Anti-dll3 antibodies and uses thereof

Abstract

Anti-DLL3 antibodies are provided, including murine antibodies, humanized antibodies, and antibodies having further optimized CDR sequences. The antibodies have excellent binding specificity and improved activity compared to other anti-DLL3 antibodies that are being developed.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480060752.9 (22) Application Date 2024.09.20 (66) Domestic Priority Data PCT / CN2023 / 120494 2023.09.21 CN PCT / CN2024 / 081042 2024.03.11 CN (85) PCT International Application Entering National Phase Date 2026.03.23 (86) PCT International Application Application Data PCT / CN2024 / 119967 2024.09.20 (87) PCT International Application Publication Data WO2025 / 061132 EN 2025.03.27 (71) Applicant Lepu Biotechnology Co., Ltd. Address 201112 2nd Floor, Building 4, No. 651, Lianheng Road, Pujiang Town, Minhang District, Shanghai (72) Inventors: Cai Zhijian, Wu Xin, Cui Feifei, Fang Lei (74) Patent Agency: Shanghai Bixing Law Firm, 31283 Patent Attorney: Shui Wenyu (51) Int.Cl. C07K 16 / 28 (2006.01) C07K 16 / 30 (2006.01) C07K 19 / 00 (2006.01) C12N 15 / 13 (2006.01) A61K 39 / 00 (2006.01) A61K 39 / 395 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title: Anti-DLL3 Antibody and Its Use (57) Abstract: Anti-DLL3 antibodies, including mouse antibodies, humanized antibodies and antibodies with further optimized CDR sequences, are provided. The antibody exhibits superior binding specificity and improved activity compared to other anti-DLL3 antibodies under development. Claims (3 pages), Description (41 pages), Sequence Listing (electronic publication), Drawings (11 pages) CN 121909211 A 2026.04.21 CN 1 21 90 92 11 A 1. An antibody or an antigen-binding fragment thereof, specific for human Delta-like ligand 3 (DLL3) protein and comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH CDR1, VH CDR2 and VH CDR3, and the VL comprises VL CDR1, VL CDR2 and VL CDR3, wherein: (a) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; the VH CDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 38 and 111-117; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 39.(a) The amino acid sequence of SEQ ID NO: 40; VL CDR2 contains the amino acid sequence of SEQ ID NO: 41; and VL CDR3 contains the amino acid sequence of SEQ ID NO: 42; (b) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 13-18; (c) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 19-24; (d) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 25-30; (e) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 25-30; CDR3 respectively comprises the amino acid sequences of SEQ ID NO: 31-36; or (f) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences of SEQ ID NO: 43-48. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: (a) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; the VH CDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 38 and 111-117; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 40; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 42. 3. The antibody or antigen-binding fragment of claim 2, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110 or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110. 4. The antibody or antigen-binding fragment of claim 2 or 3, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73. 5. The antibody or antigen-binding fragment of claim 4, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110.6. The antibody or antigen-binding fragment of claim 1, wherein (b) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 13-18. 7. The antibody or antigen-binding fragment of claim 6, wherein the VH contains the amino acid sequence of SEQ ID NO: 1 or a peptide having at least 90% sequence identity with SEQ ID NO: 1, and the VL contains the amino acid sequence of SEQ ID NO: 2 or a peptide having at least 90% sequence identity with SEQ ID NO: 2. Claims 1 / 3 page 2 CN 121909211 A 8. The antibody or antigen-binding fragment of claim 7, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 49 and a light chain containing the amino acid sequence of SEQ ID NO: 50. 9. The antibody or antigen-binding fragment of claim 1, wherein (c) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 19-24. 10. The antibody or antigen-binding fragment of claim 9, wherein the VH comprises the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 90% sequence identity with SEQ ID NO: 3, and the VL comprises the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 90% sequence identity with SEQ ID NO: 4. 11. The antibody or antigen-binding fragment of claim 10, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 51 and a light chain containing the amino acid sequence of SEQ ID NO: 52. 12. The antibody or antigen-binding fragment of claim 1, wherein (d) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 25-30. 13. The antibody or antigen-binding fragment of claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5 or a peptide having at least 90% sequence identity with SEQ ID NO: 5, and the VL comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 90% sequence identity with SEQ ID NO: 6. 14. The antibody or antigen-binding fragment of claim 13, comprising the amino acid sequence of SEQ ID NO: 25-30.15. The antibody or antigen-binding fragment of claim 1, wherein (e) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 31-36. 16. The antibody or antigen-binding fragment of claim 15, wherein the VH contains the amino acid sequence of SEQ ID NO: 7 or a peptide having at least 90% sequence identity with SEQ ID NO: 7, and the VL contains the amino acid sequence of SEQ ID NO: 8 or a peptide having at least 90% sequence identity with SEQ ID NO: 8. 17. The antibody or antigen-binding fragment of claim 16, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 55 and a light chain containing the amino acid sequence of SEQ ID NO: 56. 18. The antibody or antigen-binding fragment of claim 1, wherein (f) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 43-48. 19. The antibody or antigen-binding fragment of claim 18, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 or a peptide having at least 90% sequence identity with SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 12 or a peptide having at least 90% sequence identity with SEQ ID NO: 12. 20. The antibody or antigen-binding fragment of claim 19, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 59 and a light chain containing the amino acid sequence of SEQ ID NO: 60. 21. An antibody or antigen-binding fragment thereof, which is specific for human Delta-like ligand 3 (DLL3) protein and competitively binds to the DLL3 protein with any antibody or fragment of claim 1-20. 22. An antibody or an antigen-binding fragment thereof, which is specific for human Delta-like ligand 3 (DLL3) protein and binds to an EGF3-4 domain or an EGF6 domain. 23. The antibody or fragment thereof according to any one of claims 1-22, wherein the antibody or fragment thereof is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv. 24. The antibody or fragment thereof according to any one of claims 1-23, which is humanized. 25. A multispecific antibody comprising an antigen-binding fragment according to any one of claims 1-23 and an antigen-binding fragment specific for human Delta-like ligand 3 (DLL3) protein and an EGF3-4 domain or an EGF6 domain.Non-DLL3 target antigens have one or more antibodies or antigen-binding fragments with binding specificity. Claims 2 / 3, Page 3, CN 121909211, A 26. A chimeric antigen receptor (CAR) comprising an antigen-binding fragment according to any one of claims 1-25, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain. 27. One or more polynucleotides encoding an antibody or its antigen-binding fragment according to any one of claims 1-25 or a CAR according to claim 26. 28. The polynucleotide according to claim 27, which is one or more mRNAs. 29. The polynucleotide according to claim 28, wherein the mRNA is chemically modified. 30. A cell comprising the polynucleotide according to claim 28 or 29. 31. A composition comprising an antibody or its antigen-binding fragment according to any one of claims 1-25 or a CAR according to claim 26, and a pharmaceutically acceptable carrier. 32. A method of treating cancer in a patient in need, the method comprising administering to the patient an effective amount of an antibody or its antigen-binding fragment according to any one of claims 1-25, or a CAR according to claim 26. 33. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-25 or the CAR according to claim 26 for the preparation of a medicament for treating cancer. 34. The method according to claim 32 or the use according to claim 33, wherein the cancer is selected from the group consisting of: ovarian cancer, prostate cancer, urinary tract cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer. 35. The method according to claim 32 or the use according to claim 33, wherein the cancer is small cell lung cancer (SCLC). Claims 3 / 3 Page 4 CN 121909211 A Anti-DLL3 Antibody and Its Use Background Art

[0001] Lung cancer is the leading cause of death from malignant tumors worldwide, with small cell lung cancer (SCLC) accounting for approximately 15%. Unlike other types of cancer, SCLC is highly aggressive and can metastasize at an early stage. Clinical outcomes include frequent SCLC relapse and drug resistance. Furthermore, due to the lack of targeted therapies and challenges in early detection, the overall survival (OS) rate for SCLC is low. Although immunotherapy can be used as first-line treatment for extensive-stage small cell lung cancer (ES-SCLC), such as using a PD-L1 monoclonal antibody (durvalumab) in combination with chemotherapy, OS has not been significantly improved. Therefore, there is an urgent need for improved SCLC therapies.

[0002] Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand in the Notch ligand family. Human DLL3 has a unique structure compared to other members of this family, such as DLL1 and DLL4, consisting of a Delta / Serrate / LAG-2 (DSL) domain, six epidermal growth factor (EGF)-like repeat sequences, a 21-amino acid (aa) transmembrane domain (TM), and a 105 aa intracellular domain (ICD).

[0003] DLL3 is highly expressed on the cell surface of SCLC tumors and other neuroendocrine-originating tumor types, including glioblastoma multiforme (GBM), large cell neuroendocrine lung cancer (LCNEC), metastatic melanoma, small cell bladder cancer (SCBC), and neuroendocrine prostate cancer (NEPC). Overall, over 80% of SCLC tumors express DLL3 protein, and the expression is highly consistent across tumor cells. In contrast, only very slight expression is observed in normal tissues (e.g., neurons, pancreatic islet cells, and pituitary cells), and it is limited to the cytoplasm. In preclinical models, DLL3 expression promotes the migration and invasion of SCLC cells through a mechanism involving the control of the epithelial-mesenchymal transition protein Snail.

[0004] The unique expression pattern of DLL3 in SCLC and other neuroendocrine tumors makes it possible to develop therapeutics that specifically target these tumor types using DLL3. Currently, several clinical studies in SCLC and other neuroendocrine tumors are evaluating these DLL3-specific drugs. Several types of approaches targeting DLL3, including bispecific antibodies (BsAb), chimeric antigen receptor T-cell therapy (CAR-T), and antibody-drug conjugates (ADCs), are currently under clinical investigation.

[0005] Tarlatamab (AMG757) is a bispecific T-cell connective (BiTE) molecule with an extended half-life (targeting both DLL3 and CD3 to induce T-cell-mediated tumor lysis). In the Phase I study DeLLphi-300 (NCT03319940), it demonstrated manageable safety and encouraging durability of response in patients with relapsed / refractory SCLC. The objective response rate (ORR) was 23.4% (95% CI, 15.7 to 32.5), including two complete responses and 23 partial responses. The median duration of response (DoR) was 12.3 months (95% CI, 6.6 to 14.9). The disease control rate (DCR) was 51.4% (95% CI, 41.5 to 61.2). The median progression-free survival (PFS) and overall survival (OS) were 3.7 months (95% CI, 2.1 to 5.4) and 13.2 months (95% CI, 10.5 to not reached), respectively.

