ID NO.5 or a sequence with 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.5, and the LCDR3 sequence is the sequence shown in SEQ ID NO.6 or a sequence with 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.6.

[0084] Furthermore, the present invention provides a bispecific antibody, wherein:

[0085] (1) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 31 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 31, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 32 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 32; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 23 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 23, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 24 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 24;

[0086] (2) the first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 31 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 31, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 32 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 32; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 7 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 7, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 8 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 8;

[0087] (3) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 23 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 23, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 24 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 24; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 7 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 7, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 8 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 8.

[0088] In the present invention, the bispecific antibody further comprises a constant region sequence.

[0089] In the present invention, the bispecific antibody is an IgG1 antibody.

[0090] Preferably, the bispecific antibody of the present invention carries a CH1-CL chain exchange between the first heavy chain and the first light chain or between the second heavy chain and the second light chain.

[0091] Preferably, the Fc region of the bispecific antibody of the present invention comprises amino acid mutations that reduce ADCC, CDC and / or ADCP effects.

[0092] Further preferably, the Fc region of the bispecific antibody described in the present invention comprises amino acid mutations forming a knob and / or hole in the CH3 region to facilitate purification of the bispecific antibody; further preferably, the mutations forming the hole are Y349C, T366S, L368A and Y407V, and the mutations forming the knob are S354C and T366W.

[0093] Further preferably, the present invention provides a bispecific antibody, wherein,

[0094] (1) The bispecific antibody comprises two heavy chains and two light chains, wherein the amino acid sequence of heavy chain 1 is shown in SEQ ID NO.41, the amino acid sequence of heavy chain 2 is shown in SEQ ID NO.43, the amino acid sequence of light chain 1 is shown in SEQ ID NO.46, and the amino acid sequence of light chain 2 is shown in SEQ ID NO.48;

[0095] (2) The bispecific antibody comprises two heavy chains and two light chains, wherein the amino acid sequence of heavy chain 1 is shown in SEQ ID NO.41, the amino acid sequence of heavy chain 2 is shown in SEQ ID NO.50, the amino acid sequence of light chain 1 is shown in SEQ ID NO.46, and the amino acid sequence of light chain 2 is shown in SEQ ID NO.51;

[0096] (3) The bispecific antibody comprises two heavy chains and two light chains, wherein the amino acid sequence of heavy chain 1 is shown in SEQ ID NO.52, the amino acid sequence of heavy chain 2 is shown in SEQ ID NO.50, the amino acid sequence of light chain 1 is shown in SEQ ID NO.53, and the amino acid sequence of light chain 2 is shown in SEQ ID NO.51.

[0097] In a twelfth aspect, the present invention provides a nucleic acid molecule encoding the aforementioned bispecific antibody.

[0098] In some embodiments, the present invention provides a combination of isolated polynucleotides comprising a polynucleotide encoding a light chain of an antibody or antigen-binding fragment thereof of the present invention and a polynucleotide encoding a heavy chain of an antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the polynucleotides are operably linked to a regulatory sequence that can be recognized by a host cell transformed with the vector.

[0099] In a thirteenth aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the twelfth aspect.

[0100] In some embodiments, the expression vectors of the present invention comprise a nucleic acid molecule or a combination of polynucleotides of the present invention operably linked to regulatory sequences that allow expression of the encoded polypeptide in a host cell or a cell-free expression system. The choice of expression vector depends on the choice of host cell and can be selected so as to have the desired expression and regulatory characteristics in the selected host cell.

[0101] The nucleic acid in the vector can be operably connected to one or more expression control sequences. As used herein, "operably connected" means incorporated into a genetic construct so that the expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence composed of a region of a DNA molecule generally within 100 nucleotides upstream of the transcription start point (generally near the start site of RNA polymerase II). In order to place the coding sequence under the control of the promoter, the translation start site of the polypeptide translation reading frame must be positioned between 1 and about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, position, and level. Unlike promoters, enhancers can work when located at different distances from the transcription site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe the coding sequence into mRNA, and then the mRNA can be translated into the protein encoded by the coding sequence, the coding sequence is "operably connected" to the expression control sequence in the cell and is "under the control" of the expression control sequence.

[0102] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0103] The expression vector may include a tag sequence. The tag sequence is typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, an Fc fragment, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein, and protein A. In some embodiments, a nucleic acid molecule encoding a DLL3 fusion polypeptide is present in a vector containing a nucleic acid encoding one or more domains of an Ig heavy chain constant region, e.g., domains corresponding to the amino acid sequences of the hinge, CH2, and CH3 regions of a human immunoglobulin Cγ1 chain (Fc fragment).

[0104] In a fourteenth aspect, the present invention provides a host cell comprising the expression vector described in the thirteenth aspect.

[0105] In some embodiments, the host cell can be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic host cell can be a fungus such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, an insect cell such as S. frugiperda, a plant cell such as tobacco, or a mammalian cell such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the host cell is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell, or COS cell.

[0106] In a fifteenth aspect, the present invention provides an antibody-drug conjugate comprising the aforementioned anti-DLL3 antibody or antigen-binding fragment thereof or the aforementioned bispecific antibody.

[0107] Preferably, the antibody-drug conjugate is obtained by coupling a therapeutic agent to the aforementioned anti-DLL3 antibody or antigen-binding fragment thereof or the aforementioned bispecific antibody.

[0108] In the present invention, the therapeutic agent may be an anti-tumor drug, for example, a cytotoxic drug, an immunopotentiator or a radioisotope.

[0109] In some embodiments, types of cytotoxic drugs include tubulin inhibitors, DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics.

[0110] In some embodiments, tubulin inhibitors include, but are not limited to, auristatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (e.g., DM1, DM4, Ansamitocin, Mertansine, or dolastatin and its derivatives).

[0111] In some embodiments, the DNA topoisomerase inhibitor is a camptothecin analog or a DNA topoisomerase I inhibitor and its derivatives, for example, DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxycamptothecin, topotecan, lertotecan, belotecan, exitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3, 4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0112] In some embodiments, DNA damaging agents include, but are not limited to, calicheamicins, duocarmycins, and anthramycin derivatives such as PBD (pyrrolobenzodiazepine).

[0113] In some embodiments, antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine, or 5-fluorouracil.

[0114] In some embodiments, antitumor antibiotics include, but are not limited to, polypeptide antibiotics (eg, actinomycin D or bleomycin) or anthraquinone drugs (eg, doxorubicin or mitoxantrone hydrochloride).

[0115] In some embodiments, immune enhancers include, but are not limited to, levamisole, pidotimod, imiquimod, isoprinosine, polyinosinic-cytidylic acid, or polyinosinic-uridylic acid.

[0116] In some embodiments, the radionuclide is a diagnostic radionuclide or a therapeutic radionuclide. 68 Ga, 64 Cu, 18 F. 86 Y. 89 Zr,111 In, 99m Tc, 11 C. 123 I. 125 I and 124 According to an embodiment of the present invention, the therapeutic radionuclide is 177 Lu, 90 Y. 125 I. 131 I. 211 At 111 In, 153 Sm, 186 Re、 188 Re、 67 Cu, 225 Ac, 213 Bi, 212 Bihe 212 At least one of Pb.

[0117] In some embodiments, the antibody-drug conjugate of the present invention has the structure shown in formula (I):

[0118] A-(LD) d (I)

[0119] wherein A is the anti-DLL3 antibody or antigen-binding fragment thereof according to the first to sixth aspects of the present invention, or the bispecific antibody according to the tenth to eleventh aspects of the present invention;

[0120] L is a linker moiety, one end of which is connected to A and the other end is connected to the therapeutic agent D;

[0121] d represents the molar ratio of therapeutic agent to A (also known as DAR, i.e., drug-antibody conjugation ratio), an integer or decimal selected from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12);

[0122] When d is a decimal, it refers to the average number of linker-therapeutic agents (LD) conjugated per A.

[0123] In some embodiments, A is selected from antibody 22B12E5, antibody 25G10A9, antibody 31B6G8, antibody 33B10E8, antibody 44B8H2, Hab22B12E5, Hab31B6G8, Hab33B10E8, 33+31, 33+22, and 31+22.

[0124] In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the invention is covalently linked to a therapeutic agent via a linker.

[0125] In some embodiments, L is a cleavable linker.

[0126] In some embodiments, L is cleavable under intracellular conditions or under microenvironmental conditions.

[0127] In one embodiment, L is hydrolyzable at a pH of less than 5.5.

[0128] In some embodiments, L is cleavable by an intracellular protease.

[0129] In some embodiments, L is a cathepsin-cleavable linker.

[0130] In some embodiments, L comprises a dipeptide or a tetrapeptide.

[0131] In some embodiments, the dipeptide is selected from valine (Val)-citrulline (Cit), valine (Val)-alanine (Ala), and the tetrapeptide is selected from glycine (Gly)-glycine (Gly)-phenylalanine (Phe)-glycine (Gly).

[0132] In some embodiments, the antibody is linked to the linker via a cysteine ​​thiol group of the antibody.

[0133] In one embodiment, the antibody is linked to the linker via an amino group of the antibody, particularly an amino group of a glutamine residue.

[0134] In some embodiments, L is a cleavable linker, including, but not limited to, an acid-labile linker (e.g., a hydrazone linker), a disulfide-containing linker, a peptidase-sensitive linker (e.g., a peptide linker comprising an amino acid, e.g., valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), a photolabile linker, a dimethyl linker, a thioether linker, or a hydrophilic linker designed to avoid multidrug transporter-mediated resistance, etc.

[0135] In some embodiments, -L- is selected from -L1-L2-L3-L4-, wherein L1 is a covalent linking unit that is covalently linked to A, L2 is a stretching unit, L3 is selected from a peptide residue consisting of 2-8 amino acids, and L4 is a bond or a self-cleavable fragment.

[0136] In some embodiments, L1 is selected from Where * indicates connection with A.

[0137] In some embodiments, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2bEach independently selected from -C1-C6 alkylene-, -C1-C3 alkylene-C3-C6 cycloalkylene-, -ethynylene-C1-C6 alkylene-, -(CH2CH2O) n -、-(CH2CH2O) n C 1-3 Alkylene-, -C 1-3 Alkylene (CH2CH2O) n C 1-3 Alkylene-, -C 1-3 Alkylene-OC 1-3 Alkylene, phenyl, -phenyl-C 1-3 Alkylene-, wherein n is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0138] In some embodiments, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each is independently selected from -(CH2)5-, phenylene, -phenyl-CH2-, and -CH2-O-CH2-.

[0139] In some embodiments, L2 is selected from: (CH2CH2O) n CH2C(O)-、-(CH2CH2O) n CH2CH2-, The * position is connected to L1, and n is defined as above.

[0140] In some embodiments, L3 is selected from a peptide residue consisting of 2-8 amino acids selected from phenylalanine, valine, alanine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine.

[0141] In some embodiments, L3 is selected from:

[0142] The * position is connected to L2.

[0143] In some embodiments, L4 is selected from: a bond, -NCH2-, Wherein the * position is connected to L3, wherein R1 is selected from hydrogen,

[0144] wherein R2 is selected from:

[0145] wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), and r is preferably an integer of 8 to 24.

[0146] In some embodiments, R2 is selected from the following structures:

[0147] In some embodiments, L4 is selected from -NCH2-,

[0148] In some embodiments, L4 is selected from:

[0149] -NCH2-,

[0150] In some embodiments, L is selected from:

[0151] The * position is connected to A.

[0152] In some embodiments, L is selected from:

[0153] The * position is connected to A.

[0154] In some embodiments, D is selected from:

[0155] wherein X is selected from a bond, Wherein R3 and R4 are independently selected from H, C 1-3 Alkyl (preferably methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), or R3 and R4 together with the carbon atom to which they are connected form a 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), wherein the * position is connected to L4.

[0156] In some embodiments, X is selected from a bond,

[0157] In some embodiments, D is selected from:

[0158] In some embodiments, the antibody-drug conjugate of Formula I has the structure shown below:

[0159] In some embodiments, the antibody-drug conjugate of Formula I has the structure shown below:

[0160] wherein r is selected from an integer of 1-50, preferably an integer of 8-24, preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.

[0161] In some embodiments, in the antibody-drug conjugate of Formula I, A is selected from Hab33B10E8.

[0162] In a sixteenth aspect, the present invention provides a fusion protein comprising the anti-DLL3 antibody or antigen-binding fragment thereof according to aspects one to six.

[0163] Examples of the fusion proteins of the present invention include Fab fusion proteins, Fc fusion proteins, and single-chain antibody (scFv) fusion proteins, which are named according to the different sites to which the effector protein (eg, cytokine) is fused.

[0164] In a seventeenth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned anti-DLL3 antibody or antigen-binding fragment thereof, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate, or the aforementioned fusion protein.

[0165] In the present invention, the pharmaceutical composition described in the seventeenth aspect further includes a pharmaceutically acceptable carrier.

[0166] In an eighteenth aspect, the present invention provides use of the aforementioned anti-DLL3 antibody or antigen-binding fragment thereof, the aforementioned bispecific antibody or the aforementioned antibody-drug conjugate, the aforementioned fusion protein or the aforementioned pharmaceutical composition in the preparation of a medicament for treating tumors.

[0167] In some embodiments, the tumor is a tumor that expresses DLL3 on the surface of tumor cells (DLL3+).

