Antibody binding to DLL3 and use thereof

Abstract

The present invention relates to an antibody capable of specifically binding to DLL3 or an antigen-binding portion thereof, an antibody-drug conjugate containing the antibody or antigen-binding portion thereof, and the use thereof in the treatment of DLL3-positive tumors.

Description

Antibodies that bind to DLL3 and their applications Invention Field

[0001] This application relates to an antibody or antigen-binding portion thereof capable of specifically binding to DLL3 in humans, monkeys, and mice, and an immunoconjugate comprising the antibody or antigen-binding portion thereof. This application also relates to the use of the antibody or antigen-binding portion thereof, or the immunoconjugate thereof, in the treatment or alleviation of DLL3-related tumors such as small cell lung cancer. Background Technology

[0002] The Notch signaling pathway is a relatively conserved pathway in evolution, playing a crucial role in cell differentiation, proliferation, survival, and apoptosis. Its dysregulation can lead to diseases such as tumors. The mammalian Notch signaling pathway involves four receptors, four classical activating ligands, at least one inhibitory ligand, and non-classical ligands. These ligands and receptors interact within the same cell or between cells, determining whether the signaling pathway is activated and to what extent.

[0003] Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand, normally located in the Golgi apparatus and cytoplasmic vesicles of the cell, and only moves to the cell surface when overexpressed (Geffers I et al., (2007) Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo. J Cell Biol. 178(3):465-76). When attached to the cell surface, it exists in a single transmembrane form, containing, from the N-terminus to the C-terminus, a DSL domain, an epidermal growth factor (EGF)-like repeat, a transmembrane domain, and an intracellular domain. The DSL is a necessary structure for Notch receptor binding, while the intracellular domain is short and its function is unknown. Human DLL3 protein contains six extracellular EGF-like repeats. DLL3 typically inhibits the Notch signaling pathway by causing Notch receptors and / or activating ligands to remain intracellularly and not appear on the cell surface through intracellular interactions (Ladi E et al., (2005) The divergent DSL ligand DLL3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands. J Cell Biol. 170(6):983-92; Chapman G et al., (2011) Notch inhibition by the ligand DELTA-LIKE 3 defines the mechanism of abnormal vertebral segmentation in spondylocostal dysostosis. Hum Mol Genet. 20(5):905-916; Deng SM et al., (2017) The Notch ligand delta-like 3 promotes tumor growth and inhibits Notch signaling in lung cancer cells in mice. Biochem Biophys Res Commun.483(1):488-494).

[0004] DLL3 is a downstream agent of ASCL1, a transcription factor associated with pulmonary neuroendocrine cells, and is considered to be closely related to the development of neuroendocrine tumors, especially lung cancer (Furuta M et al., (2019) Analysis of DLL3 and ASCL1 in Surgically Resected Small Cell Lung Cancer (HOT1702). Oncologist. 24(11):e1172-e1179). Consistent with this, DLL3 is highly expressed in small cell lung cancer (SCLC) and some other neuroendocrine tumors, while it is almost undetectable on the surface of normal cells. In SCLC, DLL3 promotes SCLC development by inhibiting the Notch signaling pathway, downregulating the expression levels of HES1 and HEY1, and increasing cyclin expression, thus promoting SCLC cell proliferation (Deng SM et al., (2017) The Notch ligand delta-like 3 promotes tumor growth and inhibits Notch signaling in lung cancer cells in mice. Biochem Biophys Res Commun. 483(1):488-494). In addition, DLL3 promotes the proliferation and invasion of pituitary adenomas, as well as the growth and survival of melanomas (Wang J et al., (2017) EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2 / DLL3 signaling pathway. Tumor Biol. 39(7):1010428317706203; Ding X et al., (2019). Knockdown of Delta-like 3 restricts lipopolysaccharide-induced inflammation, migration and invasion of A2058 melanoma cells via blocking Twist1-mediated epithelial-mesenchymal transition. Life Sci. 226:149-155).However, there are also reports that DLL3 expression can induce apoptosis in hepatocellular carcinoma and play a negative regulatory role in tumor growth (Maemura K et al., (2013) Delta-like 3 is silenced by methylation and induces apoptosis in human hepatocellular carcinoma. Int J Oncol. 42(3):817-22).

[0005] Due to its abnormally high expression on the surface of related tumor cells and its anti-tumor effects, DLL3 has become a popular therapeutic target. The main types of drugs targeting DLL3 include: DLL3-targeting antibody-drug conjugates (ADCs) (such as Rova-T), DLL3-targeting T-cell binders (BiTEs, such as tarlatamab and BI 764532), and DLL3-targeting chimeric antigen receptor (CAR) therapies (such as DLL3-targeting CAR-T lymphocyte therapy and DLL3-targeting CAR-natural killer cell therapy). Rova-T failed to show a better survival advantage in a phase III clinical trial for advanced SCLC, and clinical trials in other neuroendocrine tumors such as neuroendocrine prostate cancer, or solid tumors such as melanoma, are ongoing. Talatumab is currently approved for the treatment of SCLC, but patients may suffer from adverse reactions such as cytokine release syndrome (CNS) and immune effector cell-related neurotoxicity syndrome during treatment (Su, PL et al., (2024), Chakravarthy, K., Furuya, N. et al. DLL3-guided therapies in small-cell lung cancer: from antibody-drug conjugate to precision immunotherapy and radioimmunotherapy. Mol Cancer 23:97).

[0006] Reference to any document in this application is not an admission that such document is prior art. Summary of the Invention

[0007] The inventors of this application provide novel antibodies capable of specifically binding to DLL3 protein, including IgG antibodies and nanobodies, or their antigen-binding portions, which have high binding / affinity for recombinant or expressed human, monkey, or mouse DLL3 protein on the cell surface.

[0008] The antibody or its antigen-binding moiety described in this application can be DLL3+ Endocytosis. When conjugated with cytotoxins, it can be endocytosed by DLL3. + Enthalation kills the DLL3 + The cells exhibited good anti-tumor effects both in vitro and in vivo.

[0009] Therefore, in a first aspect, this application relates to an antibody, particularly a monoclonal antibody, or its antigen-binding portion, capable of binding to a DLL3 protein (e.g., human, monkey, or mouse DLL3 protein), which may comprise i) a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3, and ii) a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 may respectively comprise SEQ ID NO:1, 2, 3, 4, 5, and 6, or ... NO:7, 8, 9, 10, 11 and 12 have amino acid sequences with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0010] In some implementations, the CDR regions of the heavy and light chain variable regions can be determined using the Kabat definition system / method.

[0011] The antibody or its antigen-binding portion may be, for example, mouse-derived, chimeric, or humanized.

[0012] In some embodiments, the heavy chain variable region may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:22, 23, 24, or 25, and the light chain variable region may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:44.