[0006] Other DLL3-based T-cell adaptors, including BI764532 from Boehringer Ingelheim and HPN328 from Harpoon Therapeutics, have also shown promising anti-tumor efficacy in their respective Phase I clinical trials. Rova-T is an ADC containing the DLL3-targeting antibody lova-tuzumab, linked to the DNA cross-linking agent pyrrolobenzodiazepine (PDB) via a linker that can be cleaved by proteases. In the Phase 3 TAHOE study comparing Rova-T with topotecan as a second-line therapy for advanced or metastatic SCLC with high DLL3 expression, Rova-T exhibited poorer overall survival (OS) and a higher toxicity rate. AMG 119, the first CAR-T cell therapy for SCLC, demonstrated a manageable safety profile and promising antitumor activity in 5 adult subjects in its Phase I study, but the inclusion statement (page 1 / 41, CN 121909211 A) is currently suspended. LB2102, an autologous CAR-T cell therapy, was recently approved by the FDA for Phase I clinical development in the treatment of adult patients with ES-SCLC. Overall, exploring different types of drugs targeting DLL3 is expected to provide more options for SCLC treatment. Summary of the Invention

[0007] In various embodiments, this disclosure provides antibodies and antigen-binding fragments specific to the human DLL3 protein. Experimental tests have shown that these newly identified antibodies bind potently and specifically to the human DLL3 protein without interacting with DLL1 and DLL4 variants. These antibodies also cross-react with cynomolgus monkey DLL3 protein, aiding in preclinical studies.

[0008] One embodiment of this disclosure provides an antibody or an antigen-binding fragment thereof that is specific to human Delta-like ligand 3 (DLL3) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH CDR1, VH CDR2 and VH CDR3, and the VL comprises VL CDR1, VL CDR2 and VL CDR3, wherein: (a) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; the VH CDR2 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 38 and 111-117; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 40; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 40.(a) The amino acid sequence of NO: 42; (b) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 13-18; (c) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 19-24; (d) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 25-30; (e) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 31-36; or (f) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 31-36; CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 43-48.

[0009] In some embodiments, (a) the VH CDR1 contains the amino acid sequence of SEQ ID NO: 37; the VH CDR2 contains the amino acid sequence selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 111-117; the VH CDR3 contains the amino acid sequence of SEQ ID NO: 39; the VL CDR1 contains the amino acid sequence of SEQ ID NO: 40; the VL CDR2 contains the amino acid sequence of SEQ ID NO: 41; and the VL CDR3 contains the amino acid sequence of SEQ ID NO: 42.

[0010] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110 or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110. In some embodiments, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73. In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104-110, and the VL comprises the amino acid sequence of SEQ ID NO: 73.

[0011] In some embodiments, (b) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise SEQ ID NO:The amino acid sequence of SEQ ID NO: 13-18.

[0012] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 1 or a peptide having at least 90% sequence identity with SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 2 or a peptide having at least 90% sequence identity with SEQ ID NO: 2.

[0013] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 49 and a light chain containing the amino acid sequence of SEQ ID NO: 50.

[0014] In some embodiments, (c) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequence of SEQ ID NO: 19-24.

[0015] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 90% sequence identity with SEQ ID NO: 3, and the VL comprises the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 90% sequence identity with SEQ ID NO: 4.

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 51 and a light chain containing the amino acid sequence of SEQ ID NO: 52.

[0017] In some embodiments, wherein (d) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 25-30.

[0018] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 5 or a peptide having at least 90% sequence identity with SEQ ID NO: 5, and the VL comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 90% sequence identity with SEQ ID NO: 6.

[0019] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 53 and a light chain containing the amino acid sequence of SEQ ID NO: 54.

[0020] In some embodiments, (e) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 31-36.

[0021] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 7 or a peptide having at least 90% sequence identity with SEQ ID NO: 7.The VL contains at least 90% sequence identity to a peptide, and the VL contains the amino acid sequence of SEQ ID NO: 8 or a peptide having at least 90% sequence identity to SEQ ID NO: 8.

[0022] In some embodiments, the antibody or its antigen-binding fragment contains a heavy chain containing the amino acid sequence of SEQ ID NO: 55 and a light chain containing the amino acid sequence of SEQ ID NO: 56.

[0023] In some embodiments, (f) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 43-48.

[0024] In some embodiments, the VH contains the amino acid sequence of SEQ ID NO: 11 or a peptide having at least 90% sequence identity to SEQ ID NO: 11, and the VL contains the amino acid sequence of SEQ ID NO: 12 or a peptide having at least 90% sequence identity to SEQ ID NO: 12.

[0025] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 59 and a light chain containing the amino acid sequence of SEQ ID NO: 60.

[0026] In one embodiment, an antibody or its antigen-binding fragment is also provided that is specific for human Delta-like ligand 3 (DLL3) protein, which competes with the antibody or fragment disclosed herein for binding to the DLL3 protein.

[0027] In one embodiment, an antibody or its antigen-binding fragment is also provided that is specific for human Delta-like ligand 3 (DLL3) protein, which binds to an EGF3-4 domain or an EGF6 domain.

[0028] A multispecific antibody is also provided that comprises the antigen-binding fragment disclosed herein and one or more antibodies or antigen-binding fragments that are specific for binding to non-DLL3 target antigens.

[0029] Yet another embodiment provides a chimeric antigen receptor (CAR) that comprises the antigen-binding fragment disclosed herein, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain.

[0030] Methods and uses for treating cancer are also provided. In some embodiments, the cancer is selected from the group consisting of: ovarian cancer, prostate cancer, urinary tract cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer. In some embodiments, the cancer is small cell lung cancer (SCLC). Specification 3 / 41 pages 7 CN 121909211 A Brief Description of the Drawings

[0031] Figures 1A-1C illustrate the binding of anti-DLL3 antibody to human / cynomolgus monkey / mouse DLL3 protein tagged with his.

[0032] Figures 2A and 2B show the ELISA binding of anti-DLL3 antibody to human paralogs DLL1 and DLL4.

[0033] Figure 3 shows the epitope domain localization results of the anti-DLL3 antibody.

[0034] Figures 4A and 4B show the cell-based binding of anti-DLL3 antibody to HEK293 cell lines expressing human or cynomolgus monkey DLL3.

[0035] Figures 5A and 5B show the cell-based binding of anti-DLL3 antibody to tumor cell lines (including SHP77 and NCI-H82).

[0036] Figures 6A and 6B show the cell-based binding of anti-DLL3 antibody to human paralogs DLL1 and DLL4 expressed on CHO-K1 cells.

[0037] Figure 7 shows the ELISA binding of anti-DLL3 humanized antibody to human DLL3 protein with his tag.

[0038] Figures 8A and 8B show cell-based binding of anti-DLL3 humanized antibodies to HEK293 cell lines expressing human or cynomolgus monkey DLL3.

[0039] Figures 9A and 9B show cell-based binding of anti-DLL3 humanized antibodies to tumor cell lines (including SHP77 and NCI-H82).

[0040] Figures 10A and 10B show cell-based binding of anti-DLL3 humanized antibodies to human paralogs DLL1 and DLL4 expressed on CHO-K1 cells.

[0041] Figures 11A and 11B show cell-based binding of anti-DLL3 humanized and PTM-de-PTM antibodies to HEK293 cells and SHP77 cells expressing human DLL3. Detailed Description

[0042] Definitions

[0043] It should be noted that the terms “a” or “an” refer to one or more of the entities described; for example, “an antibody” should be understood to represent one or more antibodies. Thus, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein.

[0044] As used herein, “antibody” or “antigen-binding moiety” refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody as well as any antigen-binding fragment or a single chain thereof. Thus, the term “antibody” includes any protein or peptide containing at least a portion of an immunoglobulin molecule having biological activity of binding an antigen. Examples of such molecules include, but are not limited to, the complementarity-determining region (CDR) of the heavy or light chain or its ligand-binding moiety, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region or any portion thereof, or at least a portion of the binding protein.

[0045] The full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. Heavy chainsThe variable domains of the light and heavy chains can be referred to as "VH" and "VL," respectively. The variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (including the light chain (LC) CDRs of LC-CDR1, LC-CDR2, and LC-CDR3, and the heavy chain (HC) CDRs of HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody-antigen binding fragments disclosed herein can be defined or identified using the following conventions: Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain (see page 4 / 41, CN 121909211 A) are inserted between flanking segments called framework regions (FRs), which are more conserved than CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are subclassed, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0046] As used herein, the term "half-antibody" refers to an immunoglobulin heavy chain associated with an immunoglobulin light chain. Those skilled in the art will readily understand that a half-antibody may encompass a fragment thereof and may also have an antigen-binding domain composed of a single variable domain, for example, derived from camelids.

[0047] As used herein, the term "single-chain half-antibody" refers to a single-chain polypeptide comprising a VL domain, optionally a CL domain, a linker, a VH domain, optionally a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the domains are positioned relative to each other in the N-terminal to C-terminal direction as follows: VL-linker-VH-hinge-CH2-CH3, VL-linker-VH-partial hinge-CH2-CH3, VL-linker-VH-hinge variant-CH2-CH3, or VL-CL-linker-VH-CH1-hinge-CH2-CH3.

[0048] Antibodies expressing "single-domain antibodies" (sdAb) or "single-variable-domain (SVD) antibodies" generally refer to antibodies in which a single variable domain (VH or VL) is capable of conferring antigen binding ability. In other words, this single variable domain does not need to bind with another...The variable structural domains interact to recognize the target antigen. Examples of single-domain antibodies include those derived from camelids (lambs and camels) and cartilaginous fish (e.g., nurse sharks), as well as those derived from human and mouse antibodies through recombinant methods (Nature [Nature] (1989) 341:544-546; Dev Comp Immunol [Developmental and Comparative Immunology] (2006) 30:43-56; Trend Biochem Sci [Trends in Biochemistry] (2001) 26:230-235; Trends Biotechnol [Trends in Biotechnology] (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; Febs Lett [Circular of the Federation of European Biochemical Societies] (1994) 339:285-290; WO 00 / 29004; WO 02 / 051870). When sdAb contains only the heavy chain, it is used interchangeably with “VHH” or “single-chain variable domain antibody” or “nanobody”.

[0049] As used herein, the term “antibody fragment” or “antigen-binding fragment” is a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and bisomal antibodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that exerts its antibody function by binding to a specific antigen to form a complex.