[0168] In some embodiments, the tumor is a tumor that highly expresses DLL3 (DLL3+) on the surface of tumor cells. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 60% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 70% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 80% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 90% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 95% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 98% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 99% of the tumor cells in a tumor cell population express DLL3.

[0169] In some embodiments, the tumor is a solid tumor.

[0170] In some embodiments, the tumor is a hematological tumor.

[0171] In some embodiments, the tumor is a malignant tumor.

[0172] In some embodiments, the tumor is cancer.

[0173] In some embodiments, the tumor is lung cancer, more preferably small cell lung cancer.

[0174] In a nineteenth aspect, the present invention provides a method for treating a tumor in an individual, particularly a tumor expressing DLL3 (DLL3+) on the surface of tumor cells, the method comprising administering to the individual a therapeutically effective amount of the aforementioned anti-DLL3 antibody or its antigen-binding fragment, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate, the aforementioned fusion protein or the aforementioned pharmaceutical composition.

[0175] In some embodiments, the tumor is a tumor that expresses DLL3 on the surface of tumor cells (DLL3+).

[0176] In some embodiments, the tumor is a tumor that highly expresses DLL3 (DLL3+) on the surface of tumor cells. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 60% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 70% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 80% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 90% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 95% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 98% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 99% of the tumor cells in a tumor cell population express DLL3.

[0177] In some embodiments, the tumor is a solid tumor.

[0178] In some embodiments, the tumor is a hematological tumor.

[0179] In some embodiments, the tumor is a malignant tumor.

[0180] In some embodiments, the tumor is cancer.

[0181] In some embodiments, the tumor is lung cancer, more preferably small cell lung cancer.

[0182] In a twentieth aspect, the present invention provides a therapeutically effective amount of the aforementioned anti-DLL3 antibody or its antigen-binding fragment, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate, the aforementioned fusion protein or the aforementioned pharmaceutical composition for treating tumors in an individual, particularly tumors expressing DLL3 (DLL3+) on the surface of tumor cells.

[0183] In some embodiments, the tumor is a tumor that expresses DLL3 on the surface of tumor cells (DLL3+).

[0184] In some embodiments, the tumor is a tumor that highly expresses DLL3 (DLL3+) on the surface of tumor cells. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 60% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 70% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 80% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 90% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 95% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 98% of the tumor cells in a tumor cell population express DLL3. In some embodiments, a tumor that highly expresses DLL3 (DLL3+) means that at least 99% of the tumor cells in a tumor cell population express DLL3.

[0185] In some embodiments, the tumor is a solid tumor.

[0186] In some embodiments, the tumor is a hematological tumor.

[0187] In some embodiments, the tumor is a malignant tumor.

[0188] In some embodiments, the tumor is cancer.

[0189] In some embodiments, the tumor is lung cancer, more preferably small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0190] Figure 1. Binding assay results of chimeric antibodies and soluble human DLL3 protein.

[0191] FIG2 shows the binding assay results of chimeric antibodies to soluble cynomolgus monkey DLL3 protein.

[0192] FIG3 shows the binding assay results of chimeric antibodies to soluble mouse DLL3 protein.

[0193] FIG4A shows the binding assay results of chimeric antibodies and soluble human DLL1 protein.

[0194] FIG4B shows the binding assay results of chimeric antibodies and soluble human DLL4 protein.

[0195] FIG5A shows the binding assay results of chimeric antibodies to human DLL3-B (Ala 176-Leu 492) truncated protein.

[0196] FIG5B shows the binding assay results of chimeric antibodies to human DLL3-C (Ser 312-Leu 492) truncated protein.

[0197] Figure 5C shows the epitope recognition results of the chimeric antibody.

[0198] FIG6A shows the experimental results of the chimeric antibody-drug conjugate killing SHP77 cells.

[0199] FIG6B shows the experimental results of the chimeric antibody-drug conjugate killing NCI-H82 cells.

[0200] FIG7 Chimeric antibody-drug conjugates inhibit the growth of xenograft tumors in NCI-H82 tumor-bearing mice.

[0201] FIG8 Chimeric antibody-drug conjugates inhibit the growth of xenograft tumors in SHP77 tumor-bearing mice.

[0202] Figure 9. Examples of molecular structures of DLL3 bi-epitope antibodies.

[0203] FIG10A shows the binding assay results of the bi-epitope antibody and soluble human DLL3 protein.

[0204] FIG10B shows the binding results of the bi-epitope antibody to soluble cynomolgus monkey DLL3 protein.

[0205] FIG10C shows the binding results of the bi-epitope antibody to soluble mouse DLL3 protein.

[0206] FIG11 shows the killing experiment results of the bi-epitope antibody DLL3 antibody-drug conjugate.

[0207] FIG12A shows the binding assay results of humanized antibodies and soluble human DLL3 protein.

[0208] FIG12B shows the binding results of humanized antibodies to soluble cynomolgus monkey DLL3 protein.

[0209] FIG12C shows the binding assay results of humanized antibodies and soluble mouse DLL3 protein.

[0210] FIG13 shows the killing experiment results of humanized DLL3 antibody-drug conjugates.

[0211] FIG14 Humanized antibody-drug conjugates inhibit the growth of xenograft tumors in NCI-H82 tumor-bearing mice.

[0212] Figure 15 Humanized antibody-drug conjugates inhibit the growth of xenografts in SHP77 tumor-bearing mice.

[0213] Figure 16 shows the single crystal diffraction pattern of compound 82.

[0214] Detailed Description of the Invention

[0215] definition

[0216] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0217] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values ​​shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum value of 1 and a maximum value of 10 (including endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0218] As used herein, the term "subject" or "individual" refers to a mammal, such as a human, but may also be other animals, such as wild animals, livestock, or experimental animals (e.g., gorillas, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

[0219] As used herein, the term "antigen" is a predetermined target to which an antibody can selectively bind. Examples of antigens include, but are not limited to, polypeptides, sugars, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds.

[0220] In a broad sense, "antibody" can refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus encompasses intact antibodies / full-length antibodies, single antibody chains, or any antigen-binding fragment of an antibody (also referred to as an "antigen-binding portion"). When "antibody" and "antigen-binding fragment / antigen-binding portion" appear in the same context, "antibody" can be understood as the intact body relative to the "antigen-binding fragment / antigen-binding portion", and the two together correspond to the broad concept of antibody.

[0221] The terms "anti-DLL3 antibody" or "antibody that binds to DLL3" include antibodies that bind to DLL3 with sufficient affinity such that the antibodies are useful as diagnostic and / or therapeutic agents when targeting DLL3. In some embodiments, the anti-DLL3 antibody binds to unrelated, non-DLL3 proteins to an extent less than about 10% of its binding to DLL3, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or an immunoassay, such as a radioimmunoassay (RIA). For an antibody that "specifically binds" or is "specific for" DLL3, in certain embodiments, the antibody binds to DLL3 with a dissociation constant (KD) of less than or equal to 500 nM, 100 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In some embodiments, an anti-DLL3 antibody binds to an epitope of the DLL3 protein that is conserved between DLL3 from different species (eg, between human and cynomolgus monkey DLL3).

[0222] A "full-length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions are further subdivided into highly variable regions known as complementarity determining regions (CDRs), interspersed with more conserved regions known as framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system. Full-length antibodies can be antibodies of any type, such as IgD, IgE, IgG, IgA or IgM (or the subclasses mentioned above), but antibodies do not need to belong to any specific class. According to the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be designated as different classes. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional structure of different classes of immunoglobulins are well known. Chimeric or humanized antibodies are also encompassed in the antibodies according to the present invention. It is well known to those skilled in the art that the complementary determining region (CDR, generally including CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. There are a variety of common definitions for the CDR amino acid sequences of VH or VL, such as the Kabat definition, the IMGT definition, the Chothia definition, etc. For the variable region amino acid sequence of a given antibody, the CDR amino acid sequences in the VH and VL amino acid sequences can generally be determined according to different definitions. In an embodiment of the present invention, the Kabat definition of CDR amino acid sequences is utilized. For the variable region amino acid sequence of a given antibody, the CDR amino acid sequences in the variable region amino acid sequence can be analyzed in a variety of ways.

[0223] The term "murine antibody" refers to an antibody that is derived from the fusion of B cells from immunized mice with myeloma cells. The mouse hybrid fusion cells are screened to produce antibodies that can both proliferate indefinitely and secrete antibodies. The antibodies are then screened, prepared, and purified. Murine antibodies are generally immunogenic and therefore require subsequent humanization.

[0224] The term "humanized antibody" refers to an antibody obtained by grafting CDR sequences derived from another mammalian species, such as the mouse germline, onto human framework sequences. In order to retain binding affinity, some residues of the framework (called FR) segment may be modified. Humanized antibodies or fragments thereof according to the present invention can be prepared by techniques known to those skilled in the art;

[0225] The term "chimeric antibody" refers to an antibody in which the variable region sequence is from one species and the constant region sequence is from another species, for example, an antibody in which the variable region sequence is from a mouse antibody and the constant region sequence is from a human antibody. Chimeric antibodies or fragments thereof according to the present invention can be prepared by using genetic recombination techniques. For example, the chimeric antibody can be produced by cloning recombinant DNA comprising a promoter and a sequence encoding the variable region of a non-human, especially mouse, monoclonal antibody according to the present invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present invention encoded by such a recombinant gene will be, for example, a mouse-human chimera, the specificity of the antibody being determined by the variable region derived from the mouse DNA, and its isotype being determined by the constant region derived from the human DNA.

[0226] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor individuals.

[0227] As used herein, the terms "antigen-binding fragment," "antigen-binding portion," or "antigen-binding region" are used interchangeably and refer to a portion of an antibody comprising amino acid residues that interact with an antigen and confer specificity and affinity to the binding agent for the antigen, particularly antibody fragments such as Fv, Fab, F(ab')2, or Fab', optionally containing chemical modifications that increase half-life, such as the addition of poly(alkylene) glycols, such as polyethylene glycol ("PEGylated") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol), wherein said fragment has DLL3 binding activity. Preferably, said antigen-binding fragment will consist of or comprise a portion of the heavy or light variable chain sequence of the antibody from which it is derived, sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived. Such antigen-binding fragments will comprise a minimum of 5 amino acids, and preferably 10, 15, 25, 50, or 100 contiguous amino acids of the antibody sequence from which it is derived. Examples of antigen-binding fragments include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which can be a bivalent fragment having two Fab' fragments connected by a disulfide bridge at the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; and (4) a VHH fragment consisting of a VH domain.

[0228] The term "single-chain antibody (scFv)" refers to a single polypeptide chain consisting of a VH domain and a VL domain connected by a peptide linker. (scFv)2 comprises two VH domains connected by a peptide linker and two VL domains, wherein the two VL domains are combined with the two VH domains via disulfide bridges.

[0229] The terms "Fc fragment," "Fc region," "Fc domain," "Fc portion," or similar terms refer to a portion of the constant region of an antibody heavy chain, including the hinge region, the CH2 segment, and the CH3 segment of the constant region. The Fc region of an anti-DLL3 antibody can be engineered or modified, including modifications related to effector functions, such as to reduce or eliminate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), by introducing one or more amino acid substitutions / mutations into the Fc region of the antibody.

[0230] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0231] The term "bispecific antibody" refers to an antibody that has the ability to bind to two antigen epitopes simultaneously. The two antigen epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have a variety of structural configurations. For example, a bispecific antibody can be composed of two Fc fragments and two antigen binding fragments fused to them respectively. Its structure is similar to that of a natural antibody, except that the two arms bind to different antigen targets or epitopes. It is also called an IgG-like bispecific antibody; non-IgG-like bispecific antibodies generally use scFv fragments or Fab fragments as basic modules. By using appropriately modified peptide chain connectors, scFv fragments or Fab fragments can form dimers, trimers, tetramers, pentamers or even higher-order oligomers.

[0232] Generally, to prepare monoclonal antibodies or functional fragments thereof, especially murine monoclonal antibodies or functional fragments thereof, reference can be made to the techniques described in the handbook "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the techniques for preparation from hybridoma cells described by Kohler and Milstein (Nature, 256: 495-497, 1975).

[0233] When describing certain embodiments involving amino acid or nucleic acid sequence mutations, the present invention uses the definition "XaaaY" (e.g., L234A, etc.), where "aaa" represents the sequence position of the amino acid or base (when a specific reference sequence exists, "aaa" represents the sequential position of the residue in the reference sequence; or it can be numbered according to a position numbering method commonly used in the art, such as the EU numbering system, etc.), "X" represents the original amino acid or base at "aaa", and "Y" represents the changed amino acid or base at "aaa". As an example, when describing the L234A mutation in the human heavy chain constant region, it means that according to the EU numbering system of the human heavy chain constant region, the leucine (L) at position 234 is mutated to alanine (A).

[0234] The term "isolated" refers to a biological component (e.g., a nucleic acid, protein (including antibodies), or organelle) that has been substantially separated or purified from other biological components (i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles) in the environment (e.g., a cell) in which the component naturally occurs. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified using standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

[0235] An "expression vector" is a vector that includes one or more expression control sequences, which are DNA sequences that control and regulate the transcription and / or translation of another DNA sequence.

[0236] The term "antibody drug conjugate" or "antibody drug conjugate" (ADC) refers to a targeting ligand such as an antibody (eg, monoclonal antibody) or antibody fragment linked to a biologically active molecule through a linker or linker.

[0237] The term "linker-drug compound" or "linker-drug compound" refers to the partial structure of an antibody-drug conjugate consisting of a linker and a biologically active compound.