[0013] In some embodiments, the heavy chain variable region may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:26, 27, 28, or 29, and the light chain variable region may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:45.

[0014] This application also relates to an antibody, particularly a monoclonal antibody, or its antigen-binding portion, capable of specifically binding to a DLL3 protein (e.g., human, monkey, or mouse DLL3 protein), which may comprise i) a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3, and ii) a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1, VH-CDR2, and VH-CDR3 can respectively bind to a protein containing SEQ ID NO. The VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable region of the amino acid sequence shown in NO:22, 23, 24, or 25 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and these VH-CDR1, VH-CDR2, and VH-CDR3 can be respectively associated with the region containing SEQ ID. The VL-CDR1, VL-CDR2, and VL-CDR3 of the heavy chain variable region of the amino acid sequence shown in NO:44 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; or the VH-CDR1, VH-CDR2, and VH-CDR3 can be respectively associated with the region containing SEQ ID. The VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable region of the amino acid sequence shown in NO:26, 27, 28, or 29 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and these VH-CDR1, VH-CDR2, and VH-CDR3 can be respectively associated with the region containing SEQ ID. The VL-CDR1, VL-CDR2, and VL-CDR3 regions of the heavy chain variable region shown in NO:45 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. The antibody or its antigen-binding moiety may be, for example, mouse-derived, chimeric, or humanized.

[0015] In some implementations, the CDRs of the heavy and light chain variable regions can be determined using the Kabat, Chothia, IMGT, AbM, or Contact definition systems / methods.

[0016] The antibody or its antigen-binding portion of this application may include a heavy chain constant region and / or a light chain constant region. The heavy chain constant region may be the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, or a functional fragment thereof (such as an Fc region). For example, the heavy chain variable region may be the human IgG1 constant region, containing, for example, the amino acid sequence shown in SEQ ID NO:46. The light chain constant region may be a κ or λ constant region, such as the human κ constant region, containing, for example, the amino acid sequence shown in SEQ ID NO:48. The N-terminus of the heavy chain constant region may be connected to the C-terminus of the heavy chain variable region or its functional fragment, and the N-terminus of the light chain constant region may be connected to the C-terminus of the light chain variable region.

[0017] In some embodiments, the antibody of this application comprises two heavy chains and two light chains, or is composed of two heavy chains and two light chains, wherein each heavy chain comprises the aforementioned heavy chain constant region sequence, heavy chain variable region sequence, and / or CDR sequence, and each light chain comprises the aforementioned light chain constant region sequence, light chain variable region sequence, and / or CDR sequence. In some embodiments, the antibody of this application may be a single-chain antibody (scFv), or may be composed of antibody fragments, such as Fab or F(ab')2 fragments.

[0018] In a second aspect, this application relates to a nanobody, particularly a monoclonal nanobody, capable of binding to a DLL3 protein (e.g., human, monkey, or mouse DLL3 protein), which may contain a variable region comprising CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 may respectively contain amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:13, 14, and 15, SEQ ID NO:16, 17, and 18, or SEQ ID NO:19, 20, and 21. The nanobody may be camel-derived or humanized.

[0019] In some implementations, the CDR of the variable region can be determined using the Kabat definition system / method.

[0020] The variable region of the nanobody may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0021] This application also relates to a nanobody, particularly a monoclonal nanobody, capable of specifically binding to a DLL3 protein (e.g., human, monkey, or mouse DLL3 protein), which may include a variable region comprising CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the variable region comprising the amino acid sequence shown in SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43. The nanobody may be, for example, camel-derived or humanized.

[0022] In some implementations, the CDR of the variable region can be determined by the Kabat, Chothia, IMGT, AbM, or Contact definition system / method.

[0023] This application also relates to a heavy chain antibody, particularly a monoclonal heavy chain antibody, or its antigen-binding portion, capable of binding to a DLL3 protein (e.g., human, monkey, or mouse DLL3 protein), which may include a heavy chain variable region comprising CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 may respectively contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:13, 14, and 15, SEQ ID NO:16, 17, and 18, or SEQ ID NO:19, 20, and 21.

[0024] In some implementations, the CDR of the heavy chain variable region can be determined using the Kabat definition system / method.

[0025] The heavy chain antibody or its antigen-binding moiety can be, for example, camel-derived, chimeric, or humanized.

[0026] The heavy chain variable region of the heavy chain antibody may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0027] This application also relates to a heavy chain antibody, particularly a monoclonal heavy chain antibody, or its antigen-binding portion, capable of specifically binding to a DLL3 protein (e.g., human, monkey, or mouse DLL3 protein), which may contain a heavy chain variable region comprising CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the CDR1, CDR2, and CDR3 of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43. The heavy chain antibody can be, for example, camel-derived, chimeric, or humanized.

[0028] In some implementations, the CDR of the heavy chain variable region can be determined by the Kabat, Chothia, IMGT, AbM, or Contact definition system / method.

[0029] The heavy chain antibody of this application may further comprise a heavy chain constant region. The heavy chain constant region may be a variable region of the IgG, IgD, IgA, IgM, or IgE heavy chain, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a functional fragment thereof (such as an Fc region). In some embodiments, the heavy chain constant region may comprise a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region may comprise both a CH2 domain and a CH3 domain. In some embodiments, the heavy chain constant region is the human IgG1 heavy chain constant region or a functional fragment thereof, for example, comprising the amino acid sequence shown in SEQ ID NO:47. The C-terminus of the heavy chain variable region may be linked to the N-terminus of the heavy chain constant region or its functional fragment.

[0030] In some embodiments, the heavy chain antibody of this application may comprise two heavy chains, or be composed of two heavy chains, wherein each heavy chain comprises the aforementioned heavy chain constant region sequence, heavy chain variable region sequence, and / or CDR sequence.

[0031] The DLL3 antibody or its antigen-binding portion thereof in this application, including IgG antibodies, nanobodies, and heavy chain antibodies or their antigen-binding portions, may be, for example, defucosylated.

[0032] In a third aspect, this application provides an immunoconjugate comprising i) the DLL3 antibody of this application (including IgG antibodies, nanobodies, and heavy chain antibodies) or its antigen-binding portion, and ii) an effector molecule. The DLL3 antibody or its antigen-binding portion may be directly conjugated to the effector molecule or conjugated to the effector molecule via a linker.

[0033] In some embodiments, the immunoconjugate may comprise i) the DLL3 antibody of this application (including IgG antibodies, nanobodies, and heavy chain antibodies) or its antigen-binding portion, ii) a linker, and iii) an effector molecule. The linker may be a cleavable linker, such as a peptide linker, like a protease-cleavable peptide linker. In some embodiments, the linker may be a maleimide (MC)-GGFG peptide.