[0050] In the case of an antibody, “Fab” refers to a monovalent antigen-binding fragment of an antibody, which consists of a light chain (including both variable and constant regions) linked by disulfide bonds to the variable region and a first constant region of a heavy chain. Fab can be obtained by digesting the antibody with papain at residues proximal to the N-terminus of the disulfide bonds between the heavy chains in the hinge region.

[0051] “Fab’” refers to a Fab fragment containing a portion of the hinge region, which can be obtained by digesting an antibody with pepsin at residues proximal to the C-terminus of the disulfide bond between the heavy chains in the hinge region. Therefore, a small number of residues in the hinge region (including one or more cysteine ​​residues) differ from those in Fab.

[0052] “F(ab)2” refers to a dimer of Fab’ containing a portion of two light chains and two heavy chains.

[0053] “Single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the immunoglobulin heavy chain (VH) and light chain (VL). In some respects, these regions are linked by short linker peptides of ten to approximately 25 amino acids in length. The linker peptides may be enriched with glycine to provide flexibility, and serine or threonine to improve solubility, and may link the N-terminus of VH to the N-terminus of VL.C-terminus, and vice versa. Despite the removal of the constant region and the introduction of linkers, the protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0054] The term antibody encompasses a wide range of polypeptide classes that can be distinguished by biochemical methods. As will be understood by those skilled in the art, heavy chains can be classified as γ, μ, α, δ, or ε (gamma, mu, alpha, delta, epsilon), including several subclasses (e.g., γ1–γ4). It is the properties of this chain that determine the “class” of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to confer functional specificity. In view of this disclosure, those skilled in the art will readily identify these categories and modified versions of the same type, which are accordingly covered within the scope of this disclosure. All immunoglobulin classes are obviously covered within the scope of this disclosure, and the following discussion will generally refer to immunoglobulin molecules of the IgG class. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with molecular weights of 53,000–70,000 Daltons. These four chains are typically linked by disulfide bonds in a “Y” configuration, wherein the light chain begins at the opening of the “Y”, wraps around the heavy chain, and extends into the variable region.

[0055] The antibodies, antigen-binding moieties, variants, or derivatives thereof disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate-derived, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0056] Light chains may be classified as κ or λ (kappa, lambda). Each heavy chain class can bind to either a κ or λ light chain. Generally, the light and heavy chains are covalently bonded to each other, and this immunoglobulin is produced by hybridoma cells, B cells, or genetically engineered...When produced by the host cell, the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In this heavy chain, the amino acid sequence extends from the N-terminus at the Y-configuration bifurcation end to the C-terminus at the bottom of each chain.

[0057] Both the light and heavy chains are divided into regions with structural and functional homology. The terms "constant" and "variable" are used functionally. In this respect, it should be understood that the variable domains (VK and VH) of both the light and heavy chains determine antigen recognition and specificity. Conversely, the constant domains (CK and CH1, CH2, or CH3) of the light and heavy chains confer important biological properties, such as secretion, transplacental activity, Fc receptor binding, complement binding, etc. By convention, the farther the constant domain is from the antigen-binding site or amino terminus of the antibody, the higher its number. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl termini of the heavy and light chains, respectively.

[0058] As described above, the variable region enables the antibody to selectively recognize and specifically bind to epitopes on antigens. That is, a subset of the antibody's VK domain and VH domain, or complementarity-determining region (CDR), binds to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen-binding site located at the end of each arm of this Y-configuration. More specifically, this antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each VH and VK chain. In some cases, such as certain immunoglobulin molecules derived from camel species or engineered based on camel immunoglobulins, the immunoglobulin molecules are complete immunoglobulin molecules that may consist only of heavy chains and lack light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0059] In naturally occurring antibodies, the six “complementarity-determining regions” or “CDRs” in each antigen-binding domain are short, discontinuous sequences of amino acids that are precisely positioned to form the antigen-binding domain when the antibody presents its three-dimensional conformation in an aqueous environment. The remaining amino acids in these antigen-binding domains, called “framework” regions, exhibit low intermolecular variability. Framework regions primarily adopt a β-sheet conformation, and CDRs form loops that connect β-sheet structures and, in some cases, form part of a β-sheet structure. Thus, the framework regions act as a scaffold that positions the CDRs in the correct orientation through interchain, non-covalent interactions. The antigen-binding domain formed by these positioned CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates the antibody's interaction with its epitope.Non-covalent binding of homologous epitopes. For any given heavy or light chain variable region, those skilled in the art can readily identify the amino acids containing the CDR and framework regions, respectively, as they have been precisely defined (see “Sequences of Proteins of Immunological Interest” Kabat, E et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0060] Where a term used and / or accepted in the art has two or more definitions, the definition of the term as used herein is intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe discontinuous antigen-binding sites found in the variable regions of both heavy and light chain polypeptides. This specific region has been described in the following references: Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J. MoI. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entirety. According to both Kabat and Chothia’s definitions of CDRs, overlap or subsets of amino acid residues are included when compared with each other. However, the application of either definition to refer to a CDR of an antibody or a variant thereof is intended to be within the scope of the terminology defined and used herein. For comparison, the corresponding amino acid residues covering these CDRs as defined in the above-cited references are shown in the table below. The exact number of residues covering a particular CDR varies depending on the sequence and size of the CDR. Those skilled in the art can generally determine which residues contain a particular CDR based on the amino acid sequence of the variable region of the antibody.

[0061]

[0062] Kabat et al. also defined a variable domain sequence numbering system applicable to any antibody. Those skilled in the art can explicitly assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data outside of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in the following literature: Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0063] In addition to the table above, the Kabat numbering system describes these CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty-third amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., after a cysteine ​​residue); comprises approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and comprises approximately 7 residues. CDR-L3 begins at approximately the thirty-third residue after the end of CDR-L2 (i.e., after a cysteine ​​residue); comprises approximately 7-11 residues and ends at sequence F or W-G-X-G, where X is any amino acid.

[0064] The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, these antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be derived from condricthoid fish (e.g., from sharks).

[0065] As used herein, the term “heavy chain constant region” includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide used in this disclosure may comprise a polypeptide chain containing a CH1 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide disclosed herein comprises a polypeptide chain containing a CH3 domain.Additionally, the antibodies used in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that heavy chain constant regions can be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0066] The heavy chain constant regions of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may contain a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may contain a hinge region partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion may contain a chimeric hinge partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0067] As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region contains at least one of a constant κ domain or a constant λ domain.

[0068] A “light chain-heavy chain pair” refers to a set of light and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0069] As previously mentioned, the subunit structures and three-dimensional spatial configurations of constant regions of various immunoglobulin classes are well known. As used herein, the term “VH domain” includes the amino-terminal variable domain of the immunoglobulin heavy chain, and the term “CH1 domain” includes the first (closest to the amino terminus) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the amino terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0070] The “CH1 domain” (also referred to as “C1” of the “H1” domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0071] As used herein, the term "hinge region" refers to the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. In IgG, this region corresponds to Glu216 to Pro230 of human IgG1, EU numbering system (Burton, Molec. Immunol. [Molecular Immunology], 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues forming the S-S bond between the heavy chains in the same position. This hinge region is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol [Journal of Immunology] 161: 4083 (1998)). Specification 8 / 41 pages 12 CN 121909211 A

[0072] As used herein, the term "CH2 domain" encompasses the extended portion of the heavy chain molecule, for example, from approximately residues 244 to 360 of the antibody using conventional numbering schemes (Kabat numbering system, residues 244 to 360; EU numbering system, residues 231 to 340; see Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique because it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

[0073] The “CH3 domain” (also known as the “C3” domain) comprises a string of residues in the Fc region from the C-terminus to the CH2 domain (i.e., from approximately amino acid residue 341 to the C-terminus of the antibody sequence, typically at amino acid residues 446 or 447 of IgG, EU numbering system).

[0074] The terms “Fc region,” “Fc domain,” or “crystallizable region fragment” are used herein to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the Fc region of the human IgG heavy chain is generally defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, a composition of a complete antibody may include a population of antibodies with all K447 residues removed, a population of antibodies without K447 residues removed, and a population of antibodies having a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0075] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group, which can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds, which, using the Kabat numbering system, are located at positions corresponding to 239 and 242 (positions 226 or 229, EU numbering system).

[0076] As used herein, the term "chimeric antibody" should be understood to mean any antibody in which the immune-reactive region or site is derived from or obtained from a first species, and the constant region (which may be whole, partial, or modified according to this disclosure) is derived from a second species. In some embodiments, the target binding region or site will be derived from a non-human source (e.g., mouse or primate), and the constant region is of human origin.

[0077] The term "humanized antibody" is used herein to describe an antibody comprising heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences have been modified to be more "human-like," i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or a variant, derivative, analog, or fragment thereof that is immune-specifically bound to a target antigen and comprises a frame (FR) region and a complementarity-determining region (CDR), wherein the frame region has a substantially identical amino acid sequence to that of a human antibody, and the complementarity-determining region has a substantially identical amino acid sequence to that of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a nonhuman antibody CDR. Humanized antibodies substantially comprise all of at least one, typically two, variable domains (Fab, Fab', F(ab')2, Fv), all or substantially all of the CDR regions within the variable domains corresponding to the CDR regions of those nonhuman immunoglobulins (i.e., donor antibodies), and all or substantially all of the frame regions being frame regions of the human immunoglobulin common sequence. In one embodiment, a humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region (Fc) of a human immunoglobulin. In some embodiments, a humanized antibody contains a light chain and at least a variable domain of a heavy chain. The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only one humanized light chain. In some embodiments, as described on page 9 / 41 of CN 121909211 A, the humanized antibody contains only one humanized heavy chain. In specific embodiments, the humanized antibody contains only the humanized variable domain of the light chain and / or the humanized heavy chain.

[0078] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody or antibody moiety binds. If two antibodies or antibody moiety competitively bind to an antigen, they may bind to the same epitope within the antigen.

[0079] "Specific binding" or "specific to..." generally means that an antibody binds to an epitope via its antigen-binding domain, and that such binding requires a certain complementarity between the antigen-binding domain and the epitope.An antibody is defined as "specifically binding" to an epitope when it binds more readily to that epitope via its antigen-binding domain than to random, unrelated epitopes. The term "specific" is used herein to identify the relative affinity of a particular antibody for a particular epitope. For example, antibody "A" may be considered more specific to a given epitope than antibody "B," or antibody "A" may be more specific to epitope "C" than to its specificity to epitope "D."