[0238] The term "linker" refers to a chemical structure fragment, represented by L, that is linked to an antibody at one end and to a cytotoxic drug at the other end. In certain embodiments of the present invention, the "linker" is formed by linking a "linker-drug compound" to an antibody. In other embodiments of the present invention, one portion of the "linker" is first linked to the antibody, and another portion is linked to the biologically active compound, and then the linker is formed by covalently linking the different portions of the linker. The linker of the present invention comprises a covalent linking unit connected to the antibody, a stretching unit, a peptide residue, and an optional self-cleaving segment.

[0239] The linker-drug compound of the present invention is connected to the antibody by conventional coupling methods in the art, including: lysine coupling, inter-light and heavy chain reductive disulfide bond coupling, and directional coupling. The present invention preferably uses inter-light and heavy chain reductive disulfide bond coupling, that is, one or more of the inter-light and heavy chain disulfide bond sites (two sites between the heavy chain and two sites between the heavy and light chains) are reduced to form a thiol group (sulfur atom of a cysteine ​​residue) for connection. In the present invention, the connecting portion of the linker L and the antibody is represented by L1, which is formed by the reaction of the L1' group in the linker compound with the antibody, for example or Wherein, * indicates connection with the sulfhydryl group of the antibody, and ** indicates connection with the spacer.

[0240] The term "extender unit" refers to the linking group between the covalent linking unit in the linker L and the polypeptide sequence, which can be any divalent organic group, such as a chemical bond, C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof; the C 1-10 Alkylene, C 2-10 Alkenylene, C2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group or a combination of two or more thereof and optionally interrupted by carbonyl, O, S, or N atoms; the C 1-10 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-10 Cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic group may be optionally replaced by C 1-6 Alkyl, C 3-6 Cycloalkyl, halogen atom, halogenated C 1-6 Alkyl substitution; preferably, the stretching unit comprises a hydrophilic segment substitution or insertion; preferably, the stretching unit is as defined in L2.

[0241] The term "peptide residue" is well known in the art and is selected from a divalent peptide group comprising 2 to 8 optionally substituted proteinogenic or non-proteinogenic amino acids, L-form or D-form amino acid residues, each of which is the same or different and is independently selected from the following amino acid residues: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valeric acid (Nva), norleucine (Nle) or analogs of the above amino acids. The peptide residues of the present invention may include substituted or derivatized amino acid residues, for example, amino acid residues whose side chain amino groups are specifically substituted as defined in CN115279417A, such as:

[0242] The term "self-cleaving fragment" is well known in the art, such as the p-aminobenzyl carbonate fragment commonly used in the art. In a specific embodiment of the present invention, the "self-cleaving fragment" comprises a modification of the "hydrophilic fragment".

[0243] The term "hydrophilic segment" is well known in the art, and the hydrophilic segment may comprise a polyethylene glycol group, such as a polyethylene glycol group with a methoxy terminal group, or further connected to other hydrophilic segments via polyethylene glycol groups. The hydrophilic segment may also comprise a polyamino acid segment, such as polyglycine or polysarcosine, which may be combined with a polyethylene glycol group. The hydrophilic segment may also be a monosaccharide, a disaccharide or an oligosaccharide, which may be a sugar in the form of a chain molecule or a cyclic molecule, and may comprise a glycosamine, a sugar acid or a phosphate sugar. Preferably, the sugar group is combined with a polyethylene glycol segment or a polyamino acid segment; further preferably, at least two sugar groups are introduced via a multivalent linking group, such as a linking group based on aspartic acid, glutamic acid or lysine. It may also comprise a polycarboxylic acid group, a polysulfonic acid group or a chelating group, etc.; the structure of the polycarboxylic acid group may be as follows:

[0244] The structure of the polysulfonic acid group is as follows:

[0245] The chelating group may be, for example, a DOTA group or a NOTA group.

[0246] The term "pharmaceutical composition" as used herein refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that is combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the ingredients contained in the pharmaceutical composition (e.g., antibodies, nucleic acid molecules, nucleic acid molecule combinations and / or conjugates according to the present invention) can be administered to an individual as a whole, or separately. When the ingredients contained in the pharmaceutical composition are administered to an individual separately, the ingredients can be administered to the individual simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or a polyalkylene glycol such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used depends, in particular, on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present invention may contain a wetting agent, an emulsifier or a buffer substance as an additive.The pharmaceutical composition or pharmaceutical preparation according to the present invention may be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

[0247] As used herein, the term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show benefit to the individual to whom it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the individual condition and severity of the condition being treated. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and is relied upon by general practitioners and other physicians, generally taking into account the condition being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0248] EC 50 EC50 refers to the concentration of a drug, antibody, or toxin that achieves 50% of its maximum biological effect after a specific exposure time. In pharmacy, in addition to characterizing the activation capacity of an agonist in vitro, it can also be used to indicate the blood concentration required to achieve half of its maximum biological effect in vivo. In some literature, EC50 is also used to characterize the potency of a compound at the cellular level (including both agonist and antagonist effects). EC50 values ​​can be determined using methods such as ELISA.

[0249] The term "fusion protein," as used herein, generally refers to a protein composed of at least two domains that are not naturally associated and are encoded by separate genes that are linked and transcribed and translated as a single protein. In the technical context of the present invention, a "fusion protein" comprising an antibody or antigen-binding fragment refers to a product obtained by fusing an antibody or antigen-binding fragment with another biologically active protein using genetic engineering techniques. Such antibody fusion proteins possess both the antigen-binding ability of an antibody and the unique biological properties of the biologically active protein to which it is fused. For example, fusion with an albumin-binding fragment can extend the in vivo half-life of the antibody.

[0250] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed fusion polypeptide that generally includes an antigen binding region (e.g., an antibody or its antigen binding portion), a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. CAR can utilize the antigen binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to the selected target in a non-MHC restricted manner. In the present invention, the antigen binding region can use the ScFv form of the antibody or bispecific antibody of the present invention.

[0251] The term "identity / homology / identity" with respect to amino acid or nucleic acid sequences is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment and introduction of gaps, if necessary, to achieve the maximum percentage identity. Methods and computer programs for alignment are well known in the art.

[0252] As used herein, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors can be benign, pre-malignant, or malignant.

[0253] As used herein, the term "malignancy" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Exemplary malignancies include carcinomas, solid tumors, melanomas, sarcomas, hematological tumors, germ cell tumors, and blastomas. More specific examples of malignancies include multiple myeloma, renal cancer, lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, gastric cancer including gastrointestinal cancer, prostate cancer, pancreatic cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, ovarian cancer, liver cancer, urinary tract cancer, hepatoma, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma and B-cell lymphoma, brain cancer and head and neck cancer and related metastases.

[0254] As used herein, the term "hematologic malignancy" refers to malignant malignancies caused by the uncontrolled growth and proliferation of abnormal cells. In most cases, the origin of these abnormal cells is the bone marrow, which is where blood cells are produced. Exemplary hematologic malignancies include various types of leukemias, multiple myeloma, and malignant lymphomas. More specific examples of hematologic malignancies include acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's leukemia, and adult T-cell leukemia, non-Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia ( macroglobulinemia), splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, B-cell chronic lymphocytic lymphoma, classical Hodgkin lymphoma, Nodular lymphocyte-predominant Hodgkin lymphoma, adult T-cell lymphoma, extranodal nasal NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Say-Sher syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disease, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, unspecified peripheral T-cell lymphoma, and anaplastic large cell lymphoma.

[0255] The term "solid tumor" as used herein refers to a tangible mass that can be felt through clinical examinations such as X-rays, CT scans, B-ultrasound or palpation. Solid tumors that have been clinically diagnosed and treated are divided into malignant and benign types. Malignant solid tumors include: Childhood Hodgkin's lymphoma: lymphocyte-predominant, nodular sclerosis, mixed cell, and lymphocyte-depleted types; Childhood non-Hodgkin's lymphoma: prelymphoblastic lymphoma, small non-cleaved cell lymphoma (Burkitt's / non-Burkitt's lymphoma), diffuse large B-cell lymphoma, and anaplastic large cell lymphoma; Childhood kidney tumors: Wilms' tumor, renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, and renal primitive neuroectodermal tumor; Childhood neuroblastoma: neuroblastoma, ganglioneuroblastoma, and ganglioneuroma; Childhood extracranial germ cell tumors: mature teratoma, immature teratoma, endodermal sinus tumor (yolk sac tumor), seminoma, dysgerminoma, choriocarcinoma, and embryonal carcinoma. Osteosarcoma and chondrosarcoma; childhood rhabdomyosarcoma: embryonal, alveolar, and pleomorphic; childhood soft tissue sarcomas: fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant neurilemoma, alveolar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, synovial sarcoma, and desmoplastic small round cell tumor; Ewing family sarcomas: Ewing sarcoma and primitive neuroectodermal tumor; childhood liver tumors: hepatoblastoma (embryonal, fetal, and undifferentiated), hepatocellular carcinoma; retinoblastoma; other tumors: posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecal carcinoid, and mesothelioma. Benign solid tumors include lymphangioma, hemangioma, and thyroglossal duct cyst. DETAILED DESCRIPTION

[0256] Example 1. Acquisition of anti-human DLL3 monoclonal antibodies

[0257] Healthy BALB / c mice were immunized with recombinant human DLL3 protein (purchased from Acro, catalog number DL3-H52H4). After the initial immunization, booster immunizations were performed every 14 days for a total of 4 immunizations. The mouse serum titer was detected by flow cytometry, and mice with high serum antibody titers were selected for cell fusion. Three days before fusion, recombinant human DLL3 protein was injected into the tail vein for shock immunization. On the day of fusion, the mice were euthanized, and the spleens of the mice were removed under sterile conditions and prepared into a single cell suspension. SP2 / 0 cells and spleen cells were mixed in a 1:1 ratio and fused by electrofusion. The fused cells were plated in a 96-well cell culture plate, cultured in a 37°C, 5% CO2 incubator, and screened using HAT culture medium.

[0258] Depending on cell growth, hybridoma supernatants were tested by ELISA after approximately 10 days. Recombinant human DLL3 protein was used for preliminary screening. Positive clones were rescreened with human DLL3 (purchased from Acro, Catalog No. DL3-H525x), cynomolgus macaque DLL3 (purchased from Acro, Catalog No. DL3-C52H3), mouse DLL3 (purchased from Acro, Catalog No. DL3-M52H9), human DLL3-B (purchased from Acro, Catalog No. DL3-H52Hy), or human DLL3-C (purchased from Acro, Catalog No. DL3-H52Hz). Five high-affinity hybridoma cell lines (22B12E5, 25G10A9, 31B6G8, 33B10E8, and 44B8H2) were selected for sequencing. By extracting RNA from these five cell lines, reverse transcription, and PCR amplification, the amino acid sequences of the variable regions of the heavy and light chains of the mouse antibodies were finally obtained. The results are as follows:

[0259] 22B12E5

[0260] Heavy chain CDR1: DYNMY SEQ ID NO.1

[0261] Heavy chain CDR2: YIDPYNGVTGFNQKFKG SEQ ID NO.2

[0262] Heavy chain CDR3: GQGGYYIDYAMDY SEQ ID NO.3

[0263] Heavy chain variable region sequence SEQ ID NO.7

[0264] Light chain CDR1: RASQDIGDTLA SEQ ID NO.4

[0265] Light chain CDR2: ATSNLDS SEQ ID NO.5

[0266] Light chain CDR3: LQYAISPYT SEQ ID NO.6

[0267] Light chain variable region sequence SEQ ID NO.8

[0268] 25G10A9

[0269] Heavy chain CDR1: TYGMS SEQ ID NO.9

[0270] Heavy chain CDR2: TIHSDGGDTYYPDSVKG SEQ ID NO.10

[0271] Heavy chain CDR3: QTGPWLLTY SEQ ID NO.11

[0272] Heavy chain variable region sequence SEQ ID NO.15

[0273] Light chain CDR1: KASQAVNTAVA SEQ ID NO.12

[0274] Light chain CDR2: WASTRHT SEQ ID NO.13

[0275] Light chain CDR3: QQHYSTPYT SEQ ID NO.14

[0276] Light chain variable region sequence SEQ ID NO.16

[0277] 31B6G8

[0278] Heavy chain CDR1: NYGMK SEQ ID NO. 17

[0279] Heavy chain CDR2: WINTYTGEPTYADDFKG SEQ ID NO. 18

[0280] Heavy chain CDR3: FSDYGGFDY SEQ ID NO.19

[0281] Heavy chain variable region sequence SEQ ID NO.23

[0282] Light chain CDR1: KASQSVSNDVA SEQ ID NO.20

[0283] Light chain CDR2: YASNRYT SEQ ID NO.21

[0284] Light chain CDR3: QQDYSSPFT SEQ ID NO. 22

[0285] Light chain variable region sequence SEQ ID NO.24

[0286] 33B10E8

[0287] Heavy chain CDR1: DYYMK SEQ ID NO. 25

[0288] Heavy chain CDR2: AFNLNNGDTFYNQKFKG SEQ ID NO. 26

[0289] Heavy chain CDR3: DVYGYGDY SEQ ID NO.27

[0290] Heavy chain variable region sequence SEQ ID NO.31

[0291] Light chain CDR1: RASKSVSTSGYSYMH SEQ ID NO. 28

[0292] Light chain CDR2: LASNLDS SEQ ID NO. 29

[0293] Light chain CDR3: QHSRELPYT SEQ ID NO.30

[0294] Light chain variable region sequence SEQ ID NO.32

[0295] 44B8H2

[0296] Heavy chain CDR1: NYWMH SEQ ID NO. 33

[0297] Heavy chain CDR2: AIYPGNSNTAYNQKFKG SEQ ID NO.34

[0298] Heavy chain CDR3: DGYYGFAY ​​SEQ ID NO.35

[0299] Heavy chain variable region sequence SEQ ID NO.39

[0300] Light chain CDR1: KASQNVGTNVA SEQ ID NO. 36

[0301] Light chain CDR2: SASYRYS SEQ ID NO. 37

[0302] Light chain CDR3: QQYKNYPYT SEQ ID NO. 38

[0303] Light chain variable region sequence SEQ ID NO.40

[0304] Note: In the above antibody sequences, the underlined CDR sequences are determined and annotated according to the Kabat numbering system.