[0034] Effector molecules can be therapeutic agents, selected from, for example, toxins (such as cytotoxins), radioactive isotopes, chemical substances (such as organic compounds), proteins, and nucleic acids. Effector molecules can also be markers, such as radioactive isotopes or dyes.

[0035] In some implementations, the effector molecule may be a therapeutic agent, such as a toxin.

[0036] In some implementations, the effector molecule may be DX-8951 (CAS No.:171335-80-1), which includes the structure shown in Formula I.

[0037] In some embodiments, the effector molecule may be a DX-8951 derivative, such as DXD (CAS No.: 1599440-33-1), which includes the structure shown in Formula II.

[0038] In some embodiments, the combination of the linker and the effector molecule can be Deruxtecan (MC-GGFG-DXD), which includes the structure shown in Formula III.

[0039] In a fourth aspect, this application provides a nucleic acid molecule that encodes the DLL3 antibody (including IgG antibody, nanobody and heavy chain antibody) of this application or its antigen-binding portion.

[0040] This application also provides an expression vector containing the nucleic acid molecule of this application.

[0041] This application also provides an isolated host cell containing the nucleic acid molecules or expression vectors of this application. In some embodiments, the host cell may be a mammalian cell, such as a CHO cell, for example a mammalian cell expressing a defucosylated antibody or its antigen-binding portion, including, but not limited to, Slc35C1 gene knockout cell lines, FUT8 knockout cell lines, mutant CHO cell line Lec13, rat fusion tumor cell line YB2 / 0, cell lines containing small interfering RNA specifically targeting the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosidase III and Golgi α-mannosidase II.

[0042] Accordingly, this application provides a method for preparing the DLL3 antibody or its antigen-binding portion using host cells, comprising: (i) expressing the DLL3 antibody or its antigen-binding portion in host cells, and (ii) isolating the DLL3 antibody or its antigen-binding portion from the host cells or their cultures.

[0043] Accordingly, this application also provides compositions comprising the DLL3 antibody of this application or its antigen-binding portion, an immunoconjugate, a nucleic acid molecule, an expression vector, or a host cell. In some embodiments, the composition may be a pharmaceutical composition and may also contain a pharmaceutically acceptable carrier.

[0044] In a fifth aspect, this application also provides a method of treating or alleviating a disease associated with DLL3 overexpression in a subject, comprising administering to the subject a therapeutically effective amount of the aforementioned pharmaceutical composition of this application. The disease associated with DLL3 overexpression can be a tumor or cancer. The tumor or cancer can be a solid tumor, such as a neuroendocrine tumor or carcinoma, including but not limited to, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), small cell bladder cancer (SCBC), glioma multiforme, metastatic castration prostate cancer, pulmonary neuroendocrine tumor, prostate cancer, and melanoma.

[0045] In some implementations, the subjects are mammals, particularly humans.

[0046] This application also provides a kill DLL3 + Cells, especially killer DLL3 + Methods involving tumor cells, including making DLL3 + Cells come into contact with the composition of this application. In some embodiments, the method includes killing DLL3 in a subject in need. + Cells, especially killer DLL3+ A method for treating tumor cells includes administering an effective amount of the composition described herein to a subject. DLL3 + Cells can be DLL3 + Neuroendocrine tumors or cancer cells, including but not limited to DLL3 + Small cell lung cancer (SCLC), DLL3 + Large cell neuroendocrine carcinoma (LCNEC), DLL3 + Gastrointestinal neuroendocrine tumor (GI-NEC), DLL3 + Small cell bladder cancer (SCBC), DLL3 + Glioblastoma multiforme, DLL3 + Metastatic castration prostate cancer, DLL3 + Pulmonary neuroendocrine tumor, DLL3 + Prostate cancer, DLL3 + Melanoma cells.

[0047] This application also provides the DLL3 antibody or its antigen-binding moiety, immunoconjugate, nucleic acid molecule, expression vector, host cell, or pharmaceutical composition of this application in the preparation of an agent for treating or alleviating DLL3-related tumors or cancers, or killing DLL3. + Uses in drugs for tumor cells. Attached Figure Description

[0048] The following detailed description, given by way of example but not intended to limit the invention to specific embodiments, can be better understood in conjunction with the accompanying drawings.

[0049] Figure 1 shows the binding affinity of the chimeric E44G4C10 antibody to cells expressing human (A), monkey (B), or mouse (C) DLL3 protein, and the binding affinity of the chimeric 47G1B2 antibody to cells expressing human (D), monkey (E), or mouse (F) DLL3 protein.

[0050] Figure 2 shows the binding affinity of chimeric antibody 43# to cells expressing human (A), monkey (B), or mouse (C) DLL3 protein, chimeric antibody 59# to cells expressing human (D), monkey (E), or mouse (F) DLL3 protein, and chimeric antibody 18# to cells expressing human (G), monkey (H), or mouse (I) DLL3 protein.

[0051] Figure 3 shows the binding affinity of the humanized E44G4C10 antibody to cells expressing human (A), monkey (B), or mouse (C) DLL3 protein, and the binding affinity of the humanized 47G1B2 antibody to cells expressing human (D), monkey (E), or mouse (F) DLL3 protein.

[0052] Figure 4 shows the binding affinity of humanized antibody 43# to cells expressing human (A), monkey (B), or mouse (C) DLL3 protein, humanized antibody 59# to cells expressing human (D), monkey (E), or mouse (F) DLL3 protein, and humanized antibody 18# to cells expressing human (G), monkey (H), or mouse (I) DLL3 protein.

[0053] Figure 5 shows the epitope competitive binding of 47G1B2VH4VL2 (A), 43-VHH5 (B), and E44G4C10VH3VL2 (C) to other humanized antibodies.

[0054] Figure 6 shows a heatmap of competition between the applicant's derived antibodies.

[0055] Figure 7 shows the binding affinity of E44G4C10VH3VL2, 47G1B2VH4VL2, 18-VHH4, 43-VHH5, and 59-VHH5 to the truncated antigen.

[0056] Figure 8 shows the binding activity of humanized antibodies E44G4C10VH3VL2, 47G1B2VH4VL2, 18-VHH4, 43-VHH5 and 59-VHH5 to SHP77 cells.

[0057] Figure 9 shows the mean fluorescence intensity (MFI, A) and percentage of luminescent cells (B) of SHP77 cells treated with each humanized antibody.

[0058] Figure 10 shows the viability of SHP77 cells treated with various humanized antibody-DXD conjugates.

[0059] Figure 11 shows the changes in tumor volume in tumor-bearing mice treated with 18-VHH4-DXD conjugate or E44G4C10VH3VL2-DXD conjugate.