[0080] As used herein, the term "treat" refers to both therapeutic treatment and preventative or preventative measures aimed at preventing or mitigating (alleviating) physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization (i.e., non-deterioration) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and mitigation (whether partial or complete), whether detectable or undetectable. "Treatment" may also refer to extended survival compared to expected survival without treatment. Those in need of treatment include those who already have a condition or disorder, those who are susceptible to a condition or disorder, or those whose condition or disorder needs to be prevented.

[0081] The terms “subject” or “individual” or “animal” or “patient” or “mammal” mean any subject in need of diagnosis, prognosis, or therapy, particularly mammal subjects. Mammal subjects include humans, domestic animals, farm animals and zoo animals, racing animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0082] As used herein, phrases such as “to a patient in need of treatment” or “subject in need of treatment” include subjects, such as mammal subjects, who will benefit from the administration of antibodies or compositions of this disclosure for, for example, detection, diagnostic procedures, and / or treatment.

[0083] Anti-DLL3 Antibodies

[0084] As demonstrated in the accompanying experimental examples, the inventors of the present invention were able to generate anti-DLL3 antibodies 9E8D8, 36B7F3, 129H2B9, 148C3A7, 310P3C5, and 362H3D3 (Table 1), all of which have a high binding affinity for human DLL3 protein. This binding is specific because they do not bind to DLL1 or DLL4.

[0085] Domain localization shows that some of these antibodies bind to certain DLL3 domains (Table A) that have not been previously targeted. For example, 129H2B9 and 148C3A7 bind to the EGF3-4 domain of human DLL3 expressed on cells (S312-E389 of UniProt NO. Q9NYJ7). 9E8D8 and 310P3C5 bind to the EGF6 domain of human DLL3 expressed on cells (UniProt NO. Q9NYJ7).The antibody specifically binds to the proximal extracellular domain of Q9NYJ7 (R429 to E465). In contrast, the baseline antibody DLL3#3 binds to the proximal extracellular domain of the membrane (F466 to L492 of UniProt NO. Q9NYJ7), and another baseline antibody DLL3-4-001 specifically binds to EGF3.

[0086] Table A. DLL3 domain sequence specification 10 / 41 pages 14 CN 121909211 A

[0087] According to one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or the antigen-binding fragment thereof has binding specificity to human DLL3 protein. In some embodiments, the antibody or the antigen-binding fragment thereof includes a heavy chain variable region (VH) containing VH CDR1, VH CDR2 and VH CDR3 and a light chain variable region (VL) containing VL CDR1, VL CDR2 and VL CDR3.

[0088] In some embodiments, an antibody or antigen-binding fragment derived from antibody 310P3C5 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 38; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 39; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 40; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41; and VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 42.

[0089] In some embodiments, VH CDR2 is PTM derisked. The tested PTM derisked versions include SEQ ID NO: 111-117 (Table 10B), wherein the N residue is substituted with A, F, H, R, V, W, or Y. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 111, 112, 113, 114, 115, 116, or 117; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 39; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 40; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41; and VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 42.

[0090] In some embodiments, VH comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110 or the amino acid sequence of SEQ ID NO: 37.The sequence 65-69 and 104-110 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity while retaining the VH CDR or its PTM re-risked version. In some embodiments, VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO: 70-73 while retaining the VL CDR or its PTM re-risked version.

[0091] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104-110, and VL comprises the amino acid sequence of SEQ ID NO: 73.

[0092] In some embodiments, an antibody that binds to the same epitope on DLL3 as 310P3C5 and its antigen-binding fragment are also provided. In some embodiments, an antibody that binds to the EGF6 domain of the DLL3 protein and its antigen-binding fragment are provided. In some embodiments, an antibody competing with 310P3C5 for binding to DLL3 and its antigen-binding fragment are also provided.

[0093] In some embodiments, an antibody or antigen-binding fragment derived from antibody 9E8D8 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 13-18.

[0094] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 1 or has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 1, while retaining the sequence of VH CDR or its PTM re-risked version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 2 or has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2, while retaining the VL CDR or its PTM re-risking version.

[0095] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 49, and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0096] In some embodiments, an antibody that binds to the same epitope on DLL3 as 9E8D8 and its antigen-binding fragment are also provided. In some embodiments, an antibody that binds to the EGF6 domain of the DLL3 protein and its antigen-binding fragment are provided.In some embodiments, an antibody competing with 9E8D8 for binding to DLL3 and its antigen-binding fragment are also provided.

[0097] In some embodiments, an antibody or antigen-binding fragment derived from antibody 36B7F3 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 19-24.

[0098] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 3 or has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 3, while retaining the VH CDR or its PTM re-risked version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 4 or has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 4, while retaining the VL CDR or its PTM re-risked version.

[0099] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 3, and VL comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 51, and the light chain comprises the amino acid sequence of SEQ ID NO: 52.

[0100] In some embodiments, an antibody and its antigen-binding fragment that binds to the same epitope on DLL3 by 36B7F3 are also provided. In some embodiments, an antibody and its antigen-binding fragment that bind to the proximal extracellular domain of the DLL3 protein are provided. In some embodiments, an antibody and its antigen-binding fragment that compete with 36B7F3 for binding to DLL3 are also provided.

[0101] In some embodiments, an antibody or antigen-binding fragment derived from antibody 129H2B9 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 25-30.

[0102] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 5, while retaining the VH CDR or its PTM re-risking version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6, while retaining the VL CDR or its PTM re-risking version.

[0103] In some embodiments, VH comprises SEQ ID NO: 5.The amino acid sequence of NO: 5, and VL includes the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain includes the amino acid sequence of SEQ ID NO: 53, and the light chain includes the amino acid sequence of SEQ ID NO: 54.

[0104] In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on DLL3 by 129H2B9 are also provided. In some embodiments, an antibody and its antigen-binding fragment that bind to the EGF3-4 domain of the DLL3 protein are provided. In some embodiments, an antibody and its antigen-binding fragment that compete with 129H2B9 for binding to DLL3 are also provided.

[0105] In some embodiments, an antibody or antigen-binding fragment derived from antibody 148C3A7 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each include the amino acid sequences of SEQ ID NO: 31-36.

[0106] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 7 or has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 7, while retaining the VH CDR or its PTM re-risking version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 8 or has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 8, while retaining the VL CDR or its PTM re-risking version.

[0107] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 7, and VL comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 55, and the light chain comprises the amino acid sequence of SEQ ID NO: 56.

[0108] In some embodiments, an antibody that binds to the same epitope on DLL3 as 148C3A7 and its antigen-binding fragment are also provided. In some embodiments, an antibody that binds to the EGF3-4 domain of the DLL3 protein and its antigen-binding fragment are provided. In some embodiments, an antibody that competes with 148C3A7 for binding to DLL3 and its antigen-binding fragment are also provided.

[0109] In some embodiments, an antibody or antigen-binding fragment derived from antibody 362H3D3 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 43-48.

[0110] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 11 while retaining the VH CDR or its PTM re-risking version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 12 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 12 while retaining the VL CDR or its PTM re-risking version.

[0111] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 59, and the light chain comprises the amino acid sequence of SEQ ID NO: 60.

[0112] In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on DLL3 as 362H3D3 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the proximal extracellular domain of the DLL3 protein are provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 362H3D3 for binding to DLL3 are also provided.

[0113] In some embodiments, antibodies and antigen-binding fragments comprising a CDR sequence derived from the CDR sequence disclosed herein, having one, two, or three amino acid substitutions, deletions, and / or additions.

[0114] Multifunctional group molecules

[0115] Multifunctional group molecules comprising antibodies or antigen-binding fragments (such as those disclosed herein) that are specific to DLL3 and one or more antibodies or antigen-binding fragments that are specific to a second antigen or a different epitope on DLL3.

[0116] In some embodiments, the second antigen is a protein expressed on immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells.

[0117] In some embodiments, the second antigen is CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

[0118] Different forms of bispecific antibodies are also provided. In some embodiments, the anti-DLL3 fragment and the second fragmentEach of these is independently selected from Fab fragments, single-chain variable fragments (scFv), or single-domain antibodies. In some embodiments, the bispecific antibody further includes an Fc fragment.

[0119] Bifunctional molecules that include not only antibodies or antigen-binding fragments are also provided. As molecules targeting tumor antigens, antibodies or antigen-binding fragments specific to DLL3 (such as those described herein) may optionally be combined with immune cytokines or ligands via a peptide head. The linked immune cytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. Such bifunctional molecules can combine immune checkpoint blockade effects with local immunomodulation at the tumor site.

[0120] Chimeric Antigen Receptor

[0121] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising an antibody or fragment thereof disclosed herein as a targeting unit. In some embodiments, the CAR comprises an antibody or fragment thereof disclosed herein, a transmembrane domain, a costimulatory domain, and an intracellular CD3ε domain.

[0122] The transmembrane domain may be designed to fuse with an extracellular domain comprising an antibody or fragment, optionally via a hinge domain. It may also fuse with an intracellular domain, such as a costimulatory domain. In some embodiments, the transmembrane domain may include a native transmembrane region of the costimulatory domain (e.g., the TM region of CD28 or 4-1BB used as a costimulatory domain) or a native transmembrane region of the hinge domain (e.g., the TM region of CD8α or CD28 used as a hinge domain).

[0123] In some embodiments, the transmembrane domain may include a sequence that crosses the cell membrane but extends into the cytoplasm and / or extracellular space of the cell. For example, a transmembrane domain may include a transmembrane sequence that may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids extending into the cytoplasm and / or extracellular space of the cell. Thus, a transmembrane domain includes a transmembrane region and may further include one or more amino acids extending into the inner or outer surface of the membrane itself; such sequences may still be considered “transmembrane domains”.

[0124] In some embodiments, the transmembrane domain is fused to a cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably between 2 and 10 amino acids in length, may form a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine duplexes (GS), glycine-serine-glycine triplets.(GSG) or alanine-alanine-alanine triplet (AAA) provides a suitable linker.