[0305] Example 2. Fortebio detects the affinity of chimeric antibodies to recombinant human DLL3 protein

[0306] The amino acid sequences of the heavy and light chain variable regions of the murine antibody obtained in Example 1 were used to construct human-mouse chimeric antibodies, with hIgG1 / Kappa being used as the constant region, and L234A / L235A / P329A / P331S (numbered according to the EU index of Kabat) amino acid mutations introduced into the Fc sequence.

[0307] Using a ProA biosensor, a DLL3 chimeric antibody was immobilized to a binding threshold of 0.3 nm. After a 120-second baseline step, the sensor was immersed in recombinant human DLL3 protein diluted in 0.02% PBST buffer. The protein concentration started at 100 nM and was then diluted three-fold over seven steps. The association and dissociation times were 120 and 300 seconds, respectively. The binding curves were analyzed using regression analysis software to calculate the antigen-antibody affinity. All antibodies exhibited comparable affinity to Rovalpituzumab (purchased from Bio-Information, B717301). The results are shown in Table 1.

[0308] Table 1: Fortebio's affinity test for chimeric antibodies and recombinant human DLL3 protein

[0309] Example 3. Cross-reactivity of antibodies binding to human, monkey, and mouse DLL3 proteins

[0310] To determine the cross-species reactivity of the antibodies, ELISA was used to determine their affinity for recombinant human, cynomolgus macaque, and mouse DLL3 proteins. Recombinant human, cynomolgus macaque, and mouse DLL3 proteins were diluted to a final concentration of 0.5 μg / ml and plated onto 96-well microtiter plates at 4°C overnight. The supernatant was discarded the next day. After blocking, the plates were then incubated with secondary antibodies and developed with TMB colorimetric buffer for 10 minutes. The reaction was terminated with 2 M HCl, and the absorbance was read at 450 nm using a microplate reader. The affinities obtained by GraphPad fitting are shown in Figures 1, 2, and 3 and Table 2. The results show that the five chimeric antibodies bind to human and cynomolgus macaque DLL3 proteins with affinities comparable to those of rovalpituzumab. 25G10A9 binds weakly to mouse DLL3, while the remaining chimeric antibodies bind to mouse DLL3 with comparable affinity to rovalpituzumab.

[0311] Table 2 Binding ability of chimeric antibodies to human, monkey and mouse DLL3 proteins

[0312] Example 4. Cross-reactivity of chimeric antibodies with human DLL1 and human DLL4 proteins

[0313] The obtained chimeric antibodies were tested for their binding ability to human DLL1 (purchased from Acro, Catalog No. DL1-H52H8) and human DLL4 (purchased from Acro, Catalog No. DL4-H5227) proteins by ELISA. Human DLL1 and human DLL4 proteins were diluted to a final concentration of 0.5 μg / ml and plated onto 96-well ELISA plates. After blocking, 4-fold serial dilutions of antibodies (0-17.1 nM) were added and reacted at room temperature for 1 hour. The supernatant was discarded, and the plates were washed five times with PBST (1‰ Tween 20). HRP-labeled goat anti-human secondary antibody was added and incubated at room temperature for 1 hour. After washing, TMB color development solution was added for 10 minutes. After terminating the reaction with 2M HCl, the absorbance at 450 nm was read on a microplate reader. The results, as shown in Figures 4A-4B, show that none of the five chimeric antibodies had significant cross-reactivity with human DLL1 and human DLL4 proteins.

[0314] Example 5 Epitope Detection of Chimeric Antibodies

[0315] 5.1 ELISA detection of chimeric antibody recognition epitope

[0316] The binding ability of chimeric antibodies to different DLL3 truncations was determined by ELISA. Human DLL3-B and DLL3-C proteins were diluted to 0.5 μg / ml and coated on an ELISA plate. After blocking, antibodies were added in a 4-fold serial dilution (0-17.1 nM) format. After incubation with secondary antibodies for 1 hour, the plates were washed and the colorimetric readings were obtained. The affinity data obtained after GraphPad fitting are shown in Figures 5A-5B and Table 3. The results showed that the chimeric antibodies 31B6G8 and rovalpituzumab bound to DLL3-B but not to DLL3-C, suggesting that the epitopes recognized by 31B6G8 and rovalpituzumab may be the same. The chimeric antibodies 22B12E5, 33B10E8, 44B8H2, and 25G10A9 bound to both DLL3-B and DLL3-C, suggesting that the epitopes recognized by rovalpituzumab are different.

[0317] Table 3. EC values ​​of chimeric antibodies binding to DLL3-B and DLL3-C proteins detected by ELISA 50 value

[0318] 5.2 Octet Epitope Pairing Detection Antibody Recognition Epitope

[0319] To further define the epitope classification of the antibodies, we used Octet epitope matching to determine the epitope classification of the chimeric antibodies 22B12E5, 33B10E8, 31B6G8, and rovalpituzumab. Human DLL3 protein was loaded onto a HIS1K sensor at 5 μg / ml and then exposed to the primary and secondary antibodies, respectively. The secondary antibody binding signal was used to determine whether the two antibodies recognized the same epitope. The data were processed using ForteBio's data analysis software 7.0. As shown in Figure 5C, 22B12E5 and 33B10E8 recognized completely different epitopes from rovalpituzumab, while 31B6G8 recognized the same epitope as rovalpituzumab. The epitopes recognized by 22B12E5, 33B10E8, and 31B6G8 were distinct.

[0320] Example 6 Binding of Chimeric Antibodies to NCI-H82 Cells

[0321] The human small cell lung cancer cell line NCI-H82 was used to evaluate the ability of the chimeric antibody to bind to cell surface DLL3. 6 Cells were plated at a density of 100 μl / well in a 96-well plate. Antibody was added at 137 nM and incubated at 4°C for 1 hour. A goat anti-human secondary antibody was added and labeled at 4°C in the dark for 45 minutes. Cells were harvested, washed, and analyzed by flow cytometry for fluorescence intensity. The results are shown in Table 4. The chimeric antibody binds to the DLL3 protein expressed on NCI-H82 cells, with higher values ​​than rovalpituzumab.

[0322] Table 4. Chimeric antibody binding assay to DLL3 on NCI-H82 cell surface

[0323] Example 7 Detection of endocytic activity of anti-DLL3 antibodies

[0324] The internalization rate of DLL3 chimeric antibody was determined by FACS, and the ability of chimeric antibody to induce cell surface DLL3 internalization was evaluated using SHP77 cell line. 6 / ml, 100 μl / well were seeded in a 96-well plate. Antibody was added at a final concentration of 34.2 nM and incubated at 4°C for 1 hour. After washing the cells with FACS buffer, the cells were incubated at 4°C and 37°C for 2 hours. Goat anti-human secondary antibody was added and labeled at 4°C in the dark for 45 minutes. After collecting and washing the cells, the fluorescence intensity of the cells was measured by flow cytometry. The internalization ratio was calculated as [(4°C reading - negative control reading) - (37°C reading - negative control reading)] / (4°C reading - negative control reading) × 100%.

[0325] The calculated antibody 2h internalization ratio is shown in Table 5. The results show that the chimeric antibody has good endocytic activity.

[0326] Table 5. Chimeric antibody 2h internalization ratio

[0327] Example 8 Binding of DLL3 Antibody-Drug Conjugates to Human DLL3-Expressing Cells

[0328] JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) was conjugated to chimeric antibodies 22B12E5, 31B6G8-ADC, 33B10E8, 44B8H2, and Rovalpituzumab using the cysteine ​​conjugation method to yield 22B12E5-ADC, 31B6G8-ADC, 33B10E8-ADC (hereinafter referred to as Cab33-ADC), 44B8H2-ADC, and Rova-ADC (positive control), respectively. The method involved adding TCEP (J&K Technology Co., Ltd.) to the DLL3 chimeric antibody and Rovalpituzumab at a molar ratio of 1:10, followed by incubation at 37°C for 2 hours. JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) was then added to the above solution at a molar ratio of 1:12, followed by incubation at 25°C for 30 minutes. Next, N-ethylmaleimide (B&K Technology Co., Ltd.) was added at a molar ratio of 1:14, and the reaction was carried out at room temperature for 20 minutes to terminate the coupling reaction. The above-mentioned antibody-drug conjugate was purified by affinity chromatography using an AKTA purifier to obtain the coupled antibody-drug conjugate (ADC) molecule, which was exchanged into PBS buffer. Rova-ADC, 22B12E5-ADC, 31B6G8-ADC, 33B10E8-ADC, and 44B8H2-ADC were all normally coupled to the JSSW-001 molecule by SEC\LC-MS and other tests. The free small molecule content of the coupled ADC was less than 0.0015%, the SEC purity was greater than 95%, and the DAR value of the coupling was about 8. The DAR value and free small molecule content of the coupled ADC met the requirements.

[0329] The ability of the antibody-drug conjugate to bind to cell surface DLL3 was evaluated using the cancer cell line NCI-H82. 6Cells were plated at a density of 100 μl / well in a 96-well plate. Antibody was added at 137 nM and incubated at 4°C for 1 hour. A goat anti-human secondary antibody was added and labeled for 45 minutes at 4°C in the dark. Cells were harvested, washed, and analyzed for fluorescence intensity by flow cytometry. The results are shown in Table 6. The ability of the DLL3 antibody-drug conjugates to bind to DLL3 on the surface of NCI-H82 cells was essentially the same as that of the corresponding chimeric antibodies. With the exception of the 31B6G8-ADC, all other ADCs were superior to the Rova-ADC.

[0330] Table 6. Binding experiments of DLL3 antibody-drug conjugates to NCI-H82 cells

[0331] Example 9 In vitro cytotoxicity evaluation of ADC samples

[0332] Small cell lung cancer SHP77 and NCI-H82 cells with high DLL3 expression were selected as cell lines for in vitro activity detection to observe the dose-effect of different antibody-drug conjugates on cell killing. 4 / ml density, 50μl / well inoculated in 96-well plates. Add 3-fold serial dilution of antibody to a final concentration of 0-400nM, incubate for 6 days and then add Cell Titer Luminescent Cell Viability Assay Reagent was shaken at room temperature for 10 minutes to mix thoroughly. The data was read by a microplate reader and the survival rate of the whole cell group without drug was taken as 100%. The survival rate = (test group / blank control group) × 100% and the IC was calculated. 50 The results are shown in Figures 6A-6B and Table 7. The results show that the ADCs disclosed in the present invention have significant in vitro cell killing effects on cells with high DLL3 expression, and 33B10E8 and 22B12E5 have the strongest in vitro cytotoxic activity.

[0333] Table 7. Killing experiments of DLL3 antibody-drug conjugates

[0334] Example 10 Biological Activity of DLL3 Antibody-Drug Conjugates in Animals

[0335] 10.1 In vivo antitumor activity of ADC drugs in the NCI-H82 model

[0336] The monotherapy efficacy of chimeric antibody ADC molecules 31B6G8-ADC (abbreviated as 31B6G8 in Figure 7 and Table 8), 22B12E5-ADC (abbreviated as 22B12E5 in Figure 7 and Table 8), 33B10E8-ADC (abbreviated as 33B10E8 in Figure 7 and Table 8), and Rova-ADC (abbreviated as PC(ROVA) in Figure 7 and Table 8) were evaluated in tumor xenograft models. 6 NCI-H82 human small cell lung cancer cells were inoculated into Nu / nu mice. When the tumor volume reached 100-150 mm 3 At 4 hr, 2 mg / kg of 31B6G8-ADC, 22B12E5-ADC, 33B10E8-ADC, and Rova-ADC were intravenously injected, along with a 0.9% sodium chloride injection control (0.9% INJ NS), for a total of one dose. Tumor volume was calculated at the end of the trial. Calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter). Compared with the solvent group, all groups inhibited tumor growth, and 22B12E5-ADC significantly inhibited the volume of the transplanted tumor (P < 0.05). See Figure 7 and Table 8 for details.