[0060] Figure 12 shows the concentration changes of 18-VHH4-DXD conjugate and E44G4C10VH3VL2-DXD conjugate in mouse serum. Detailed Implementation

[0061] Unless otherwise specified, the terms used herein have their common meanings as found in dictionaries, textbooks, and technical reference books, or as commonly understood by those skilled in the art. The following descriptions of some terms are for the purpose of understanding this application only and are not intended to impose any particular limitations on these terms, unless otherwise specified.

[0062] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form of the object referred to, unless the context clearly specifies otherwise.

[0063] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0064] The terms "comprising" or "including" mean that the stated elements, integers, or steps are included, but do not exclude the inclusion of any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. The terms "consisting of" or "comprises of" generally mean that only the stated elements, integers, or steps are included, without the addition of other elements, integers, or steps.

[0065] The term "DLL3" refers to delta-like ligand 3, an inhibitory Notch ligand. This term includes variants, homologs, orthogonal homologs, and parallel homologs. "Human DLL3" refers to a DLL3 protein having a human amino acid sequence, including, for example, the amino acid sequence shown in SEQ ID NO:49. "Monkey DLL3" refers to a DLL3 protein having a monkey amino acid sequence, including, for example, the amino acid sequence shown in SEQ ID NO:50. "Mouse DLL3" refers to a DLL3 protein having a mouse amino acid sequence, including, for example, the amino acid sequence shown in SEQ ID NO:51.

[0066] The terms “optional” or “optionally” in this document mean that a component or step is not mandatory or necessary to include, that is, a component or step may be included in some cases and may not be included in others.

[0067] The term "antibody" as used herein is intended to include IgG, IgA, IgD, IgE, and IgM antibodies, heavy chain antibodies, and nanobodies, as well as any antigen-binding portion (i.e., antigen-binding part). "Whole antibody" or "full-length antibody" refers to a glycoprotein that contains the complete structure of an immunoglobulin; for example, an IgG whole antibody contains two long chains and two light chains, and a full-length heavy chain antibody contains two heavy chains.

[0068] In some cases, "antibody" in this article refers to IgG, IgA, IgD, IgE, and IgM antibodies, especially IgG antibodies. Each heavy chain can be modified by heavy chain variant regions (abbreviated as V). H It consists of three structural domains: C and the heavy chain constant region. The heavy chain constant region consists of three structural domains, namely C H1 C H2 and C H3 Each light chain can be composed of a light chain variable region (V for short). L It consists of a light chain constant region and a light chain constant region. The light chain constant region consists of a structural domain C. L Composition. V H and V LThe region can also be divided into highly variable regions called complementarity-determining regions (CDRs), which are separated by more conservative framework regions (FRs). Each V H and V L Composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody heavy chain can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system, and can also prolong the antibody's in vivo half-life. The "functional fragment" of the heavy chain constant region refers to the portion of the constant region that retains certain functions, such as guiding antibody binding to host tissues or factors to induce, for example, ADCC, CDC, ADCP, or prolonging antibody half-life.

[0069] In some contexts, "antibody" as used herein refers to a heavy chain antibody or its antigen-binding portion. The term "heavy chain antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks a light chain. Naturally occurring heavy chain antibodies are found in camel-dwelling animals (camels, llamas, or alpacas). Individual camel-derived heavy chain antibodies may contain a variable region and a constant region of the heavy chain; the variable region is called the V region. H H domain, V H H fragment, single-domain antibody, or nanobody (sdAb). V H H interacts with the antigen. V H The heavy chain constant region (H) contains three complementation-determining regions (CDRs) and four framework regions (FRs), arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain constant region may contain a hinge region, a CH2 domain, and a CH3 domain. A missing CH1 domain can be replaced by an extended hinge region. In chimeric or humanized heavy chain antibodies, the heavy chain constant region may contain human IgG, such as the IgG1, IgG2, or IgG4 constant region. The heavy chain constant region can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system, and can also prolong the antibody's in vivo half-life. A "functional fragment" of the heavy chain constant region refers to the portion of the constant region that retains certain functions, such as guiding antibody binding to host tissues or factors to induce, for example, ADCC, CDC, ADCP, or prolonging antibody half-life.

[0070] It has been confirmed that the antigen-binding function of an antibody can be implemented by fragments (antigen-binding portions) of the full-length antibody, including, but not limited to, (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; and (iii) fragments consisting of V... H and C H1 (iv) Fd fragments composed of antibody single-arm V L and V H The Fv segment is composed of (v) and V. H H-component sdAb fragments (Ward et al., (1989) Nature 341:544-546); (vi) separated complementarity-determining regions (CDRs), etc.

[0071] In this article, "specific binding to DLL3" means binding to DLL3 but not binding to non-DLL3 proteins, or binding to DLL3 with a binding strength at least 1, 5, 10, 50, or 100 times higher than the binding strength to non-DLL3 proteins.

[0072] The term "camel-derived antibody" refers to an antibody whose variable region backbone and CDR region are derived from camel genotype immunoglobulin sequences. The camel-derived antibody of this application may contain amino acid residues not encoded by camel genotype immunoglobulin sequences, for example, mutations introduced through in vitro random mutations, point mutations, or in vivo somatic mutations. However, the term "camel-derived antibody" does not include antibodies that insert CDR sequences derived from other mammalian species into the camel backbone sequence. Correspondingly, "mouse-derived antibody" refers to an antibody whose variable region backbone and CDR region are derived from mouse genotype immunoglobulin sequences, and may contain amino acid residues not encoded by camel genotype immunoglobulin sequences, for example, mutations introduced through in vitro random mutations, point mutations, or in vivo somatic mutations.

[0073] The term "chimeric antibody" refers to an antibody obtained by combining non-human genetic material (such as camel or mouse genetic material) with human genetic material. Or, more generally, a chimeric antibody is an antibody that combines genetic material from one species with genetic material from another species.

[0074] The term "humanized antibody" refers to an antibody derived from a non-human species (e.g., camel or mouse) whose protein sequence has been modified to increase its similarity to antibodies naturally generated in the human body.

[0075] The term "EC" 50 "50% maximum effect concentration" is also called the molecular concentration that causes 50% of the maximum effect.

[0076] The term "IC" 50"Half-inhibition concentration" refers to the concentration of molecules required to inhibit a specified biological process by half.

[0077] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, are preferred.

[0078] The term "effective amount" refers to the amount of the antibody or its antigen-binding portion, or immunoconjugate of this application used to achieve the intended result. The term "therapeutic effective amount" refers to the amount of the antibody or its antigen-binding portion, or immunoconjugate of this application used to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). Therapeutic effective amounts are related to the disease being treated, and the actual effective amount can be readily determined by those skilled in the art.