[0125] In some embodiments, the CAR further includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between the transmembrane domain and the activation domain. Examples of co-stimulatory domains include, but are not limited to, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), and CD79A. CD79B (B-cell antigen receptor complex-associated α chain), CD84 (SLAMF5), CD96 (tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KTR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (T FSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (KG2D), CD319 (SLAMF7), CD335 (K-p46), CD336 (K-p44), CD337 (K-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF 18), molecule that can induce T cell co-stimulatory activity.(ICOS), LFA-1 (CD11a / CD18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2R β, IL-2R γ, IL-7R α, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc γ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor, Toll ligand receptor and fragments or combinations thereof. Specification 14 / 41 pages 18 CN 121909211 A

[0126] In some embodiments, the cytoplasmic portion of the CAR further includes a signal transduction / activation domain. In one embodiment, the signal transduction / activation domain is a CD3ε domain, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the CD3ε domain.

[0127] Polynucleotides, mRNA, and methods of expressing or preparing antibodies

[0128] This disclosure also provides polynucleotide or nucleic acid molecules encoding antibodies, variants or derivatives thereof, or CARs disclosed herein. The polynucleotides disclosed herein may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on different polynucleotide molecules. Furthermore, the polynucleotides disclosed herein may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on a separate polynucleotide molecule.

[0129] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA may be introduced into target cells to express an antibody or a fragment thereof.

[0130] The mRNA may be synthesized according to any of a variety of known methods. For example, the mRNA may be synthesized via in vitro transcription (IVT). In short, IVT typically uses a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions will vary depending on the specific application.

[0131] In some embodiments, to prepare mRNA encoding an antibody, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter (e.g., a T3, T7, or SP6 promoter) for in vitro transcription, followed by the desired nucleotide sequence for encoding the desired antibody (e.g., encoding a heavy or light chain) and a termination signal.

[0132] The mRNA sequence encoding the desired antibody (e.g., encoding a heavy or light chain) can be determined using standard methods and...Incorporated into a DNA template. For example, virtual reverse translation is performed based on a degenerate genetic code, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence). Then, an optimization algorithm can be used to select appropriate codons. Typically, the G / C content can be optimized on the one hand to achieve the highest possible G / C content, and on the other hand, the frequency of tRNA can be considered as much as possible based on the use of codons. The optimized RNA sequence can be constructed and displayed, for example, by means of a suitable display device, and compared with the original (wild-type) sequence. Secondary structures can also be analyzed to calculate the stability and instability characteristics of the RNA, or its regions can be calculated separately.

[0133] mRNA can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to enhance stability. Modification of mRNA can include, for example, modification of the nucleotides of RNA. Thus, modified mRNA can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleoside analogs or derivatives that are purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl- Guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl- 2-Thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, huaistin, and acylaminophosphates, thiophosphates, peptide nucleotides, methylphosphonates, 7-denitroguanine, 5-methylcytosine, and inosine. (Instructions 15 / 41 pages 19 CN 121909211 A)The preparation of analogues is known to those skilled in the art, for example, from U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0134] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain RNA backbone modifications. Typically, backbone modifications are modifications that chemically modify the phosphate groups of the backbone of the nucleotides contained in the RNA. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylaminophosphates, aminophosphates, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphate)), borophosphates, positively charged guanidine groups, etc., which means replacing the phosphodiester bond with other anionic, cationic, or neutral groups.

[0135] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain sugar modifications. Typical sugar modification refers to the chemical modification of the sugars in the nucleotides it contains, including but not limited to sugar modifications selected from the group consisting of: 2'-deoxy-2'-fluoro-oligonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligonucleotides, 2'-deoxy-2'-C-alkyl oligonucleotides (2'-O-methylcytidine 5'-... Triphosphates, 2'-methyluridine 5'-triphosphates, 2'-C-alkyl oligonucleotides and their isomers (2'-cytarabine 5'-triphosphate, 2'-cytarabine 5'-triphosphate) or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0136] In some embodiments, mRNA (e.g., mRNA encoding the heavy and light chains) may contain modifications of nucleotide bases (base modifications). Modified nucleotides containing base modifications are also called base-modified nucleotides. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine ribonucleoside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, and 5-iodineCytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine nucleoside 5'-triphosphate, 7-deazoadenosine 5'-triphosphate, 7-deazoguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole ribonucleoside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate, or xanthine nucleoside 5'-triphosphate.

[0137] Typically, mRNA synthesis involves adding a “cap” at the N-terminus (5’) and a “tail” at the C-terminus (3’). The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the “tail” is to protect the mRNA from degradation by exonucleases.

[0138] Thus, in some embodiments, the mRNA (e.g., mRNA encoding both heavy and light chains) includes a 5’ cap structure. The 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5’ nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphates via guanylate transferase, creating a 5’5’5 triphosphate bond; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5') ppp (5'(A,G(5')ppp(5)A and G(5)ppp(5')G.

[0139] Thus, in some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) includes a 3' poly(A) tail structure. The poly(A) tail at the 3' end of the mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) includes a 3' poly(C) tail structure. A suitable poly(C) tail at the 3' end of the mRNA typically comprises about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly(C) tail may be added to or may replace the poly(A) tail.

[0140] Therefore, in some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the length of the 5' untranslated region can be about 50 to 500 nucleotides (e.g., about 50 to 400 nucleotides, about 50 to 300 nucleotides, about 50 to 200 nucleotides, or about 50 to 100 nucleotides).

[0141] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) includes a sequence encoding a signal peptide, such as those described herein. In a particular embodiment, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the sequence encoding the signal peptide is directly or indirectly linked at the N-terminus to a sequence encoding the heavy or light chain.

[0142] The techniques of the present invention can be used to deliver any antibody known in the art and antibodies that can be generated against a desired antigen using standard methods. This invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or compositions, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0143] Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptide disclosed herein are fully human. Fully human antibodies can be prepared using techniques described in the art and as described herein. For example, fully human antibodies against specific antigens can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigen challenge, but whose endogenous loci have been deactivated. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents: 6,150,584; 6,458,592; 6,420,140, ​​the contents of which are incorporated herein by reference in their entirety.

[0144] Treatment and Use

[0145] As described herein, the antibodies, variants, derivatives, or antibody-drug conjugates disclosed herein can be used in certain therapeutic and diagnostic methods.

[0146] This disclosure further relates to antibody-based therapies involving the administration of the disclosed antibodies, fragments, or antibody-drug conjugates to patients (e.g., animals, mammals, and humans) to treat one or more of the disorders or conditions described herein. Therapeutic compounds disclosed herein include, but are not limited to, the disclosed antibodies (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides (including variants and derivatives thereof as described herein) encoding the disclosed antibodies.

[0147] The disclosed antibodies can also be used to treat or inhibit cancer. As provided above, DLL3 can be overexpressed in tumor cells, particularly liver, stomach, pancreas, esophagus, ovary, and lung tumors. Inhibition of DLL3 has been shown to be useful in the treatment of tumors.

[0148] Therefore, in some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method involves administering an effective amount of the antibody, fragment, or antibody-drug conjugate disclosed herein to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient overexpresses DLL3.

[0149] Cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy, are also provided in this disclosure. Suitable cells may be used that are transduced with or contacted with a CAR encoding a CAR comprising the anti-DLL3 antibody disclosed herein (or alternatively engineered to express the anti-DLL3 antibody disclosed herein). After such contact or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer as disclosed herein. The cells (e.g., T cells) may be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, but are not limited thereto. Specification 17 / 41 pages 21 CN 121909211 A

[0150] In some embodiments, the cells are isolated from the cancer patient's own body. In some embodiments, the cells are provided by a donor or from a cell bank. When cells are isolated from cancer patients, adverse immune responses can be minimized.

[0151] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, and cutaneous T-cell lymphoma. In some embodiments, the cancer is small cell lung cancer (SCLC).

[0152] Additional diseases or conditions associated with increased cell survival (which can be treated, prevented, diagnosed, and / or predicted by the antibodies disclosed herein or variants or derivatives thereof) include, but are not limited to, the progression and / or metastasis of malignancies and related disorders, such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic)). Leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, nipple carcinoma.Adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

[0153] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody or its variant or derivative used, the patient's age, weight, overall health condition, sex, diet and time of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of healthcare professionals regarding such factors is within the general technical scope of this field. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0154] Methods of administering antibodies, antibody fragments, or antibody-drug conjugates include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered by any convenient route, such as by infusion or bolus injection, absorption through the epithelial or mucosal lining of the skin (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Thus, pharmaceutical compositions containing the antigen-binding peptides disclosed herein can be administered orally, rectally, parenterally, intracisional, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0155] As used herein, the term “parenterally” refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0156] Administration can be systemic or local. Additionally, it is desirable to introduce the antibodies disclosed herein into the central nervous system via any suitable route, including intraventricular injection and intrathecal injection; intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir (such as an Ommaya reservoir). Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer, and formulations containing nebulizing agents.

[0157] It is desirable to apply the antigen-binding peptides or compositions disclosed herein locally to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion during surgery, local application (e.g., in conjunction with postoperative wound dressings), by injection, via catheter, via suppository, or via implants that are porous or non-porous.Or gel-like materials, including membranes, such as sialastic membranes or fibers. Preferably, care must be taken to use materials that the protein (including antibodies) of this disclosure is not absorbed when administering them.

[0158] The amount of antibodies, fragments or antibody-drug conjugates of this disclosure that are effective in treating, inhibiting and preventing inflammatory, immune or malignant diseases, disorders or conditions can be determined by standard clinical techniques. In addition, in vitro assays may optionally be used to help determine the ideal dose range. The precise dose to be used in the formulation will also depend on the route of administration and the severity of the disease, disorder or condition, and should be determined based on the physician's judgment and the individual patient's situation. The effective dose can be deduced from dose-response curves derived from in vitro or animal model testing systems.

[0159] As a general recommendation, the dose of antibodies, fragments or antibody-drug conjugates of this disclosure administered to a patient is typically between 0.001 mg / kg and 100 mg / kg of patient body weight, between 0.01 mg / kg and 20 mg / kg of patient body weight, or between 0.5 mg / kg and 10 mg / kg of patient body weight. Generally, due to the immune response to exogenous peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, the dosage of human antibodies can usually be reduced and the frequency of administration decreased. Furthermore, the dosage and frequency of administration of the antibodies disclosed herein can be reduced by modifications (e.g., lipidation) to enhance antibody uptake and tissue penetration (e.g., into the brain).

[0160] In another embodiment, the compositions disclosed herein are administered in combination with cytokines. Cytokines that can be administered with the compositions disclosed herein include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0161] In another embodiment, the compositions disclosed herein are administered in combination with other therapeutic or prophylactic regimens (e.g., radiotherapy).