[0337] Table 8. Tumor parameters of each group after 19 days of administration of DLL3-ADC (chimeric antibody)

[0338] 10.2 In vivo antitumor activity of ADC drugs in the SHP77 model

[0339] The monotherapy efficacy of chimeric antibody ADC molecules 31B6G8-ADC (abbreviated as 31B6G8 in FIG8 and Table 9), 22B12E5-ADC (abbreviated as 22B12E5 in FIG8 and Table 9), 33B10E8-ADC (abbreviated as 33B10E8 in FIG8 and Table 9), 44B8H2-ADC (abbreviated as 44B8H2 in FIG8 and Table 9), and Rova-ADC (abbreviated as PC(ROVA) in FIG8 and Table 9) were evaluated in tumor xenograft models. 7 SHP77 cells were inoculated into NCG mice. When the tumor volume reached 100-150 mm 3At 1 mg / kg, 31B6G8-ADC, 22B12E5-ADC, 33B10E8-ADC, 44B8H2-ADC, and Rova-ADC were intravenously injected once, along with a 0.9% sodium chloride injection as a control. The in vivo anti-tumor effect of DLL3-ADC was investigated. At the end of the trial, compared to the vehicle group, the 31B6G8-ADC group showed a 63.8% tumor inhibition and a tumor volume of 726.9±237.8, the 22B12E5-ADC group showed a 76.4% tumor inhibition and a tumor volume of 519.3±156.8, the 33B10E8-ADC group showed a 79.5% tumor inhibition and a tumor volume of 466.7±115.9, the 44B8H2-ADC group showed a 57.3% tumor inhibition and a tumor volume of 834.7±150.5, and the Rova-ADC (positive control) group showed a 23.0% tumor inhibition and a tumor volume of 1408.7±183.6. With the exception of the Rova-ADC (positive control), all other groups significantly inhibited the volume of the transplanted tumors (P<0.05). See Figure 8 and Table 9 for details.

[0340] Table 9. Tumor parameters of each group after 19 days of administration of DLL3-ADC (chimeric antibody)

[0341] Example 11 Obtaining DLL3 Bi-epitope Antibodies

[0342] Through epitope analysis, 22B12E5, 31B6G8, and 33B10E8 were found to bind distinct epitopes. Their variable regions were used to construct bi-epitope molecules, employing a symmetrical 1:1 structure. The heavy chain was constructed using the knob-in-hole technique to generate two different heavy chains, while the light chain was constructed using Crossmab technology. The constant region employed hIgG1 / kappa, and the Fc sequence was modified with amino acid mutations L234A / L235A / P329A / P331S (numbering according to the EU Kabat index). This resulted in three bi-epitope antibody molecules: 33B10E8 + 22B12E5 (33 + 22), 31B6G8 + 22B12E5 (31 + 22), and 33B10E8 + 31B6G8 (33 + 31). The antibody structures are shown in Figure 9 and the sequences are shown in Table 10.

[0343] Table 10. Sequences of dual-complementary epitope antibodies

[0344] Example 12 DLL3 bi-epitope antibody affinity detection

[0345] First, the affinity of the bi-epitope antibody for recombinant human, cynomolgus macaque, and mouse DLL3 proteins was determined by ELISA. DLL3 protein was diluted to 5 μg / mL and coated on an ELISA plate. After blocking, a 4-fold serial dilution of the antibody (0-13.4 nM) was added. After incubation with the secondary antibody, the color was developed and the results were read. The affinities obtained by GraphPad fitting are shown in Figures 10A-10C and Table 11.

[0346] Table 11. EC of ELISA for detection of affinity of bicomplementary epitope antibodies 50 value

[0347] Next, we used Fortebio to test the affinity of the bi-epitope antibodies. The experimental method was the same as in Example 2. The results are shown in Table 12. The above results indicate that the affinity of all bi-epitope antibodies is comparable to or better than that of Rovalpituzumab.

[0348] Table 12: Fortebio's affinity test for bi-epitope antibodies to human DLL3 protein

[0349] Example 13 Binding of bi-epitope antibodies to cells expressing human DLL3

[0350] SHP77 cells were used to evaluate the binding ability of the bi-epitope antibody to cell surface DLL3. The cells were cultured at 2×10 6 The cells were plated at a density of 100 μl / well in a 96-well plate. 137 nM of antibody was added and incubated at 4°C for 1 hour. A goat anti-human secondary antibody was added and the cells were labeled for 45 minutes at 4°C in the dark. Cells were harvested, washed, and then analyzed for fluorescence intensity using a flow cytometer. The results are shown in Table 13, demonstrating that the bi-epitope antibody binds better to cells overexpressing DLL3 than the control molecule, rovalpituzumab.

[0351] Table 13. Binding detection of dual-epitope antibodies to cell surface DLL3 protein

[0352] Example 14 Endocytic activity of bi-epitope antibodies

[0353] The internalization rate of DLL3 bi-epitope antibody was determined by FACS, and the ability of bi-epitope antibody to induce cell surface DLL3 internalization was evaluated using SHP77 cells. 6100 μl / well of the culture medium was plated at a density of 100 μl / ml in a 96-well plate. 34.2 nM antibody was added and incubated at 4°C for 1 hour. After washing with FACS buffer, the cells were incubated at 4°C and 37°C for 2 hours, respectively. Goat anti-human secondary antibody was added and labeled at 4°C in the dark for 45 minutes. Cells were harvested, washed, and then measured for fluorescence intensity using a flow cytometer. The internalization ratio was calculated as [(4°C reading - negative control reading) - (37°C reading - negative control reading)] / (4°C reading - negative control reading) × 100%.

[0354] The calculated 2h endocytosis ratio of the antibodies is shown in Table 14. The results showed that the dual-epitope antibody had good endocytosis activity, and the endocytosis ability of 31+22 was better than that of 33+31, 33+22 and Rovalpituzumab.

[0355] Table 14. DLL3 dual-epitope antibody 2h endocytosis experiment

[0356] Example 15 Cytotoxicity Study of DLL3 Bi-epitope Antibody-Drug Conjugate

[0357] JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) was conjugated to the bi-epitope antibody using the cysteine ​​conjugation method, as in Example 8, to obtain 33+31ADC, 31+22ADC, and 33+22ADC, respectively. The DAR value of the conjugation was 8. SHP77 cells with high DLL3 expression were selected as the cell line for in vitro activity detection, as in Example 9. After the data were read by the microplate reader, the survival rate of the whole cell group without drug addition was taken as 100% for data processing and calculation of EC 50 The results are shown in Figure 11 and Table 15. The results showed that the dual-epitope ADC had a significant in vitro cell killing effect on DLL3 high-expressing cells SHP77, EC 50 The value reaches pM level.

[0358] Table 15. Killing experiments of DLL3 antibody-drug conjugates

[0359] Example 16 Humanization of DLL3 Antibody

[0360] Using Discovery Studio and Antibody Modeling was performed for homology modeling. Through structural simulation and rational design, the human framework region closest to the mouse antibody framework region was obtained. The CDRs of the light and heavy chains were transplanted onto the framework sequences of the matching human light and heavy chain genes, respectively, to obtain humanized antibodies Hab31B6G8, Hab33B10E8, and Hab22B12E5. Then, the humanized antibodies Hab31B6G8, Hab33B10E8, and Hab22B12E5 were obtained through Discovery Studio and Antibody Modeling: A 3D model was constructed and analyzed to determine if any framework positions where mouse amino acids were substituted with human amino acids affected binding and / or CDR conformation. Back mutations were performed and the resulting sequences are shown in Table 16.

[0361] Table 16. Sequences with VH backmutations (CDR numbering is Kabat numbering)

[0362] Example 17 Affinity detection of humanized anti-DLL3 antibodies

[0363] The humanized antibody variable region obtained in Example 16 was combined with the human IgG1 / kappa constant region to construct a complete antibody for expression and purification. The amino acid mutations L234A / L235A / P329A / P331S (numbering according to the EU Kabat index) were introduced into the Fc sequence. The affinity of the humanized antibody for human DLL3 protein was tested using Fortebio, using the same method as in Example 2. The antigen-antibody affinity data was calculated by regression analysis based on the binding curves. The results are shown in Table 17.

[0364] Table 17: Fortebio's affinity test for humanized antibodies and human DLL3 protein

[0365] Next, the affinity of the humanized antibody to recombinant human, cynomolgus monkey, and mouse DLL3 proteins was tested by ELISA. The affinity obtained after GraphPad fitting is shown in Figures 12A-12C and Table 18.

[0366] Table 18. EC values ​​of humanized antibody affinity detected by ELISA 50 value

[0367] The above results indicate that the humanized antibodies Hab33B10E8 and Hab31B6G8 exhibited affinities that were substantially equivalent to those of Rovalpituzumab.

[0368] Example 18 Binding of humanized antibodies to cells expressing human DLL3

[0369] The ability of humanized antibodies to bind to cell surface DLL3 was evaluated using SHP77 / NCI-H82 cells. 6The cells were plated at a density of 100 μl / well in a 96-well plate. 66.7 nM of antibody was added and incubated at 4°C for 1 hour. A goat anti-human secondary antibody was added and the cells were labeled for 45 minutes at 4°C in the dark. Cells were harvested, washed, and then analyzed for fluorescence intensity using a flow cytometer. The experimental results are shown in Table 19. The results demonstrate that the humanized antibodies bind to DLL3 protein expressed in the cell lines. The Hab33B10E8 antibody showed superior affinity for cell surface DLL3 recognition to the control molecule, rovalpituzumab.

[0370] Table 19. Humanized antibody binding to cell surface DLL3

[0371] Example 19 Detection of endocytic activity of humanized antibodies

[0372] The internalization rate of DLL3 humanized antibody was determined by FACS, and the human small cell lung cancer cell lines SHP77 and NCI-H82 were used to evaluate the ability of humanized antibody to induce cell surface DLL3 internalization. 6 100 μl / well of the culture medium was plated at a density of 100 μl / ml in a 96-well plate. Antibody was added at a final concentration of 34.2 nM and incubated at 4°C for 1 hour. After washing the cells with FACS buffer, the cells were incubated at 4°C and 37°C for 2 hours. Goat anti-human secondary antibody was added and labeled at 4°C in the dark for 45 minutes. Cells were harvested, washed, and then analyzed by flow cytometry for fluorescence intensity. The internalization ratio was calculated as [(4°C reading - negative control reading) - (37°C reading - negative control reading)] / (4°C reading - negative control reading) × 100%.

[0373] The calculated 2h endocytosis ratios of the antibodies are shown in Table 20. The results showed that the endocytosis activity of Hab33B10E8 was better than that of Rovalpituzumab in SHP77 cells, and the endocytosis activities of Hab33B10E8 and Hab22B12E5 were better than those of Rovalpituzumab in NCI-H82 cells.

[0374] Table 20. Humanized antibody 2h endocytosis experiment

[0375] Example 20 In vitro cytotoxicity evaluation of humanized ADC samples

[0376] Based on the same method as in Example 8, JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) was conjugated with humanized antibodies Hab22B12E5, Hab31B6G8, and Hab33B10E8 using the cysteine ​​conjugation method to obtain Hab22B12E5-ADC, Hab31B6G8-ADC, and Hab33B10E8-ADC, respectively. The small cell lung cancer cell line SHP77 with high DLL3 expression was selected for in vitro activity testing to observe the dose-effect of different antibody-drug conjugates on cell killing. The cells were cultured at a rate of 2×10 4 / ml density, 50μl / well inoculated in 96-well plates. Add 3-fold serial dilution of antibody to a final concentration of 0-400nM. After six days of co-incubation, add Cell Titer Luminescent Cell Viability Assay Reagent was shaken at room temperature for 10 minutes to mix thoroughly. The data was read by a microplate reader, and the viability of the whole cell group without drug addition was taken as 100% for data processing and calculation of IC 50 The results are shown in Figure 13 and Table 21. The results show that the humanized ADCs have obvious in vitro cell killing effects on DLL3 high-expressing cells, and Hab33B10E8-ADC and Hab22B12E5-ADC have the strongest in vitro cytotoxic activity.

[0377] Table 21. Killing experiments of DLL3 antibody-drug conjugates Note: NA means EC was not fitted 50 value.

[0378] Example 21 Biological Activity of DLL3 Humanized Antibody-Drug Conjugates in Animals

[0379] 21.1 Efficacy Test of Humanized ADC Samples on Human Small Cell Lung Cancer NCI-H82 Cell Xenografts

[0380] The monotherapy efficacy of Hab22B12E5-ADC, Hab33B10E8-ADC and Rova-ADC (positive control) was evaluated in a tumor xenograft model. 6 NCI-H82 human small cell lung cancer cells were inoculated into NU / NU mice. On day 12 after transplantation, a single intravenous injection of 2 mg / kg of Hab22B12E5-ADC, Hab33B10E8-ADC, and Rova-ADC (positive control) was administered, along with 0.9% sodium chloride injection as a control, to investigate the in vivo anti-tumor activity of the humanized DLL3-ADC.

[0381] At the endpoint, compared to the vehicle group, the Hab22B12E5-ADC group achieved 48.3% tumor inhibition and a tumor volume of 966.0±377.1; the Hab33B10E8-ADC group achieved 86.3% tumor inhibition and a tumor volume of 351.1±216.8; and the Rova-ADC (positive control) group achieved 74.0% tumor inhibition and a tumor volume of 599.1±495.6. Compared to the vehicle group, all groups significantly inhibited the volume of transplanted tumors (P<0.05), with Hab33B10E8-ADC demonstrating superior tumor inhibition compared to the control Rova-ADC molecule. See Figure 14 and Table 22 for details.

[0382] Table 22. Tumor parameters of each group after 14 days of administration of DLL3-ADC (humanized antibody) # The DAR of Rova-ADC (Yangshen) is 7.9

[0383] 21.2 Efficacy Test of Humanized ADC Samples on Human Small Cell Lung Cancer SHP77 Cell Xenografts

[0384] The monotherapy efficacy of Hab22B12E5-ADC, Hab31B6G8-ADC, Hab33B10E8-ADC, chimeric antibody 33B10E8-ADC (Cab33-ADC) and Rova-ADC (Yangshen) was evaluated in a tumor xenograft model. 7 SHP77 human small cell lung cancer cells were inoculated into NCG mice. On day 6 after transplantation, a single intravenous injection of 1 mg / kg of Hab22B12E5-ADC, Hab31B6G8-ADC, Hab33B10E8-ADC, Cab33-ADC, and Rova-ADC (positive control) was administered, along with 0.9% sodium chloride injection as a control. The in vivo anti-tumor activity of the humanized DLL3-ADC was evaluated.