[0079] The term "identity" or "sequence identity" as used herein refers to the percentage of nucleotides / amino acids in a sequence that are identical to those in a reference sequence after sequence alignment. If necessary, spaces are introduced in the sequence alignment to achieve the maximum percentage of sequence similarity between the two sequences. Those skilled in the art can use various methods, such as computer software, to perform pairwise or multiple sequence alignments to determine the percentage of sequence similarity between two or more nucleic acid or amino acid sequences. Such computer software includes, for example, ClustalOmega, T-coffee, Kalign, and MAFFT.

[0080] In this article, "effect molecules" refer to molecules capable of producing a certain effect. Examples include molecules that produce labeling effects, such as dyes or reflective isotopes, or molecules that produce therapeutic effects, such as drugs (chemical, protein, or nucleic acid-based) and toxins. These effect molecules can be directly or via a linker coupled to antibodies or their antigen-binding sites, thereby labeling the antigen when the antibody or its antigen-binding site binds to it, or entering the cell expressing the antigen along with the antibody or its antigen-binding site to exert cytotoxic effects on the cell.

[0081] This application first provides novel antibodies that specifically bind to DLL3, including IgG antibodies, nanobodies, and heavy chain antibodies, or their antigen-binding portions. These antibodies exhibit high binding affinity to recombinant human, monkey, or mouse DLL3 proteins expressed on cell surfaces.

[0082] The antibody or its antigen-binding moiety described in this application can be DLL3 + Endocytosis. When conjugated with cytotoxins, it can be endocytosed by DLL3. + Enthalation kills the DLL3 +The cells exhibited good anti-tumor effects both in vitro and in vivo.

[0083] Furthermore, the antibody or its antigen-binding portion of this application, when carrying a heavy chain constant region that binds to immune system cells or complement system components, also has the potential to induce the immune system's response to DLL3 via mechanisms such as ADCC, CDC, and ADCP. + The antibody or its antigen-binding portion may also be prepared into a bispecific or multispecific molecule, or into a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

[0084] The variable region CDR in the antibody or its antigen-binding portion described in this application, including the heavy chain variable region CDR and the light chain variable region CDR, can be determined by the Kabat definition system / method, or by, for example, the Chothia, IMGT, AbM or Contact definition system / method.

[0085] The SEQ ID NOs of the heavy chain / light chain variable region sequences of the IgG antibody or its antigen-binding portion, and the CDR sequences determined by the Kabat definition system / method, are listed in Table 1. The SEQ ID NOs of the variable region sequences of the nanobodies or heavy chain antibodies of this application, and the CDR sequences determined by the Kabat definition system / method, are listed in Table 2.

[0086] The IgG antibody or heavy chain antibody of this application, or its antigen-binding portion, may include a heavy chain constant region and / or a light chain constant region, where applicable. The heavy chain constant region may, for example, enable the antibody to bind to host tissues or factors to guide, for example, ADCC, CDC, ADCP, or to prolong the half-life of the antibody or its antigen-binding portion.

[0087] The antibody or its antigen-binding portion of this application may contain one or more conserved modifications, such as in the CDR region or other regions. It is known in the art that some conserved sequence modifications do not result in the loss of antigen binding. As used herein, the term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter molecular properties, such as binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or molecule of this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue sets include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Antibodies or molecules obtained after conserved sequence modification can be functionally tested using the functional assays described herein.

[0088] The antibody in this application can be used in V H and / or V L Genetic modifications are made to the backbone residues of antibodies to, for example, alter antibody properties. Backbone modifications include mutations in one or more residues in the backbone region, or even one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also known as "deimmunization," and is described in more detail in US Patent Publication 20030153043.

[0089] In addition to modifications within the backbone or CDR region, the antibodies of this application can be genetically modified to include gene modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cytotoxicity. Furthermore, the antibodies of this application can be chemically modified (e.g., by attaching one or more chemical functional groups to the antibody), or modified to alter its glycosylation, to change one or more functional properties of the antibody.

[0090] In one implementation, C H1 The hinge region is modified, altered, for example, by increasing or decreasing the number of cysteine ​​residues in the hinge region. This method is further described in U.S. Patent 5,677,425. Modification of C... H1Cysteine ​​residues in the hinge region can, for example, promote the assembly of heavy and light chains or increase / decrease antibody stability.

[0091] In another embodiment, the Fc hinge region of the antibody is mutated to increase or decrease the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the C13C ... H2 -C H3 The linker region thus weakens the SpA binding affinity of the antibody relative to the natural Fc-hinge domain SpA binding. This method is described in more detail in U.S. Patent 6,165,745.

[0092] In another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for the antigen. Such glycosylation modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that location. Such deglycosylation can increase the antibody's affinity for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0093] Furthermore, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced fucose residues, or antibodies with increased bisecting GlcNac structures. The altered glycosylation forms have been shown to increase the ADCC activity of the antibodies. Such glycosylation modifications can be performed, for example, by expressing the antibody in host cells with altered glycosylation systems. Cells with altered glycosylation systems are known in the art, including, but not limited to, Slc35c1 gene knockout cell lines, FUT8 knockout cell lines, mutant CHO cell line Lec13, rat fusion tumor cell line YB2 / 0, cell lines containing small interfering RNA specifically targeting the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosyltransferase III and Golgi α-mannosidase II. These can be used as host cells for expressing the recombinant antibody of this application to prepare antibodies with altered glycosylation. Slc35c1 gene knockout cell lines, for example, utilize the fucose knockout platform technology independently developed by Tianguangshi, see the CHO cell line with accession number CGMCC No. 14287 in US Patent No. 10377833B2.

[0094] Another modification to the antibody described herein is polyethylene glycol (PEGylation). Antibodies can be PEGylated, for example, to increase the antibody's biological (e.g., serum) half-life. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that attach one or more PEG groups to the antibody or antibody fragment. Preferably, PEGylation is carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any form of PEG used to derive other proteins, such as mono(C1-C1) PEG. 10 Alkyl- or aryl-oxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of this application. See, for example, EP 0 401 384.

[0095] The antibody in this application may not contain an asparagine isomer site. Deamidation of asparagine may occur in the NG or DG sequence, creating isoaspartic residues, which introduce kinks into the polypeptide chain and reduce its stability (isoaspartic effect).