[0162] Compositions

[0163] The disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody, fragment, or antibody-drug conjugate, and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0164] In certain embodiments, the term "pharmaceuticalally acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more particularly in humans. Furthermore, "pharmaceuticalally acceptable carrier" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0165] The term "carrier" refers to a diluent, adjuvant, excipient, or medium administered with a therapeutic agent. Such a drugThe carrier can be a sterile liquid, such as water and oil, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetate, citrate, or phosphate. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for regulating tension, such as sodium chloride or dextran, are also considered. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions may be formulated into suppositories with conventional binders and carriers (such as triglycerides). Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in E. W. Martin's specification, page 19 / 41, 23 CN 121909211 A Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide (preferably in purified form) and an appropriate amount of carrier to provide a suitable form of administration to the patient. The formulation should be suitable for the method of administration. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0166] In one embodiment, the composition is formulated according to conventional procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic buffer solutions. If necessary, the composition may also include a solubilizer and a local anesthetic, such as lidocaine, to reduce pain at the injection site. Generally, the components are provided individually or in combination in unit dosage forms, for example, as lyophilized powders or anhydrous concentrates in sealed containers (such as ampoules or pouches), with the amount of active agent indicated. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, a single ampoule of sterile water for injection or saline can be provided to allow for mixing of the components prior to administration.

[0167] Examples

[0168] Example 1. Generation of mouse anti-human DLL3 antibody

[0169] This example describes the generation of mouse anti-human DLL3 monoclonal antibodies using hybridoma technology.

[0170] Immunogens: Two immunogens were used in the mouse immunization process. One consisted of the extracellular domain (ECD, UniProt NO. Q9NYJ7, A27-L492) of a human DLL3 protein fused with a human Fc fragment (hDLL3-hFc, Acro Bio, catalog number DL3-H5255). The other was an ECD of a human DLL3 protein fused with a his tag (hDLL3-his, Acro Bio, catalog number DL3-H52H4).

[0171] Mouse immunization protocol: To generate mouse monoclonal antibodies against human DLL3, BALB / c and C57BL / 6 mice were immunized intraperitoneally or subcutaneously every two weeks with hDLL3-hFc or hDLL3-his protein. Serum titers of immunized mice were monitored by ELISA against human hDLL3-his protein. After several rounds of immunization, mice with sufficient titers of hDLL3-his protein were enhanced and selected for fusion.

[0172] Cell fusion and hybridoma screening: Spleen cells from selected mice were fused with the mouse myeloma cell line Sp2 / 0 by electrofusion. These hybridoma cells were then plated in 96-well microplates and secreted mouse antibodies in the supernatant. In the initial screening process, positive clones were screened in a high-throughput manner using proteins that bind to hDLL3-his as detected by ELISA, and cells that bind to the ECD of human DLL3 overexpressed on HEK293 cells (HEK293-hDLL3, customized by Genomeditech) or the constitutive DLL3-expressing SCLC cell line SHP77 (ATCC, catalog number CRL-2195) as detected by FACS. Confirmatory screening was then performed, and clones that nonspecifically bound to human DLL1 his-tagged fusion protein (hDLL1-his, Sino Biologial, catalog number 11635-H08H) or human DLL4 his-tagged fusion protein (hDLL4-his, Sino Biologial, catalog number 10171-H08H) were filtered out by ELISA. Clones binding to cynomolgus monkey DLL3 his-tagged fusion protein (cynoDLL3-his, Acro Bio, catalog number DL3-C52H3) were identified by ELISA.

[0173] Subcloning and sequencing: Positive primary clones from each fusion that met the above criteria were subcloned by limiting dilution to ensure that the hybridoma subclones originated from a single parental cell. The screening criteria for subclones are the same as those for the above primary fusions.The same clones were used. Subclones that specifically bind to hDLL3 and cynoDLL3 but not to hDLL1 and hDLL4 were screened for subsequent sequencing.

[0174] The obtained mouse antibody variable region sequence was fused with the constant region of human IgG1 to generate a chimeric DLL3 mAb. The DNA sequence of the chimeric antibody was cloned into the pcDNA3.4 plasmid and expressed in CHO-K1 cells, and then the antibody was purified using a protein A affinity chromatography column or beads. The purified chimeric antibody was subjected to continuous in vitro screening to determine affinity, binding ability, specificity and species cross-reactivity.

[0175] Based on the performance of the screening test, six chimeric mAbs (including 9E8D8, 36B7F3, 129H2B9, 148C3A7, 310P3C5 and 362H3D3) were selected for further analysis. The amino acid sequences of the variable regions of the selected chimeric DLL3 antibodies are provided in Table 1 below, and the CDR sequences are summarized in Table 2. Table 3 lists the heavy and light chain sequences of all antibodies. Two benchmark DLL3 mAbs, DLL3-4-001 (CC) (sequences sourced from the IMGT database) and DLL3#3 (see WO 2019234220), are the DLL3 binding elements of two T-cell adaptor (TCE) drugs, talatazumab and BI765432, which are currently undergoing clinical evaluation.

[0176] Table 1. Variable region sequence of DLL3 chimeric mAb (underlined CDR) Specification 21 / 41 page 25 CN 121909211 A

[0177] Table 2. CDR sequence (Kabat number) Specification 22 / 41 page 26 CN 121909211 A

[0178] Table 3. Sequence of DLL3 chimeric mAb Specification 23 / 41 page 27 CN 121909211 A Specification 24 / 41 page 28 CN 121909211 A

[0179] Example 2. Protein binding activity of chimeric monoclonal antibody targeting DLL3

[0180] 2.1. ELISA detection of binding to human DLL3

[0181] In order to determine the binding ability of chimeric mAb to human DLL3 protein, the following ELISA-based binding assay was performed. In short, hDLL3-his protein was diluted to 1 μg / mL with DPBS buffer and adsorbed into the wells of a 96-well microplate and incubated overnight at 4°C. After blocking the wells with 1% bovine serum albumin (BSA) to prevent nonspecific binding, DLL3 chimeric Ab, the baseline antibody DLL3-4-001 (CC), and DLL3#3 or an isotype control were titrated at 3-fold dilutions starting at 100 nM and added to the pre-adsorbed plate.The hDLL3-his protein was added to the wells. The mixture was incubated at room temperature (RT) for 1 hour. The bound DLL3 mAb was recognized by the detection antibody against human IgG Fc conjugated to horseradish peroxidase (HRP) (Jackson Immuno, catalog number 109-035-008). The substrate of HRP, tetramethylbenzidine (TMB), was added to the wells to visualize the binding signal. After sufficient color development, stop solution was added to the wells. The absorbance of the signal was detected at 450 nm using an Envision multi-label microplate reader (PerkinElmer). A graph was generated by fitting a four-parameter nonlinear regression curve in Graphpad Prism 9 software and statistical analysis was performed.

[0182] As shown in Figure 1A, all DLL3 chimeric Abs effectively bound to human DLL3 protein. Table 4 summarizes the EC50 values ​​of the binding curves of each antibody.

[0183] 2.2 ELISA Detection of Binding to Cynomolgus Monkey and Mouse DLL3 Proteins

[0184] To determine the cross-reactivity of the chimeric Abs with cynomolgus monkey and mouse DLL3, ELISA binding assays were performed as described above. Recombinant cynoDLL3-his (KACTUS, catalog number DLL-CM103) and mouse DLL3 his-tagged protein (mDLL3-his, KACTUS, catalog number DLL-MM103) were used as coating antigens at a concentration of 1 μg / mL.

[0185] As shown in Figures 1B and 1C, all DLL3 chimeric Abs effectively bound to cynomolgus monkey DLL3 and mouse DLL3 proteins.

[0186] Table 4 summarizes the EC50 values ​​of the binding curves for each antibody.

[0187] 2.3. ELISA Detection of Binding to Human DLL1 and DLL4

[0188] Since the sequences of family member proteins DLL1 and DLL4 are highly similar to those of DLL3, it is necessary to determine the specificity of the DLL3 chimeric antibodies.

[0189] To determine the binding affinity of the chimeric Ab to human DLL1 and DLL4 proteins, an ELISA binding assay was performed as described above. hDLL1-his protein (Sino Biological, catalog number 11635-H08H) and hDLL4-his protein (Sino Biological, catalog number 10171-H08H) were used as coating antigens at a concentration of 1 μg / mL. DLL1 mAb pidilizumab (CAS No. 1036730-42-3) and DLL4 mAb (Fab) MLCK-2 (patent No. WO 2015005632, ABL) were used.Bio (Bio Inc.) was used as a positive control.

[0190] As shown in Figures 2A and 2B, most DLL3 chimeric Abs showed negligible binding to hDLL1 or hDLL4 proteins, similar to the baseline antibody. However, 362H3D3 showed detectable binding to both human DLL1 and DLL4 proteins, which may represent cross-reactivity of this DLL3 Ab with the other two family members.

[0191] Table 4. Binding activity of DLL3 antibody to antigen protein

[0192] --: No binding specification 26 / 41 pages 30 CN 121909211 A

[0193] 2.4. Affinity measurement

[0194] The binding affinity of the chimeric antibody to human DLL3 protein was determined using a Biacroe™ 8K. Briefly, the antibody (1 or 2 μg / ml) was captured using a Pro-A chip. A single dose (50 or 100 nM) of human DLL3-his protein was injected onto the captured antibody at a flow rate of 30 μL / min. Antigen association was performed for 120 s, followed by dissociation for 150–400 s. Data analysis was conducted using Biacore™ 8K evaluation software. The results showed that all chimeric DLL3 antibodies exhibited high affinity for human DLL3, similar to the baseline antibody (Table 5).