[0385] Compared to the vehicle group, the Hab22B12E5-ADC group resulted in 65.9% tumor inhibition and a tumor volume of 459.1±160.4, the Hab31B6G8-ADC group resulted in 100.6% tumor inhibition and a tumor volume of 101.8±20.5, the Hab33B10E8-ADC group resulted in 91.0% tumor inhibition and a tumor volume of 201.7±102.5, and the Cab33-ADC (chimeric) group resulted in 85.7% tumor inhibition and a tumor volume of 255.1±60.8. The Rova-ADC (positive control) group reduced tumor growth, resulting in 50.8% tumor inhibition and a tumor volume of 613.7±252.1. Compared to the vehicle group, all groups significantly inhibited the volume of the transplanted tumors (P<0.05). All molecules outperformed the control Rova-ADC. See Figure 15 and Table 23 for details.

[0386] Table 23. Tumor parameters of each group after 14 days of administration of DLL3-ADC (humanized antibody) # The DAR of Rova-ADC (Yangshen) is 7.9 ## Cab33-ADC has a DAR of 6.4

[0387] Example 22: Preparation of Antibody Drug Conjugate (ADC) Hab33B10E8-045

[0388] 22.1 Preparation of Compound 045

[0389] 1. Preparation of intermediate compound 18

[0390] Compound 11 (47.5 g, 189 mmol) and methanol (250 mL) were added to a reaction flask. 80% hydrazine hydrate (35.4 g, 567 mmol) was slowly added at room temperature. The temperature was raised to 70°C and the reaction was refluxed for 6 hours. After cooling, white crystals precipitated. The remaining solid was filtered and washed with methanol (20 mL × 3) to obtain a white solid 12 (47.47 g, 100% yield). MS (ESI): (M+H) + Calculated value 252.1, experimental value 252.2.

[0391] Compound 12 (47.4 g, 189 mmol), potassium hydroxide (12.7 g, 227 mmol), and ethanol (400 mL) were added to a reaction flask and stirred at room temperature to dissolve. Carbon disulfide (17 g, 283 mmol) was slowly added, and the temperature was raised to 100°C and refluxed for 5 hours to complete the reaction. After removing the solvent under reduced pressure, water (50 mL) was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The mixture was extracted with dichloromethane (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 13 (49.9 g, 90% yield). MS (ESI): (M+H) + Calculated value 294.1, experimental value 294.3.

[0392] Compound 13 (5.86 g, 20 mmol), triethylamine (2.42 g, 24 mmol), and tetrahydrofuran (36 mL) were added to the reaction flask. Methyl iodide (3.12 g, 22 mmol) was then added to the reaction solution. The reaction was stirred at 25°C for 1.5 hours until completion. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 14 (4.05 g, 66% yield). MS (ESI): (M+H) + Calculated value 308.1, experimental value 308.2.

[0393] Compound 14 (3.3 g, 10.6 mmol) and ethyl acetate (20 mL) were added to a reaction flask, and a solution of hydrogen chloride in ethyl acetate (2.7 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours. The solvent was removed under reduced pressure to obtain a crude product 15, which was directly used for the next reaction.

[0394] The crude product 15, diglycolic anhydride 6 (1.35 g, 11.7 mmol), triethylamine (2.14 g, 21.2 mmol), and tetrahydrofuran (30 mL) were added to a reaction flask and reacted at 25°C for 1.5 hours. The solvent was removed under reduced pressure, and ether was added to the residue. After filtration, the residue was washed with water and ether to obtain compound 17 (3.4 g, yield 99%). MS (ESI): (M+H) + Calculated value 324.1, experimental value 324.2.

[0395] Compound 17 (3.4 g, 10.5 mmol) and glacial acetic acid (20 mL) were added to a reaction flask. After dissolution, potassium permanganate (2.48 g, 15.7 mmol) was added at 0°C and the temperature was returned to 25°C for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless. The mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 18 (2.4 g, yield 65%). MS (ESI): (M+H) + Calculated value 356.0, experimental value 356.0.

[0396] 2. Preparation of Compound 045

[0397] At 0°C, 1-(4-aminophenyl)-3-butyn-1-ol (4997 mg, 31 mmol), N-fluorenylmethoxycarbonyl-L-alanine (9651 mg, 31 mmol), EEDQ (11498 mg, 46.5 mmol), and ultra-dry DCM (90 mL) were added to a reaction flask. After 3 hours of reaction, the solvent was removed under reduced pressure, and the residue was slurried in 500 mL of MTBE to afford compound 72 (9007 mg, 64% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value 455.2, experimental value 455.2.

[0398] Chiral resolution of intermediate 72 (retention time 13.55 min, 16.29 min): Compound 72 (50 g) was separated by SFC to afford 81 (19 g, retention time 13.55 min) and 82 (20 g, retention time 16.29 min). SFC separation method: Column type: DAICEL CHIRALCEL OD (250 mm-50 mm, 10 μm); Mobile phase: A: CO2, B: CO2-ACN / i-PrOH (0.1% NH3H2O); Isocratic elution: B in A for 50%; Flow rate: 200 mL / min; Detector: PDA; Column temperature: 25°C; Back pressure: 100 Bar.

[0399] HPLC method: Instrument information: Thermo liquid chromatograph (ADC-U3000-01); Chromatographic column: ID (4.6*150 mm, 5 μm); column temperature: 25°C; injection plate temperature: 25°C; mobile phase: A: 10 mM NH4FA; B: ACN; flow rate: 0.8 mL / min; detection wavelength: 254 nm; injection volume: 2 μl;

[0400] Gradient conditions:

[0401] The structure and configuration of compound 82 were confirmed by X-RAY. The single crystal diffraction pattern of compound 82 is shown in Figure 16:

[0402] At 0°C, compound 82 (3632 mg, 8 mmol) and 90 mL of commercially available THF were added to a reaction flask. DBU (1215 mg, 8 mmol) was slowly added with stirring, and the reaction was allowed to proceed for half an hour before being allowed to cool to room temperature. After TLC monitoring of the disappearance of the starting material, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to afford compound 83 (1707 mg, 92% yield). MS (ESI): (M+H) + Calculated value 233.1, experimental value 233.2.

[0403] Compound 83 (1624 mg, 7 mmol), Fmoc-Val-OSu (362 mg, 8.4 mmol), and N,N-dimethylformamide (150 mL) were added to a reaction flask, stirred, and DIEA (1158 uL, 7 mmol) was slowly added dropwise. The reaction was continued at 25°C for 12 hours. The solvent was removed under reduced pressure, and 50 mL of EA and 50 mL of PE were added to slurry, resulting in the precipitation of a white solid. This reaction was repeated three times to obtain compound 84. MS (ESI): (M+H) + Calculated value 554.3, experimental value 554.4.

[0404] Compound 84 (1661 mg, 3 mmol), p-nitrophenyl chloroformate (1206 mg, 6 mmol), and THF (150 mL) were added to a reaction flask, stirred, and Py (474 ​​uL, 6 mmol) was added dropwise. The mixture was reacted at 65°C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 98:2) to obtain compound 85 (1831 mg, 85% yield). MS (ESI): (M+H) + Calculated value 719.8, experimental value 719.9.

[0405] Compound 85 (1436 mg, 2 mmol), isotecan mesylate 86 (1168.2 mg, 2.2 mmol), and N,N-dimethylformamide (100 mL) were added to a reaction flask, stirred, and DIEA (695 uL, 4 mmol) was slowly added dropwise. The reaction was continued at 25°C for 20 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to obtain compound 87 (1664 mg, 82% yield). MS (ESI): (M+H) + Calculated value 1015.4, experimental value 1015.1.

[0406] At 0°C, 87 (1522 mg, 1.5 mmol) and 40 mL of commercially available THF were added to a reaction flask. DBU (228 mg, 1.5 mmol) was slowly added with stirring. After half an hour of reaction, the mixture was brought to room temperature and the reaction was continued for 40 minutes until the starting material disappeared on TLC. The reaction solution was concentrated in vacuo to remove THF, and 15 mL of DCM and 300 mL of PE were added to produce a large amount of yellow-green solid. Solid 88 (1177 mg, 99% yield) was filtered out using a Buchner funnel. MS (ESI): (M+H) + Calculated value 793.3, experimental value 793.6.

[0407] Compound 88 (1031 mg, 1.3 mmol), 18 (554 mg, 1.56 mmol), EDCI (498 mg, 2.6 mmol), HOBT (263 mg, 1.95 mmol), and N,N-dimethylformamide (20 mL) were added to a reaction flask and reacted at 25°C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by C18 purification (60% ACN / 0.05% formic acid in H2O) and lyophilized to obtain compound 89 (1322 mg, 90% yield). MS (ESI): (M+H) + Calculated value 1130.4, experimental value 1130.7.

[0408] Compound 89 (903 mg, 0.8 mmol), compound 80 (702 mg, 1.2 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (34.7 mg, 0.08 mmol) and cuprous bromide (11.4 mg, 0.08 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and THF / DMF / H2O (3.5 mL:0.6 mL:0.4 mL) was added. The reaction was continued at 25°C for 0.5 h. The residue was purified by C18 preparative purification (62% ACN / 0.05% formic acid in H2O) to afford compound 045 (1276 mg, 93% yield). MS (ESI): (M+H) + Calculated value 1715.7, experimental value 1716.2. 1H NMR (600MHz, DMSO) δ10.71 (s, 1H), 10.08 (s, 1H), 8.48 (d, J = 6.7Hz, 1H), 8.17 ( d,J=8.6Hz,3H),8.09(d,J=8.8Hz,1H),8.03(d,J=8.7Hz,2H),7.89(s,1H),7. 81(d,J=10.8Hz,1H),7.66(d,J=8.4Hz,2H),7.42–7.36(m,3H),6.61(s,1H),6 .02(t,J=6.8Hz,1H),5.55(s,2H),5.38-5.26(m,3H),4.55–4.47(m,3H),4.46– 4.41(m,1H),4.36(q,J=15.6Hz,2H),4.28(s,2H),3.80(s,5H),3.62–3.55(m, 36H),3.52–3.47(m,9H),3.44–3.36(m,2H),3.30-2.29(m,1H),3.19–3.09(m, 1H),2.42(s,3H),2.28–2.19(m,1H),2.17(s,3H),2.17–2.10(m,1H),2.04–1. 89 (m, 2H), 1.42 (d, J = 7.0Hz, 3H), 1.02 (d, J = 6.7Hz, 3H), 0.98 (t, J = 7.2Hz, 6H).

[0409] 22.2 Preparation of Antibody-Drug Conjugate (ADC) Hab33B10E8-045

[0410] The concentration of antibody Hab33B10E8 was adjusted to 10 g / L using phosphate buffer solution (pH 7.0). The antibody solution after liquid exchange was placed in a centrifuge tube, and 10.0 equivalents of 5 mM TCEP (Adamas-beta, Ltd.) were added and reacted at 37 ° C for 1 hour to reduce the disulfide bonds between the antibody chains to sulfhydryl groups. Subsequently, a 20 mM solution of compound 045 dissolved in DMSO was added (12 equivalents of 045 for each antibody). The reaction was carried out at 25 ° C for 1 hour. After the coupling solution was passed through an ultrafiltration membrane bag (Cobetter, Ltd.) to remove residual linker toxins and stored in PBS solution at pH 7.4. The average drug loading of each antibody was measured by reverse phase chromatography to be approximately 8.

[0411] Example 23: Enzyme-linked immunosorbent assay to determine the binding activity of Hab33B10E8-045

[0412] The affinity of the antibody to the recombinant human DLL3 protein was determined by ELISA. Human DLL3 protein (Kaixia Biotechnology, catalog number: DLL-HM103) was diluted to a final concentration of 0.1 μg / ml and plated onto a 96-well ELISA plate at 4°C overnight. The supernatant was discarded the next day. After blocking, 4-fold serial dilutions of the antibody (0-100 nM) were added. After incubation with the secondary antibody, the plate was developed with TMB colorimetric solution for 10 minutes. After adding 2 M HCl to terminate the reaction, the absorbance at 450 nm was read on a microplate reader to calculate the EC value. 50 As shown in Table 24, the binding affinity of Hab33B10E8-045 to human DLL3 protein was substantially equivalent to that of unconjugated Hab33B10E8.

[0413] Table 24. Binding activity of Hab33B10E8-045 determined by enzyme-linked immunosorbent assay

[0414] Example 24: In vitro cytotoxicity of Hab33B10E8-045

[0415] The in vitro cytotoxicity mediated by Hab33B10E8-045 was evaluated in DLL3-positive cell lines NCI-H82, SHP77, NCI-H526, and NCI-H209, and in DLL3-negative cell lines A431 and NCI-H69. Cells were harvested, incubated with serial dilutions of Hab33B10E8-045, and then incubated at 37°C. Viability was determined after 6 days using CTLPlus. The data were read and analyzed on an EnVision 2105 (PerkinElmer) to determine the IC 50 (half-maximal inhibitory concentration) value.