[0096] On the other hand, the DLL3 antibody or its antigen-binding portion of this application can be conjugated to an effector molecule to form an immune crosslink, such as an antibody-drug crosslink (ADC). The effector molecule can be a therapeutic agent selected from, for example, toxins (such as cytotoxins), radioisotopes, chemicals (such as organic compounds), proteins, and nucleic acids. Suitable therapeutic agents include, but are not limited to, cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalating agents, DNA crosslinking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. The effector molecule can also be a marker, such as a radioisotope or dye. The effector molecule used in the examples is a DX-8951 derivative, a cytotoxic molecule. In an ADC, the antibody or its antigen-binding portion can be directly conjugated to the effector molecule or conjugated via a linker. The conjugation site can be random or specific. When an antibody or its antigen-binding moiety can be directly conjugated to an effector molecule, the conjugation site is usually random, and conjugation can be achieved by incubating the antibody or its antigen-binding moiety with the effector molecule. When an antibody or its antigen-binding moiety is conjugated via a linker, the linker determines the conjugation site. Linkers for immunoconjugates, especially ADCs, are mainly divided into two categories: cleavable linkers and non-cleavable linkers. Cleavable linkers are designed to remain stable in the bloodstream and then release the payload within the cell. Types of cleavable linkers include enzyme-cleavable peptide linkers, acid-sensitive hydrazone linkers, and glutathione-sensitive disulfide linkers. Non-cleavable linkers, such as SMCCs, generally rely on intracellular lysosomal degradation to release the drug payload. Peptide linkers are a type of protease-cleavable linker. Peptide linkers have been developed as key components of antibody-drug conjugates, playing a crucial role in the overall success of ADC drugs due to their excellent plasma stability and controlled payload release mechanism. Well-designed linkers help antibodies selectively deliver cytotoxic drugs to tumor cells and accurately release the drug at the tumor site. The ADC constructed in the embodiments of this application includes a linker, specifically a maleimide (MC)-GGFG peptide.

[0097] The DLL3 antibody or its antigen-binding portion of this application can be linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to form a bispecific molecule. The term "bispecific molecule" includes molecules having three or more specificities.

[0098] This application also provides a chimeric antigen receptor comprising the DLL3 antibody of this application or its antigen-binding portion. A chimeric antigen receptor generally comprises (a) an extracellular antigen-binding domain containing the antibody; (b) a transmembrane domain; and (c) an intracellular signal transduction domain.

[0099] Oncolytic viruses preferentially infect and kill cancer cells. The DLL3 antibody or its antigen-binding portion of the present invention can be used in conjunction with oncolytic viruses. Furthermore, oncolytic viruses encoding the DLL3 antibody or its antigen-binding portion of the present application can be introduced into the human body.

[0100] On the other hand, this application provides nucleic acid molecules encoding the antibody of this application or its antigen-binding portion. Nucleic acids can be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “isolated” or “substantially pure” after purification from other cellular components or other contaminants such as other cellular nucleic acids or proteins using standard techniques. The nucleic acid of this application can be, for example, DNA or RNA, and may or may not contain intron sequences.

[0101] DNA molecules encoding the antibody or its antigen-binding portion of this application can be inserted into one or more expression vectors, operatively linking them to transcriptional and translational regulatory sequences. In this context, the term "operatively linking" means that the nucleic acid molecule of this application is linked to the vector, thereby allowing the transcriptional and translational control sequences within the vector to perform their intended functions of regulating the transcription and translation of the nucleic acid molecule. "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Regulatory sequences for mammalian host cell expression include viral elements that guide high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses such as the adenovirus major late promoter (AdMLP), and polyomaviruses. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or β-globin promoters, can be used. Additionally, regulatory elements may consist of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and long terminal repeats of human T-cell leukemia virus type I. The expression vector and expression control sequence were selected to be compatible with the expression host cells used.

[0102] Expression vectors can be transfected into host cells using standard techniques. The term "transfection" encompasses various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextrose transfection. While expressing the antibodies described in this application in prokaryotic or eukaryotic host cells is theoretically feasible, antibodies can be expressed in eukaryotic cells, particularly mammalian host cells, because eukaryotic cells, especially mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies. Examples of expression vectors available for this application include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), transformable artificial chromosomes (TAC), mammalian artificial chromosomes (MAC), and artificially attached chromosomes (HAEC).

[0103] In another aspect, the present invention provides a composition comprising the DLL3 antibody (including IgG antibody, heavy chain antibody) of this application or its antigen-binding moiety, an immunoconjugate, a nucleic acid molecule, an expression vector, or a host cell. In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier. The composition may optionally comprise one or more other pharmaceutically active ingredients, such as another anticancer agent.

[0104] Pharmaceutical compositions may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspenders, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof.

[0105] The pharmaceutical composition may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or bolus). Depending on the route of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate it. “Parenteral administration” refers to a method other than intestinal and topical application, typically administered by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, sub-bursular, subarachnoid, spinal, supradural, and intrasternal injections and boluses. Alternatively, the pharmaceutical composition of this application may be administered via non-parenteral routes, such as topical, epidermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical application.

[0106] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high concentrations of drugs.

[0107] The amount of active ingredient prepared together with the carrier material into a single dosage form will vary depending on the therapeutic subject and specific administration mode, and is essentially the amount of the composition that produces the therapeutic effect. In percentage terms, this amount is approximately 0.01% to approximately 99% of the active ingredient bound to a pharmaceutically acceptable carrier.

[0108] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, rapid infusion can be administered, multiple fractions can be administered over time, or the dose can be proportionally reduced or increased depending on the severity of the treatment condition. Particularly advantageous are parenteral compositions formulated in convenient and uniformly dose-unit formats. A dose-unit format refers to physically separate units suitable for a single dose to the therapeutic subject; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect when used with the pharmaceutical carrier. Alternatively, antibodies can be administered as sustained-release formulations, in which case the required frequency of administration is reduced.

[0109] The pharmaceutical composition can be a sustained-release agent, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable and biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The pharmaceutical composition can be administered via medical devices, such as needle-free subcutaneous injection devices, microinfusion pumps, transdermal drug delivery devices, injection devices, and permeation devices. In some embodiments, the pharmaceutical composition of the present invention can be formulated to ensure suitable in vivo distribution. For example, to ensure that the pharmaceutical composition of this application crosses the blood-brain barrier, it can be formulated in liposomes, which may additionally contain targeting functional groups to enhance selective delivery to specific cells or organs.

[0110] The antibody or its antigen-binding moiety, or immunoconjugate (when the effector molecule is a therapeutic agent, etc.) of this application can be used to kill DLL3. + Cells, or for the treatment or relief of DLL3-related cancers, such as cancers that overexpress DLL3.

[0111] Furthermore, the antibody or its antigen-binding moiety, or the immunoconjugate (when the effector molecule is a dye, etc.) of this application can target and label DLL3. + Cells or tissues, especially tumor cells or tissues.

[0112] This application provides a combination therapy in which the pharmaceutical composition of this application is administered together with one or more other therapeutic agents, such as immune enhancers. The combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions wherein each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially. Furthermore, if multiple administrations of the combination therapy are performed, and the agents are administered sequentially, the order of sequential administration at each time point can be reversed or kept the same; sequential administration can be combined with simultaneous administration or any combination thereof.