[0195] Table 5. Chimeric antibody affinity ranking results by Biacore™

[0196] 2.5. Domain localization of chimeric DLL3 antibodies

[0197] To evaluate the exact binding domain of the chimeric mAb to human DLL3, the following cell-based binding assays were performed. Briefly, these assays used HEK293 cells with a series of overlapping ECDs stably expressing human DLL3. Human DLL3 protein (UniProt NO. Q9NYJ7, A27-K618) has a unique extracellular structure (A27-L492), which consists of a DSL domain, six EGF-like repeat sequences, and an unstructured proximal extracellular region (MPER) adjacent to the transmembrane domain (TM). Therefore, cell lines expressing TM and ICD conjugates of DLL3 were constructed (custom-made by Genomeditech). These cell lines included EGF1 and EGF2 (EGF1+2, A216-E310), EGF2 and EGF3 (EGF2+3, G274-E351), EGF3 and EGF4 (EGF3+4, S312-E389), EGF4 and EGF5 (EGF4+5, R353-R427), EGF5 and EGF6 (EGF5+6, D393-E465), and EGF6 and MPER (EGF6-MPER, R429-L492).The specified DLL3 chimeric antibody, benchmark antibody, or isotype control was diluted to 100 nM in a 96-well microplate and incubated with 5 x 10⁴ specified cells at 4°C for 30 min. The antibodies binding to the cell surface antigen were detected using the goat anti-human IgG (H+L) cross-adsorption secondary antibody Alexa Fluor™ 488 (Thermo Fisher Scientific, catalog number A-11013) at a dilution of 1:2000. Cells were analyzed using a MACSQuant® Analyzer 16 flow cytometer (Miltenyi Biotec BV & Co. KG). Data analysis was performed using Flowjo 10.0 software. Graphs were generated and statistical analysis was performed using a four-parameter nonlinear regression curve fitted in Graphpad Prism 9 software.

[0198] As shown in Figure 3 and Table 6, these DLL3 chimeric antibodies bind to different ECD domains of human DLL3 expressed on HEK293 cells. More specifically, 129H2B9 and 148C3A7 bind to the EGF3-4 domain of human DLL3 expressed on cells. 9E8D8 and 310P3C5 specifically bind to the EGF6 domain of human DLL3 expressed on cells. 36B7 and 362H3D3 bind to the proximal extracellular domain, which is similar to the benchmark antibody DLL3#3. Another benchmark antibody, DLL3-4-001 (CC), specifically binds to EGF3, consistent with previously reported data. Instructions for Use, Page 27 / 41, CN 121909211 A

[0199] Table 6. Domain Binding Properties of Chimeric Antibodies

[0200] Example 3. Cell Binding Activity of Chimeric Monoclonal Antibodies Targeting DLL3

[0201] 3.1 Binding Ability with Human DLL3 Overexpressed on HEK-293 Cells

[0202] To evaluate the binding activity of chimeric mAbs with human DLL3 expressed on cells, the following cell-based binding assays were performed. Briefly, HEK293 cells (HEK293-hDLL3) stably expressing high levels of human DLL3 ECD were used in these assays. The designated DLL3 chimeric Ab, reference antibody, or isotype control was diluted 3-fold starting at a concentration of 50 nM in staining buffer (PBS buffer containing 2% FBS). The antibody dilutions were incubated with 5 x 10⁴ indicator cells in 96-well microplates at 4°C for 30 min. Antibodies binding to cell surface antigens were detected using the goat anti-human IgG (H+L) cross-adsorption secondary antibody Alexa Fluor™ 488 (Thermo Fisher Scientific, catalog number A-11013).The dilution rate was 1:2000. Cells were analyzed using a MACSQuant® Analyzer 16 flow cytometer (Miltenyi Biotec BV & Co. KG). Data analysis was performed using Flowjo 10.0 software. Graphs were generated and statistical analysis was performed using four-parameter nonlinear regression curve fitting in Graphpad Prism 9 software.

[0203] As shown in Figure 4A, all DLL3 chimeric antibodies bound efficiently to human DLL3 expressed on HEK293 cells in a dose-dependent manner. Notably, compared to the baseline antibodies DLL3-4-001 (CC) and DLL3#3, the chimeric antibodies 129H2B9, 148C3A7, 310P3C5, and 362H3D3 showed higher binding potency to human DLL3 expressed on HEK293 cells, as indicated by improved EC50 compared to DLL3-4-001 (CC) or improved maximum binding signal compared to DLL3#3. The maximum binding potency of 9E8D8 and 36B7F3 to human DLL3 expressed on HEK293 cells was comparable to the maximum binding potency of EGF3 to the baseline DLL3-4-001 (CC).

[0204] Table 7 summarizes the EC50 values ​​of binding potency to human DLL3 expressed on cells.

[0205] 3.2 Binding potency to cyno DLL3 overexpressed on cells

[0206] To evaluate the species cross-reactivity of the chimeric mAb to cynomolgus monkey DLL3 expressed on cells, the following cell-based binding assay was performed according to the previously described protocol. Briefly, HEK293 cells stably expressing full-length human cyno DLL3 (HEK293-cynoDLL3) (custom-made by Cusabio) were constructed. Specified DLL3 chimeric Abs, baseline antibodies, or isotype controls were tested in this assay.

[0207] As shown in Figure 4B, all DLL3 chimeric antibodies effectively bound to cyno DLL3 expressed on HEK293 cells, with binding efficacy comparable to their binding efficacy to human DLL3.

[0208] Table 7 summarizes the EC50 of the binding ability to cynomolgus monkey DLL3 expressed on cells.

[0209] 3.3 Binding to Human DLL3 Expressed on Tumor Cells Specification 28 / 41 pages 32 CN 121909211 A

[0210] To evaluate the binding activity of the chimeric mAbs to human DLL3 expressed on tumor cells, the human SCLC cell lines SHP77 and NCI-H82, which express low levels of DLL3, were used in a cell-based binding assay according to the protocol described in Example 3.1.

[0211] As shown in Figures 5A and 5B, all DLL3 chimeric antibodies effectively bound to human DLL3 expressed on SCLC tumor cell lines SHP77 or NCI-H82. Interestingly, unlike the binding trend on HEK293 cells overexpressing DLL3, in these two tumor cell lines expressing DLL3, DLL3 mAbs 129H2B9 and 148C3A7, which bind to the distal membrane region, showed significantly better binding efficacy than mAbs that bind to the proximal membrane region (including 9E8D8, 36B7F3, 310P3C5, and 362H3D3), indicating that the distal membrane conjugates have stronger binding ability (Figures 5A and 5B).

[0212] 3.4 Binding ability with human DLL1 and DLL4 overexpressed on cells

[0213] To exclude non-specific binding of chimeric mAbs to human DLL1 and DLL4 expressed on cells, cell-based binding assays were used as described above. In short, CHO-K1 cells stably expressing full-length human DLL1 and DLL4 (CHO-K1-hDLL1 and CHO-K1-hDLL4, Genomeditech) were constructed. The sequences of human DLL1 and DLL4 used in this example were derived from UniProt (O00548 and Q9NR61). Protein expression was confirmed by FACS analysis using anti-human DLL1 (R&D Systems, MAB1818) and anti-human DLL4 (R&D Systems, MAB1506) antibodies. Designated DLL3 chimeric antibodies, baseline antibodies, or isotype controls were tested in the assay.

[0214] As shown in Figure 6A, in contrast to the significant binding ability of the positive control DLL1 mAb pidiliterizumab to DLL1, no specific binding to human DLL1 expressed on CHO-K1 cells was detected in most DLL3 chimeric antibodies (including 9E8D8, 36B7F3, 129H2B9, 148C3A7, 310P3C5, and two benchmark antibodies). 362H3D3 only showed weak binding to DLL1 overexpressed on CHO-K1 cells at the highest dose level of 100 nM. Furthermore, similar to the two benchmark antibodies DLL3-4-01 (CC) and DLL3#3, the binding of all DLL3 chimeric antibodies to DLL4 overexpressed on CHO-K1 cells was negligible, which is in contrast to the significant binding strength of the DLL4-specific positive control antibody MLCK-2 (Figure 6B). Overall, all chimeric antibodies exhibited high specificity in binding to human DLL3 expressed on cells.

[0215] Table 7. Binding characteristics of chimeric antibodies to different antigens

[0216] --: No binding

[0217] Example 4. Humanization of chimeric DLL3 antibodies

[0218] The variable region of the 310P3C5 chimeric antibody was selected for humanization. (Instructions for use, pages 29 / 41, 33 CN 121909211 A)

[0219] In short, the amino acid sequences of VH and VL were compared with existing human Ig gene sequence databases to determine the most compatible human germline Ig gene sequence. Then, the CDRs of the chimeric antibody heavy and light chains were transplanted into candidate germlines. A 3D model of the CDR-transplanted antibody was generated using a Molecular Operating Environment (MOE) to determine if any key human amino acids in the frame region must be reverted to their corresponding mouse amino acids to maintain CDR conformation and antibody function.

[0220] For the heavy chain of 310P3C5, the candidate germline sequence is the IGKV1-18 O1 or IGKV1-3 O1 gene. For the light chain of 310P3C5, the candidate germline sequence is the IGKV1-12 O1 or IGKV1-33 O1 gene. For the heavy chain, M48I, R67Q, V68A, M70L, T72V, and T74K in the IGKV1-18 O1 framework and R38K, M48I, V68A, I70L, R72V, T74K, and S84R in the IGKV1-3 O1 framework participated in reversion mutations. For the light chain, Y49S, Y49S, and T69K in the IGKV1-33 O1 and IGKV1-12 O1 framework regions participated in reversion mutations, respectively.

[0221] Different combinations of reversion mutation sites were selected to generate the variable region of the humanized antibody. Table 8A lists the sequences of the variable regions of the heavy and light chains of the 310P3C5 humanized antibody. Table 8B lists the pairings of VH and VL of a single humanized antibody. Then, the variable region of the humanized antibody was fused with the constant region of human IgG1 for antibody production and functional characterization. Table 8C lists the complete sequences of the heavy and light chains of the humanized antibody.

[0222] Table 8A. Variable region sequence of humanized antibody (underline / bold indicates CDR; bold / italic indicates reversion mutation) Specification 30 / 41 pages 34 CN 121909211 A

[0223] Table 8B. Pairing of humanized antibody VH and VL Specification 31 / 41 pages 35 CN 121909211 A

[0224] Table 8C. Sequence of heavy and light chains of humanized antibody Specification 32 / 41 pages 36 CN 121909211 A Specification 33 / 41 pages 37 CN 121909211 A Specification 34 / 41 pages 38 CN 121909211 A Specification 35 / 41 pages 39 CN 121909211 A Specification 36 / 41 pages 40 CN 121909211 A

[0225] Example 5. Antigen-binding properties of humanized antibodies

[0226] 5.1 Binding with recombinant human DLL3

[0227] To evaluate antigen-binding activity, humanized antibodies were tested by ELISA as described above. As shown in Figure 7, the binding efficacy of all humanized 310P3C5 antibodies to human DLL3 protein was comparable to that of their parental chimeric antibodies.