[0416] Experimental process: The cell density of the test cells was adjusted to 6.7×10 4 / mL, and inoculated into 96-well plates at 75μL per well, and cultured in a 37°C, 5% CO2 incubator overnight. Add 5-fold gradient dilution of Hab33B10E8-045 (0-100nM) for a total of 9 concentration gradients, and add 75μL per well to the cells. The blank control is the culture medium of the corresponding cells. Set up 2 replicates for each concentration. After the cells were cultured in a 37°C, 5% CO2 incubator for 6 days, 50μL CTLPlus (manufacturer: Biyuntian, product number: C0068XL) luminescent reagent was added and incubated in the dark for 10 minutes at room temperature. Chemiluminescence detection was performed on the enzyme reader EnVision 2105. Data analysis and collation: The blank control was used as the zero killing control, and the inhibition rate was calculated as follows: Inhibition rate (%) = (1-test group / blank control group) × 100%. Use GraphPad Prism to process and analyze data and calculate IC 50The results are shown in Table 25. The results showed that Hab33B10E8-045 had good in vitro cytotoxicity against DLL3-positive cell lines.

[0417] Table 25. In vitro cytotoxicity of Hab33B10E8-045

[0418] Example 25: Pharmacodynamic evaluation of Hab33B10E8-045 in a human small cell lung cancer SHP77 xenograft mouse model

[0419] 10 cells were collected from the DLL3-positive cell line SHP77 7 The cells were inoculated into the right forelimb axilla of nu / nu nude mice (7 mice per group). When the average tumor volume of the mice reached 100 mm 3 The mice were randomly divided into groups and the drug was administered by a single intravenous injection. The day of grouping was set as day 0, and drug administration began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter), the pharmacodynamic evaluation of the test drug is shown in Table 26.

[0420] Table 26. Pharmacodynamic evaluation of the experimental drugs in the lung cancer xenograft SHP-P77 mouse model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1-(Ti–T0) / (Ci-C0)] × 100, where T0 and C0 are the mean tumor volumes of the drug-treated group and the vehicle control group on the day of grouping (Day 0), and Ti and Ci are the mean tumor volumes of the drug-treated group and the vehicle control group on Day 22, respectively; c. Compared with the tumor volume of the vehicle control group;

[0421] Compared with the vehicle control group, the ADC of the present invention significantly inhibited tumor growth in the SHP-77 model at a dose of 1 mg / kg, with a tumor growth inhibition rate (TGI) of 98.1%. There was no animal mortality or significant weight loss in the treatment group, and no obvious drug toxicity was observed. During the treatment period, the mice tolerated the ADC of the present invention well.

[0422] Example 26: Pharmacodynamic evaluation of Hab33B10E8-045 in human small cell lung cancer NCI-H526 and NCI-H69 xenograft mouse models

[0423] 10 cells were collected from the DLL3-positive cell line NCI-H5267 The cells were inoculated into the right forelimb axilla of nu / nu nude mice (3 mice per group). 3 (1-2 groups), the mice were randomly divided into groups and dosed by a single intravenous injection. The day of grouping was set as day 0, and the dosing began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter). The pharmacodynamic evaluation of the test drug is shown in Table 27.

[0424] Table 27. Pharmacodynamic evaluation of the experimental drugs in the human small cell lung cancer xenograft NCI-H526 mouse model

[0425] 10 cells were collected from the DLL3-positive cell line NCI-H69 7 The cells were inoculated into the right forelimb axilla of nu / nu mice (3 mice per group). When the average tumor volume of the mice reached 140 mm 3 (1-2 groups), the mice were randomly divided into groups and dosed by a single intravenous injection. The day of grouping was set as day 0, and the dosing began on day 0. After tumor inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Tumor volume calculation formula: tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter). The pharmacodynamic evaluation of the test drug is shown in Table 28.

[0426] Table 28. Pharmacodynamic evaluation of the experimental drugs in the human small cell lung cancer xenograft NCI-H69 mouse model

[0427] In the NCI-H526 and NCI-H69 models, the ADC of the present invention significantly inhibited tumor growth in both NCI-H526 and NCI-H69 models at a dose of 3 mg / kg, compared with the vehicle control group, with tumor growth inhibition rates (TGI) of 113.0% and 76.5%, respectively. There were no animal deaths or significant animal weight loss in each treatment group, and no obvious drug toxicity reactions were observed. During the treatment period, the mice tolerated the ADC of the present invention well.

[0428] Example 27: Evaluation of the toxicokinetics and tolerability of ADC in cynomolgus monkeys

[0429] Cynomolgus monkeys (2 female) were selected and Hab33B10E8-045 (5 mg / mL, 30-minute injection) was administered intravenously at dose levels of 10, 30, and 30 mg / kg every 2 weeks (3 doses in total). Parameters assessed during the study included general observations, body weight, food intake, body temperature, electrocardiogram (lead II), clinical pathology (hematology, blood biochemistry, coagulation), and macroscopic and microscopic examinations of a large number of tissues. Toxicokinetic samples were collected at 0, 24, 72, 120, 336, and 504 hours after each dose. TK samples were analyzed using the MesoScale Discovery (MSD) electrochemiluminescence platform (total monoclonal antibody, total ADC) and LC-MS / MS (free exitecan). The experiments showed that Hab33B10E8-045 was well tolerated with an MTD of 30 mg / kg, and a higher MTD may exist. In a dose study, cynomolgus monkeys administered 10, 30, and 30 mg / kg of Hab33B10E8-045 showed no general abnormalities. No toxicological abnormalities were observed in body weight, temperature, coagulation, or urinalysis. All changes were reversible before each dose. Hab33B10E8-045 was well tolerated in cynomolgus monkeys and exhibited a stable pharmacokinetic profile.

[0430] Table 29. Toxicokinetics and tolerability of Hab33B10E8-045 in cynomolgus monkeys

Claims

1. An anti-DLL3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein: (1) the sequence of HCDR1 is the sequence shown in SEQ ID NO. 25 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 25, the sequence of HCDR2 is the sequence shown in SEQ ID NO. 26 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 26, and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 27 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 27; (2) the sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.1, the sequence of HCDR2 is the sequence shown in SEQ ID NO.2 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.2, and the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.3; (3) the sequence of HCDR1 is the sequence set forth in SEQ ID NO. 9 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto, the sequence of HCDR2 is the sequence set forth in SEQ ID NO. 10 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto, and the sequence of HCDR3 is the sequence set forth in SEQ ID NO. 11 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto; (4) the sequence of HCDR1 is the sequence shown in SEQ ID NO. 17 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 17, the sequence of HCDR2 is the sequence shown in SEQ ID NO. 18 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 18, and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 19 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 19; or (5) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 33 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 33, the sequence of HCDR2 is the sequence shown in SEQ ID NO. 34 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 34, and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 35 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO.

35.

2. An anti-DLL3 antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein: (1) the sequence of LCDR1 is the sequence shown in SEQ ID NO. 28 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 28, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 29 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 29, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 30 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 30; (2) the sequence of LCDR1 is the sequence shown in SEQ ID NO. 4 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 4, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 5 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 5, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 6 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 6; (3) the sequence of LCDR1 is the sequence shown in SEQ ID NO. 12 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 12, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 13 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 13, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 14 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 14; (4) the sequence of LCDR1 is the sequence shown in SEQ ID NO. 20 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 20, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 21 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 21, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 22 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 22; or (5) The sequence of LCDR1 is the sequence shown in SEQ ID NO. 36 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 36, the sequence of LCDR2 is the sequence shown in SEQ ID NO. 37 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 37, and the sequence of LCDR3 is the sequence shown in SEQ ID NO. 38 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.

38.

3. An anti-DLL3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein: (1) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 25 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 25, the sequence of HCDR2 is the sequence shown in SEQ ID NO. 26 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 26, and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 27 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 27, the sequence of LCDR1 is the sequence shown in SEQ ID NO. 28 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 28, and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 29 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. The sequence of SEQ ID NO. 29 is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 29, and the sequence of LCDR3 is the sequence of SEQ ID NO. 30 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 30; (2) The sequence of HCDR1 is the sequence shown in SEQ ID NO.1 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.1, the sequence of HCDR2 is the sequence shown in SEQ ID NO.2 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.2, and the sequence of HCDR3 is the sequence shown in SEQ ID NO.3 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.3, the sequence of LCDR1 is the sequence shown in SEQ ID NO.4 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.4, and the sequence of LCDR2 is the sequence shown in SEQ ID NO.5 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. The sequence of LCDR3 is the sequence of SEQ ID NO. 6 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence of SEQ ID NO. 6; (3) the sequence of HCDR1 is the sequence set forth in SEQ ID NO. 9 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 9, the sequence of HCDR2 is the sequence set forth in SEQ ID NO. 10 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 10, and the sequence of HCDR3 is the sequence set forth in SEQ ID NO. 11 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 11, the sequence of LCDR1 is the sequence set forth in SEQ ID NO. 12 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 12, and the sequence of LCDR2 is the sequence set forth in SEQ ID NO. 13 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. The sequence of SEQ ID NO. 13 is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 13, and the sequence of LCDR3 is the sequence of SEQ ID NO. 14 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 14; (4) the sequence of HCDR1 is the sequence set forth in SEQ ID NO. 17 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 17, the sequence of HCDR2 is the sequence set forth in SEQ ID NO. 18 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 18, and the sequence of HCDR3 is the sequence set forth in SEQ ID NO. 19 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 19, the sequence of LCDR1 is the sequence set forth in SEQ ID NO. 20 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. 20, and the sequence of LCDR2 is the sequence set forth in SEQ ID NO. 21 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO. The sequence of LCDR3 is the sequence of SEQ ID NO. 22 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO. 21; or (5) The sequence of HCDR1 is the sequence shown in SEQ ID NO. 33 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 33, the sequence of HCDR2 is the sequence shown in SEQ ID NO. 34 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 34, and the sequence of HCDR3 is the sequence shown in SEQ ID NO. 35 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 35, the sequence of LCDR1 is the sequence shown in SEQ ID NO. 36 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 36, and the sequence of LCDR2 is the sequence shown in SEQ ID NO. 37 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. The sequence shown in NO.37 has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity, and the sequence of LCDR3 is the sequence shown in SEQ ID NO.38 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.

38.

4. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The anti-DLL3 antibody or antigen-binding fragment thereof is a murine, chimeric, humanized or fully human antibody or antigen-binding fragment. Preferably, the anti-DLL3 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment.

5. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The anti-DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein: (1) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 31 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 31, or the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 58 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 58; (2) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 7 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 7, or the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 54 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 54; (3) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 15 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 15; (4) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 23 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 23, or the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 56 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 56; or (5) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 39 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO.

39.

6. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The anti-DLL3 antibody or antigen-binding fragment thereof comprises a light chain variable region, wherein: (1) The light chain variable region sequence is the sequence shown in SEQ ID NO. 32 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 32, or the light chain variable region sequence is the sequence shown in SEQ ID NO. 59 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 59; (2) the light chain variable region sequence is the sequence shown in SEQ ID NO. 8 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 8, or the light chain variable region sequence is the sequence shown in SEQ ID NO. 55 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 55; (3) The light chain variable region sequence is the sequence shown in SEQ ID NO. 16 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 16; (4) the light chain variable region sequence is the sequence shown in SEQ ID NO. 24 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 24, or the light chain variable region sequence is the sequence shown in SEQ ID NO. 57 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 57; (5) The light chain variable region sequence is the sequence shown in SEQ ID NO. 40 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO.

40.

7. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The anti-DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: (1) the heavy chain variable region sequence is the sequence shown in SEQ ID NO.31 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.31, and the light chain variable region sequence is the sequence shown in SEQ ID NO.32 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.32; or the heavy chain variable region sequence is the sequence shown in SEQ ID NO.58 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.58, and the light chain variable region sequence is the sequence shown in SEQ ID NO.59 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.59; (2) the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 7 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 8 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto; or the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 54 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 55 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity thereto; (3) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 15 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 15, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 16 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 16; (4) the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 23 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 23, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 24 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 24; or the heavy chain variable region sequence is the sequence shown in SEQ ID NO. 56 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 56, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 57 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 57; (5) The heavy chain variable region sequence is the sequence shown in SEQ ID NO. 39 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO. 39, and the light chain variable region sequence is the sequence shown in SEQ ID NO. 40 or a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence shown in SEQ ID NO.

40.

8. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein The antigen-binding fragment is Fv, VHH, scFv, Fab, Fab' or (Fab')2.

9. The anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein The anti-DLL3 antibody is a full-length antibody.

10. The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 9, wherein The anti-DLL3 antibody is an IgG antibody, preferably, the anti-DLL3 antibody is an IgG1 antibody.

11. The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 10, wherein The Fc region of the anti-DLL3 antibody comprises amino acid mutations that reduce ADCC, CDC and / or ADCP effects.

12. The anti-DLL3 antibody or antigen-binding fragment thereof according to claim 11, wherein The amino acid mutation is one or more amino acid mutations selected from the group consisting of L234A, L235A, P329A and P331S.

13. A nucleic acid molecule encoding the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. An expression vector comprising the nucleic acid molecule of claim 13.

15. A host cell comprising the expression vector according to claim 14.

16. A bispecific antibody comprising a first antigen-binding site and a second antigen-binding site, wherein one of the antigen-binding sites comprises the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

17. A bispecific antibody comprising a first antigen-binding site and a second antigen-binding site, wherein one antigen-binding site comprises an anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and the other antigen-binding site comprises another anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

18. The bispecific antibody according to claim 17, characterized in that The first antigen binding site and the second antigen binding site bind to different epitopes of the same antigen.