[0113] Various aspects and implementations of this application will be discussed with reference to the accompanying drawings and the following embodiments. Other aspects and implementations will be apparent to those skilled in the art. All documents described herein are incorporated herein by reference in their entirety. Although this application has been described in conjunction with exemplary embodiments, many equivalent modifications and variations were apparent to those skilled in the art at the time of this application. Therefore, the exemplary embodiments of this application are exemplary and not limiting. Various changes can be made to the described embodiments without departing from the spirit and scope of this application.

[0114] Example

[0115] Example 1. Preparation and screening of mouse-derived DLL3 monoclonal antibodies

[0116] mouse immunization

[0117] Ten BALB / c mice (Vitollivan, China) were immunized with recombinant human DLL3-his (Cat#:DL3-H52H4, Bipsy, China) every two weeks, 50 μg per mouse per injection, for a total of five injections. Human DLL3-his protein was emulsified using equal volumes of complete Freund's adjuvant (Cat#:F5881-10*10ML, Sigma, USA), incomplete Freund's adjuvant (Cat#:F5506-6*10ML, Sigma, USA), or PBS via ultrasonic emulsification. All ten mice were used for the next step of hybridoma cell line preparation.

[0118] Preparation of hybridoma cell lines

[0119] Hybridoma cell lines were prepared using hybridoma fusion technology, with slight modifications to the protocol.

[0120] Four days after the final immunization, mice were sacrificed, and spleens were harvested and prepared into single-cell suspensions in PBS. Spleen cells were washed three times with DMEM medium (Cat#: SH30243.01B, Hyclone, USA). Mouse myeloma cells in logarithmic growth phase SP2 / 0 (Cat#: CRL-1581, ATCC, USA) were mixed with the isolated mouse spleen cells at a 1:4 ratio and washed twice with DMEM. Cell fusion was performed using PEG (Cat#: P7181, Sigma, USA). The fused cells were washed three times with DMEM and resuspended in cell growth medium (RPMI 1640 (Cat#: C22400500CP, Gibco, USA) + 10% FBS + 1×HAT (Cat#: H0262, Sigma, USA)). The cell suspension was plated in 96-well plates at 200 μl per well, incubated at 5×10⁻⁶ cm⁻¹. 4 Cells were cultured in each well at 37°C in a humidified cell culture incubator with 5% CO2 for 7 days. Afterward, the culture medium was replaced with fresh medium (DMEM + 10% FBS + 1X HAT). Two to three days later, the cell culture supernatant was aspirated, and hybridoma cells were selected by ELISA.

[0121] Hybridoma cell lines were screened using ELISA.

[0122] Using human DLL3-his (Cat#:DL3-H52H4, Bipsys, China), hybridoma clones binding to human DLL3 were screened using high-throughput ELISA binding assays. Two hybridoma cell lines with specific binding affinity to human DLL3 were identified by the ELISA assay. These two hybridoma clones underwent two rounds of subcloning. During subcloning, multiple subclones (n>3) of each clone were selected and characterized using the aforementioned ELISA assay. The subclones obtained through this step were identified as monoclonal hybridoma cell lines. Ultimately, E44G4C10 and 47G1B2 were obtained, exhibiting high binding affinity to human DLL3.

[0123] Hybridoma cells that produced both clones were placed in T175 cell culture flasks containing 100 ml of fresh serum-free hybridoma medium (Cat#: 12045-076 Gibco, USA) and 1% HT supplement (Cat#: 11067-030 Gibco) and cultured at 37°C in a 5% CO2 incubator for 10 days. The cultures were collected, centrifuged at 3500 rpm for 5 min, and filtered through a 0.22 μm filter to remove cell debris. Monoclonal antibodies were enriched and purified using a pre-equilibrated protein-A affinity column (Cat#: 17040501, GE, USA). Elution was then performed with elution buffer (20 mM citrate, pH 3.0–3.5). The antibodies were then stored in PBS (pH 7.0), and antibody concentrations were detected using NanoDrop. The isotypes of purified antibodies were determined using a rapid typing kit for κ and λ- mice (Cat#:26179, Thermal, USA) and a mouse monoclonal antibody typing kit (Cat#:IS02-1KT, Sigma, USA), with the detection procedures consistent with the kit instructions. E44G4C10 produced IgG1 / κ antibody with an expression titer of 29.15 mg / L; 47G1B2 produced IgG1 / κ antibody with an expression titer of 38.47 g / L.

[0124] Example 2. Preparation and screening of camel-derived DLL3 monoclonal antibodies

[0125] Camel Immunity

[0126] Recombinant human DLL3-his (Cat#:DL3-H52H4, Bipsys, China) was injected into Bactrian camels in Xinjiang once a week, 200 μg each time, for a total of 6 injections. The human DLL3-his protein was ultrasonically emulsified with an equal volume of complete Freund's adjuvant (Cat#:F5881-10*10ML, Sigma, USA) or incomplete Freund's adjuvant (Cat#:F5506-6*10ML, Sigma, USA). One week after the sixth immunization, serum was collected from the neck of Bactrian camels in Xinjiang for the next step of phage library preparation.

[0127] Preparation of phage libraries

[0128] One week after the completion of six immunizations, 100 ml of serum was collected from the neck of Bactrian camels in Xinjiang. Total RNA was extracted, cDNA was synthesized, and V was amplified using nested PCR. H H. 20 μg of pMECS phage display vector and 10 μg of V were digested with restriction endonucleases PstⅠ and NotⅠ. H H, and ligated two fragments. The ligation product was transformed into electrocompetent TG1 cells to construct a nanobody phage display library and determine the library size, which was approximately 1 × 10⁻⁶. 9 .

[0129] Enrichment and screening of phage libraries

[0130] 200 μL of recombinant TG1 cells were cultured in 2×TY medium. During this period, 40 μL of helper phage VCSM13 was added to infect the TG1 cells, and the cells were cultured overnight to amplify the phage. The next day, the phage was precipitated using PEG / NaCl and collected by centrifugation. 200 μg of DLL3 protein dissolved in 100 mM pH 8.2 NaHCO3 was coupled to an ELISA plate and incubated overnight at 4°C. A negative control was also set up. The next day, 100 μL of 3% BSA was added, and the plate was blocked at room temperature for 2 h. After 2 h, 100 μL of amplified phage (2×10⁻⁶) was added. 11 The TFU-immunized camel nanobody phage display gene library was incubated at room temperature for 1 hour. The plate was washed five times with PBS + 0.05% Tween-20 to remove unbound phages. Phages specifically bound to the DLL3 protein were dissociated using trypsin at a final concentration of 25 mg / ml and used to infect *E. coli* TG1 cells in logarithmic growth phase. The cells were incubated at 37°C for 1 hour to generate and collect phages for the next round of screening. This screening process was repeated three times to progressively enrich the phages.