[0228] 5.2 Affinity ranking of humanized antibodies using Biacore™

[0229] To explore whether humanized antibodies could maintain their binding kinetics, single-dose affinity ranking was performed using Biacore™. Antibodies (2 μg / ml) were captured using a protein A chip. 100 nM of human DLL3-his protein was injected onto the captured antibodies at a flow rate of 30 μL / min for 120 s. Antigen dissociation was allowed for 400 s. Experiments were performed on a Biacore™ 8K. Data analysis was performed using Biacore™ 8K evaluation software.

[0230] The results in Table 9 show that several humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14 and 310P3C5-z15, have comparable affinity to their chimeric antibodies.

[0231] Table 9. Affinity ranking results of humanized antibodies. Specification 37 / 41 pages 41 CN 121909211 A

[0232] 5.3 Binding to HEK293 cells overexpressing human DLL3 and cynomolgus monkey DLL3

[0233] To evaluate the binding characteristics with human DLL3 and cynomolgus monkey DLL3 overexpressed on the cells, the selected humanized antibodies were analyzed by FACS according to the protocol described in the foregoing examples.

[0234] As shown in Figures 8A and 8B, compared with the chimeric antibody, all selected humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14, and 310P3C5-z15, showed binding to human DLL3 overexpressed on HEK293 cells, which was significantly better than the two benchmark antibodies DLL3-4-001 (CC) and DLL3#3. Furthermore, all these humanized mAbs also bound to cyno DLL3 expressed on HEK293 cells.

[0235] 5.4 Binding to Human DLL3 Expressed on Cancer Cell Lines

[0236] To evaluate the binding characteristics to human DLL3 expressed on cancer cells, the selected humanized antibodies were analyzed by FACS according to the previously described protocol.

[0237] As shown in Figures 9A and 9B, compared with chimeric antibodies, including 310P3C5-z4, 310P3C5-z8, and 310P3C5-All selected humanized antibodies, including z12, 310P3C5-z14, and 310P3C5-z15, showed binding to endogenously expressed human DLL3 on tumor cells SHP77 and NCI-H82, which was superior to the baseline antibody DLL3#3.

[0238] 5.5 Binding to overexpressed human DLL1 and DLL4 on CHO-K1 cells

[0239] To rule out non-specific binding of humanized antibodies to expressed human DLL1 and DLL4 on cells, the selected humanized antibodies were analyzed by FACS according to the protocol described in Example 42, CN 121909211 A, pages 38 / 41 of the aforementioned specification.

[0240] As shown in Figures 10A and 10B, all humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14, and 310P3C5-z15, showed negligible binding to human DLL1 and DLL4 overexpressed on CHO-K1 cells.

[0241] Example 6. Optimization of Humanized Antibodies

[0242] In some cases, humanized antibodies are further optimized to improve their exploitability, including long-term stability, manufacturability, and low heterogeneity. One key factor, post-translational modifications (PTMs), such as deamidation, isomerization, glycosylation, and oxidation, can affect exploitability and further impair the potency, efficacy, and safety of therapeutic antibodies. In this example, computational tools were used to predict PTM-prone sites to facilitate the engineering of antibodies with better exploitability.

[0243] 6.1 Design of PTM Site Removal

[0244] In this case, MOE was used to predict the PTM-prone site. Amino acid N55, located in the CDR region of the 310P3C5 antibody heavy chain, was identified as a potential deamidation site. Therefore, this amino acid was replaced with A, F, H, R, V, W, or Y. Table 10A shows the variable region sequence of the PTM-removed humanized antibody, and Table 10B shows the modified VH CDR2 sequence. The variable region of the humanized antibody was then fused with the constant region of human IgG1 for antibody production and functional characterization.

[0245] Table 10A. Sequences of PTM-removed humanized antibody variable regions (underlined CDRs; bold / italicized indicates predicted PTM sites and mutations) Specification 39 / 41 pages 43 CN 121909211 A

[0246] Table 10B. Optimized VH CDR2 sequences Specification 40 / 41 pages 44 CN 121909211 A

[0247] 6.2 Binding of PTM-removed humanized antibody to HEK293 cells expressing human DLL3

[0248] In order to evaluate the binding characteristics with human DLL3 expressed on the cells, the protocol described in the foregoing examples was followed.FACS analysis was performed on PTM-removed humanized antibodies.

[0249] As shown in Figures 11A and 11B, all selected PTM-removed humanized antibodies showed binding to human DLL3 overexpressed on HEK-293 cells or endogenously expressed in the tumor cell line SHP77, compared to the parental humanized antibody 310P3C5-z15.

[0250]

[0251] The scope of this disclosure is not limited to the particular embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations of this disclosure, provided they are within the scope of the appended claims and their equivalents.

[0252] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated to be incorporated by reference. Instruction manual page 41 / 41 45 CN 121909211 A Figure 1A Figure 1B Instruction manual drawing page 1 / 11 46 CN 121909211 A Figure 1C Figure 2A Instruction manual drawing page 2 / 11 47 CN 121909211 A Figure 2B Figure 3 Instruction manual drawing page 3 / 11 48 CN 121909211 A Figure 4A Figure 4B Instruction manual drawing page 4 / 11 49 CN 121909211 A Figure 5A Figure 5B Instruction manual drawing page 5 / 11 50 CN 121909211 A Figure 6A Figure 6B Instruction manual drawing page 6 / 11 51 CN 121909211 A Figure 7 Figure 8A Instruction manual drawing page 7 / 11 52 CN 121909211 A Figure 8B Figure 9A Instruction manual drawing page 8 / 11 53 CN 121909211 A Figure 9B Figure 10A Appendix to the Instruction Manual, Page 9 / 11, 54 CN 121909211 A Figure 10B Figure 11A Appendix to the Instruction Manual, Page 10 / 11, 55 CN 121909211 A Figure 11B Appendix to the Instruction Manual, Page 11 / 11, 56 CN 121909211 A

Claims

1. An antibody or an antigen-binding fragment thereof, which is specific for human Delta-like ligand 3 (DLL3) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3, and the VL comprises VL CDR1, VL CDR2, and VL CDR3, wherein: (a) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 37; The VH CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and 111-117; The VH CDR3 contains the amino acid sequence of SEQ ID NO: 39; The VL CDR1 contains the amino acid sequence of SEQ ID NO: 40; The VL CDR2 contains the amino acid sequence of SEQ ID NO: 41; and The VL CDR3 contains the amino acid sequence of SEQ ID NO: 42; (b) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO:13-18; (c) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO:19-24; (d) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequences of SEQ ID NO:25-30; (e) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO:31-36; or (f) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO:43-48.

2. The antibody or its antigen-binding fragment according to claim 1, wherein: (a) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 37; The VH CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and 111-117; The VH CDR3 contains the amino acid sequence of SEQ ID NO: 39; The VL CDR1 contains the amino acid sequence of SEQ ID NO: 40; The VL CDR2 contains the amino acid sequence of SEQ ID NO: 41; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:

42.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110 or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110.

4. The antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104-110, and the VL comprises the amino acid sequence of SEQ ID NO:

73.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein (b) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 13-18.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 or a peptide having at least 90% sequence identity with SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 2 or a peptide having at least 90% sequence identity with SEQ ID NO:

2.

8. The antibody or antigen-binding fragment thereof according to claim 7, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 49 and a light chain containing the amino acid sequence of SEQ ID NO:

50.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein (c) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 19-24.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the VH comprises the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 90% sequence identity with SEQ ID NO: 3, and the VL comprises the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 90% sequence identity with SEQ ID NO:

4.

11. The antibody or antigen-binding fragment thereof according to claim 10, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 51 and a light chain containing the amino acid sequence of SEQ ID NO:

52.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein (d) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 25-30.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5 or a peptide having at least 90% sequence identity with SEQ ID NO: 5, and the VL comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 90% sequence identity with SEQ ID NO:

6.

14. The antibody or antigen-binding fragment thereof according to claim 13, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 53 and a light chain containing the amino acid sequence of SEQ ID NO:

54.

15. The antibody or antigen-binding fragment thereof according to claim 1, wherein (e) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 31-36.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 or a peptide having at least 90% sequence identity with SEQ ID NO: 7, and the VL comprises the amino acid sequence of SEQ ID NO: 8 or a peptide having at least 90% sequence identity with SEQ ID NO:

8.

17. The antibody or antigen-binding fragment thereof according to claim 16, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 55 and a light chain containing the amino acid sequence of SEQ ID NO:

56.

18. The antibody or antigen-binding fragment thereof according to claim 1, wherein (f) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 43-48.

19. The antibody or antigen-binding fragment thereof according to claim 18, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 or a peptide having at least 90% sequence identity with SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 12 or a peptide having at least 90% sequence identity with SEQ ID NO:

12.

20. The antibody or antigen-binding fragment thereof according to claim 19, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 59 and a light chain containing the amino acid sequence of SEQ ID NO:

60.

21. An antibody or an antigen-binding fragment thereof, which is specific for human Delta-like ligand 3 (DLL3) protein and competitively binds to the DLL3 protein with an antibody or a fragment thereof according to any one of claims 1-20.

22. An antibody or an antigen-binding fragment thereof, which is specific for human Delta-like ligand 3 (DLL3) protein and binds to an EGF3-4 domain or an EGF6 domain.

23. The antibody or fragment thereof according to any one of claims 1-22, wherein the antibody or fragment thereof is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

24. The antibody or fragment thereof according to any one of claims 1-23, wherein it is humanized.

25. A multispecific antibody comprising an antigen-binding fragment according to any one of claims 1-23 and one or more antibodies or antigen-binding fragments having binding specificity to non-DLL3 target antigens.

26. A chimeric antigen receptor (CAR) comprising an antigen-binding fragment, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain according to any one of claims 1-25.

27. One or more polynucleotides encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-25 or a CAR according to claim 26.

28. The polynucleotide of claim 27, wherein it is one or more mRNAs.

29. The polynucleotide of claim 28, wherein the mRNA is chemically modified.

30. A cell comprising the polynucleotide according to claim 28 or 29.

31. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-25 or a CAR according to claim 26, and a pharmaceutically acceptable carrier.

32. A method of treating cancer in a patient in need, the method comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-25, or a CAR according to claim 26.

33. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-25 or the CAR according to claim 26 for the preparation of a medicament for treating cancer.

34. The method of claim 32 or the use of claim 33, wherein the cancer is selected from the group consisting of: ovarian cancer, prostate cancer, urinary tract cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer.

35. The method of claim 32 or the use of claim 33, wherein the cancer is small cell lung cancer (SCLC).

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