19. The bispecific antibody according to claim 18, characterized in that (1) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 25 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 25, the HCDR2 sequence is the sequence shown in SEQ ID NO. 26 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 26, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 27 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 27; in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 28 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 28, and the LCDR2 sequence is the sequence shown in SEQ ID NO. 29 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. NO.29, and the LCDR3 sequence is the sequence shown in SEQ ID NO.30 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.30; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.17 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.17, the HCDR2 sequence is the sequence shown in SEQ ID NO.18 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.18, and the HCDR3 sequence is the sequence shown in SEQ ID NO.19 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. No. 19, wherein the LCDR1 sequence is SEQ ID NO. 20 or a sequence having 85%, 90%, 95%, 98% or 99% identity thereto; the LCDR2 sequence is SEQ ID NO. 21 or a sequence having 85%, 90%, 95%, 98% or 99% identity thereto; and the LCDR3 sequence is SEQ ID NO. 22 or a sequence having 85%, 90%, 95%, 98% or 99% identity thereto; (2) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO. 25 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 25, the HCDR2 sequence is the sequence shown in SEQ ID NO. 26 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 26, and the HCDR3 sequence is the sequence shown in SEQ ID NO. 27 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 27; in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO. 28 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 28, and the LCDR2 sequence is the sequence shown in SEQ ID NO. 29 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. NO.29, and the LCDR3 sequence is the sequence shown in SEQ ID NO.30 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.30; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.1 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.1, the HCDR2 sequence is the sequence shown in SEQ ID NO.2 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.2, and the HCDR3 sequence is the sequence shown in SEQ ID NO.3 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. NO.3, in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.4 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.4, the LCDR2 sequence is the sequence shown in SEQ ID NO.5 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.5, and the LCDR3 sequence is the sequence shown in SEQ ID NO.6 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.6; or (3) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.17 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.17, the HCDR2 sequence is the sequence shown in SEQ ID NO.18 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.18, and the HCDR3 sequence is the sequence shown in SEQ ID NO.19 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.19; in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.20 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.20, and the LCDR2 sequence is the sequence shown in SEQ ID NO.21 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. NO.21, and the LCDR3 sequence is the sequence shown in SEQ ID NO.22 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.22; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein in the heavy chain variable region, the HCDR1 sequence is the sequence shown in SEQ ID NO.1 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.1, the HCDR2 sequence is the sequence shown in SEQ ID NO.2 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.2, and the HCDR3 sequence is the sequence shown in SEQ ID NO.3 or a sequence with 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO.

3. NO.3 has 85%, 90%, 95%, 98% or 99% identity with the sequence shown in NO.3; in the light chain variable region, the LCDR1 sequence is the sequence shown in SEQ ID NO.4 or a sequence with 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.4, the LCDR2 sequence is the sequence shown in SEQ ID NO.5 or a sequence with 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.5, and the LCDR3 sequence is the sequence shown in SEQ ID NO.6 or a sequence with 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.

6.

20. The bispecific antibody according to claim 19, characterized in that The bispecific antibody is an IgG-like bispecific antibody, in which the left arm is the first antigen binding site and the right arm is the second antigen binding site.

21. The bispecific antibody according to claim 20, wherein (1) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 31 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 31, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 32 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 32; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 23 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 23, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 24 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 24; (2) the first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 31 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 31, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 32 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 32; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 7 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 7, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 8 or a sequence having 85%, 90%, 95%, 98% or 99% identity to the sequence shown in SEQ ID NO. 8; (3) The first antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 23 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 23, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 24 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 24; the second antigen-binding site comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region sequence is a sequence shown in SEQ ID NO. 7 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO. 7, and the light chain variable region sequence is a sequence shown in SEQ ID NO. 8 or a sequence having 85%, 90%, 95%, 98% or 99% identity with the sequence shown in SEQ ID NO.

8.

22. The bispecific antibody according to any one of claims 20 to 21, characterized in that The bispecific antibody is an IgG1 antibody; preferably, the Fc region of the bispecific antibody comprises amino acid mutations that reduce ADCC, CDC and / or ADCP effects; and / or amino acid mutations that form a knob and / or a hole to facilitate purification of the bispecific antibody; preferably, the bispecific antibody carries a CH1-CL chain exchange between the first heavy chain and the first light chain or between the second heavy chain and the second light chain; preferably, the Fc region of the bispecific antibody comprises amino acid mutations that form a knob and / or a hole located in the CH3 region to facilitate purification of the bispecific antibody; further preferably, the mutations that form the hole are Y349C, T366S, L368A and Y407V, and the mutations that form the knob are S354C and T366W.

23. The bispecific antibody of claim 22, wherein: (1) The bispecific antibody comprises two heavy chains and two light chains, wherein the amino acid sequence of heavy chain 1 is shown in SEQ ID NO.41, the amino acid sequence of heavy chain 2 is shown in SEQ ID NO.43, the amino acid sequence of light chain 1 is shown in SEQ ID NO.46, and the amino acid sequence of light chain 2 is shown in SEQ ID NO.48; (2) The bispecific antibody comprises two heavy chains and two light chains, wherein the amino acid sequence of heavy chain 1 is shown in SEQ ID NO.41, the amino acid sequence of heavy chain 2 is shown in SEQ ID NO.50, the amino acid sequence of light chain 1 is shown in SEQ ID NO.46, and the amino acid sequence of light chain 2 is shown in SEQ ID NO.51; (3) The bispecific antibody comprises two heavy chains and two light chains, wherein the amino acid sequence of heavy chain 1 is shown in SEQ ID NO.52, the amino acid sequence of heavy chain 2 is shown in SEQ ID NO.50, the amino acid sequence of light chain 1 is shown in SEQ ID NO.53, and the amino acid sequence of light chain 2 is shown in SEQ ID NO.

51.

24. A nucleic acid molecule encoding the bispecific antibody according to any one of claims 16 to 23.

25. An expression vector comprising the nucleic acid molecule of claim 24.

26. A host cell comprising the expression vector of claim 25.

27. An antibody-drug conjugate comprising the anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-12 or the bispecific antibody of any one of claims 16-23.

28. The antibody-drug conjugate of claim 27, wherein The antibody-drug conjugate is obtained by coupling a therapeutic agent to the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the bispecific antibody according to any one of claims 16 to 23.

29. The antibody-drug conjugate of claim 28, wherein The therapeutic agent is selected from the group consisting of a cytotoxic drug, an immunopotentiator or a radioisotope; preferably, the therapeutic agent is a cytotoxic drug selected from a microtubule inhibitor, a DNA topoisomerase inhibitor, a DNA damaging agent, an antimetabolite or an antitumor antibiotic; more preferably, the therapeutic agent is selected from DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyecliptin, topotecan, lertotecan, belotecan, exitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]- DNA topoisomerase inhibitors of 4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride or N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

30. The antibody-drug conjugate of claim 29, wherein The antibody or antigen-binding fragment thereof is conjugated to the therapeutic agent via a linker, which can be a cleavable linker or a non-cleavable linker.

31. The antibody-drug conjugate of claim 27, which has the structure shown in formula (I): A-(LD) d (I) wherein A is the anti-DLL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the bispecific antibody according to any one of claims 16 to 23; L is a linker moiety, one end of which is connected to A and the other end is connected to the therapeutic agent D; d represents the average molar ratio of therapeutic agent to A (also known as DAR, i.e., drug-antibody conjugation ratio), which is an integer or decimal selected from 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); when d is a decimal, it refers to the average number of linkers-therapeutic agents (LD) conjugated to each A.

32. The antibody-drug conjugate of claim 29, wherein A is selected from the group consisting of antibody 22B12E5, antibody 25G10A9, antibody 31B6G8, antibody 33B10E8, antibody 44B8H2, Hab22B12E5, Hab31B6G8, Hab33B10E8, 33+31, 33+22, and 31+22; The antibody 22B12E5 has a heavy chain variable region sequence of SEQ ID NO. 7 and a light chain variable region sequence of SEQ ID NO. 8; The antibody 25G10A9 has a heavy chain variable region sequence of SEQ ID NO. 15 and a light chain variable region sequence of SEQ ID NO. 16; Among them, antibody 31B6G8 has a heavy chain variable region sequence of SEQ ID NO.23 and a light chain variable region sequence of SEQ ID NO.24; The antibody 33B10E8 has a heavy chain variable region sequence of SEQ ID NO. 31 and a light chain variable region sequence of SEQ ID NO. 32; The antibody 44B8H2 has a heavy chain variable region sequence of SEQ ID NO. 39 and a light chain variable region sequence of SEQ ID NO. 40; The antibody Hab22B12E5 has a heavy chain variable region sequence of SEQ ID NO.54 and a light chain variable region sequence of SEQ ID NO.55; The antibody Hab31B6G8 has a heavy chain variable region sequence of SEQ ID NO.56 and a light chain variable region sequence of SEQ ID NO.57; The antibody Hab33B10E8 has a heavy chain variable region sequence of SEQ ID NO.58 and a light chain variable region sequence of SEQ ID NO.59; wherein antibody 33+31 has heavy chain 1 of SEQ ID NO.41, heavy chain 2 of SEQ ID NO.43, light chain 1 of SEQ ID NO.46, and light chain 2 of SEQ ID NO.48; wherein antibody 33+22 has heavy chain 1 of SEQ ID NO.41, heavy chain 2 of SEQ ID NO.50, light chain 1 of SEQ ID NO.46, and light chain 2 of SEQ ID NO.51; Among them, antibody 31+22 has heavy chain 1 of SEQ ID NO.52, heavy chain 2 of SEQ ID NO.50, light chain 1 of SEQ ID NO.53 and light chain 2 of SEQ ID NO.

51.

33. The antibody-drug conjugate of any one of claims 31-32, wherein -L- is selected from -L1-L2-L3-L4-, wherein L1 is a covalent linking unit that is covalently linked to A, L2 is an extension unit, L3 is selected from a peptide residue consisting of 2-8 amino acids, and L4 is a bond or a self-cleavable fragment.

34. The antibody-drug conjugate of claim 33, wherein L1 is selected from Where * indicates connection with A; L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from -C1-C6 alkylene-, -C1-C3 alkylene-C3-C6 cycloalkylene-, -ethynylene-C1-C6 alkylene-, -(CH2CH2O) n -、-(CH2CH2O) n C 1-3 Alkylene-, -C1-C3 alkylene (CH2CH2O) n -C1-C3 alkylene-, -C1-C3 alkylene-O-C1-C3 alkylene, phenyl, -phenyl-C1-C3 alkylene-, wherein n is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); L3 is selected from a peptide residue consisting of 2-8 amino acids selected from phenylalanine, valine, alanine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine; L4 is selected from: bond, -NCH2-, wherein R1 is selected from hydrogen, wherein R2 is selected from: wherein r, s, t, and u are each independently selected from an integer of 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

35. The antibody-drug conjugate of any one of claims 33-34, wherein: L2 is selected from: (CH2CH2O) n CH2C(O)-、-(CH2CH2O) n CH2CH2-、 The * position is connected to L1, and n is defined as in claim 34.

36. The antibody-drug conjugate of any one of claims 33 to 35, wherein: L3 is selected from: Among them * Set to connect to L2.

37. The antibody-drug conjugate of any one of claims 33 to 36, wherein: L4 is selected from: -NCH2-, wherein r is selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

38. The antibody-drug conjugate of claim 34, wherein L is selected from: Where * indicates connection with A.

39. The antibody-drug conjugate of any one of claims 33 to 38, wherein D is selected from the group consisting of: wherein X is selected from a bond, Wherein R3 and R4 are independently selected from H, C 1-3 Alkyl (preferably methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), or R3 and R4 together with the carbon atom to which they are connected form a 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), wherein the * position is connected to L4.

40. The antibody-drug conjugate of any one of claims 33 to 39, wherein D is selected from the group consisting of:

41. The antibody-drug conjugate of any one of claims 27 to 40, which is selected from the following structures:

42. A fusion protein comprising the anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-12.

43. A chimeric antigen receptor polypeptide (CAR) comprising the anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-12, or the bispecific antibody of any one of claims 16-23.

44. A genetically modified cell, characterized in that The cells express the chimeric antigen receptor (CAR) polypeptide of claim 43, and the cells are selected from autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and red blood cells.

45. A pharmaceutical composition comprising the anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-12, the bispecific antibody of any one of claims 16-23, or the antibody-drug conjugate of any one of claims 27-41, the fusion protein of claim 42, the chimeric antigen receptor polypeptide of claim 43 or a nucleic acid molecule expressing the same, or the genetically modified cell of claim 44.

46. Use of the anti-DLL3 antibody or antigen-binding fragment thereof of any one of claims 1-12, the bispecific antibody of any one of claims 16-23, the antibody-drug conjugate of any one of claims 27-41, the fusion protein of claim 42, the chimeric antigen receptor polypeptide or the nucleic acid molecule expressing the same of claim 43, the genetically modified cell of claim 44, or the pharmaceutical composition of claim 45 in the preparation of a medicament for treating an individual tumor; preferably, the tumor is a tumor expressing DLL3 (DLL3+) on the surface of tumor cells; more preferably, the tumor is a solid tumor or hematological tumor associated with high DLL3 expression; further preferably, the tumor is lung cancer; more preferably, the tumor is small cell lung cancer.

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