[0131] From the cell culture plates after three rounds of screening, 200 single colonies were selected and inoculated into 96-well plates containing TB medium with 100 μg / mL ampicillin, with a blank control included. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM, and the plates were incubated overnight at 28°C. Crude antibodies were obtained using the osmotic rupture method and transferred to antigen-coated ELISA plates, incubated at room temperature for 1 hour. Unbound antibodies were washed away with PBST, and 100 μL of mouse anti-HA tag antibody (purchased from Covance) diluted 1:2000 was added, incubated at room temperature for 1 hour. Unbound mouse anti-HA tag antibody was washed away with PBST, and 100 μL of anti-mouse alkaline phosphatase conjugate (purchased from Sigma) diluted 1:2000 was added, incubated at room temperature for 1 hour. Unbound alkaline phosphatase conjugates were washed away with PBST, and alkaline phosphatase chromogenic solution was added. After reacting for 5-10 minutes, the absorbance was read at 450 nm using a microplate reader. A sample well was considered a positive clone if its OD value was more than 5 times greater than that of the control well. The bacteria in the positive clone wells were then transferred to LB medium containing 100 μg / μL ampicillin for plasmid extraction and sequencing.

[0132] The gene sequences of each clone were analyzed using the sequence alignment software Vector NTI. Clones with the same FR1, FR2, FR3, FR4, CDR1, CDR2, and CDR3 sequences were considered as the same clone, while clones with different sequences were considered as different clones. Finally, phage-positive clones No. 18, No. 43, and No. 59 that bound DLL3 were obtained.

[0133] Example 3. Preparation of chimeric antibodies

[0134] The heavy and light chain constant regions of the mouse-derived antibody obtained above were replaced with the human IgG1 constant region (SEQ ID NO:46) and the human κ constant region (SEQ ID NO:48), respectively, to form a chimeric antibody. The DLL3 camel-derived antibody was fused with the human IgG1 constant region (CH2-CH3, SEQ ID NO:47) to form a chimeric antibody. Each chimeric antibody was expressed in CHO-K1 cells and purified according to the steps in Example 1.

[0135] Example 4. Chimeric antibody binds to human, monkey, or mouse DLL3 on the cell surface.

[0136] Stable cell lines overexpressing human, monkey, or mouse DLL3 were constructed using CHO-K1 or NCI-H520 cells. In short, the cDNA sequences of human, monkey, or mouse DLL3 (amino acid sequences as shown in SEQ ID NOs:49, 50, and 51, respectively) were synthesized and cloned into the pLV-EGFP(2A)-Puro vector (Beijing Yingmaoshengye Biotechnology Co., Ltd., China). The resulting pLV-EGFP(2A)-Puro-DLL3, along with psPAX and pMD2.G plasmids, were transfected into CHO-K1 or NCI-H520 cells (Nanjing Kebai Co., Ltd., China) via liposome transfection to generate lentivirus. The specific transfection method was completely consistent with the instructions of the Lipofectamine 3000 kit (Thermo Fisher Scientific, USA). Three days after transfection, lentivirus was harvested from the cell culture medium (DMEM medium (Cat#:SH30022.01, Gibco) supplemented with 10% FBS (Cat#:FND500, Excell)) of CHO-K1 or NCI-H520 cells. CHO-K1 or NCI-H520 cells were then transfected with the lentivirus, and these cells were cultured for 7 days in DMEM + 10% FBS medium containing 0.2 μg / ml purine toxin (Cat#:A11138-03, Gibco) to obtain CHO-K1 or NCI-H520 cells stably expressing human, monkey, or mouse DLL3, and were designated CHO-K1 / human DLL3, CHO-K1 / monkey DLL3, CHO-K1 / mouse DLL3, and NCI-H520 / human DLL3 cells, respectively.

[0137] Using the stable CHO-K1 and NCI-H520 cells overexpressing human, monkey, or mouse DLL3 prepared above, FACS cell binding detection was performed on the chimeric antibody. Simply put, 100 μl of 10... 5 CHO-K1 or NCI-H520 cells were plated in 96-well plates, and 50 μl of serially diluted DLL3 chimeric antibody was added. After incubation at 4°C for 1 hour, the 96-well plates were washed three times with PBST. Then, 500-fold diluted PE-goat anti-human IgG (Cat#:PA1-86078, Invitrogen) was added. After incubation at 4°C for 1 hour, the 96-well plates were washed three times with PBS, and cell fluorescence was detected using a FACS (BD) detector.

[0138] Data showed that the E44G4C10 chimeric antibody exhibited high binding affinity to human (Fig. 1, A), monkey (Fig. 1, B), and mouse (Fig. 1, C) DLL3 expressed on CHO-K1 cells; the 47G1B2 chimeric antibody also exhibited high binding affinity to human (Fig. 1, D), monkey (Fig. 1, E), and mouse (Fig. 1, F) DLL3 expressed on CHO-K1 cells. The 43# chimeric antibody exhibited high binding affinity to human (Fig. 2, A), monkey (Fig. 2, B), and mouse (Fig. 2, C) DLL3 on the surface of CHO-K1 cells; the 59# chimeric antibody exhibited high binding affinity to human (Fig. 2, D), monkey (Fig. 2, E), and mouse (Fig. 2, F) DLL3; and the 18# chimeric antibody also exhibited high binding affinity to human (Fig. 2, G), monkey (Fig. 2, H), and mouse (Fig. 2, I) DLL3.

[0139] Example 5. Affinity determination of chimeric antibody with DLL3

[0140] via BIAcore TM The binding affinity of the chimeric antibody to human DLL3 was quantitatively determined using 8K (GE Life Sciences, USA). Specifically, 100-200 RU (reaction units) of human DLL3-his (Cat#:DL3-H52H4, Bipsy, China) was coupled to a CM5 biochip (Cat#:BR-1005-30, GE Life Sciences, USA), followed by blocking unreacted groups on the chip with 1M aminoethanol. Serially diluted antibodies (from 0.3 μM to 10 μM) were injected into SPR reaction solution (HBS-EP buffer, pH 7.4, Cat#:BR-1006-69, GE Life Sciences, USA) at a rate controlled at 30 μL / min. When calculating antibody binding affinity, the RU of the blank control wells was subtracted. Binding rate (kJ / kJ) was calculated. a ) and dissociation rate (k d The equilibrium dissociation constant K was calculated using the formula for the 1:1 paired model in the BIA evaluation software. D via k d / k a Calculated.

[0141] Table 3. Binding affinity of chimeric antibodies to human DLL3

[0142] via BIAcore TM The measured binding affinity of the antibodies is shown in Table 3. It can be seen that the chimeric antibody prepared from mouse-derived antibody and nanobody has a high affinity for human DLL3.

[0143] Example 6. Humanization of DLL3 antibody

[0144] First, humanization modification was performed on E44G4C10 and 47G1B2. Humanization modification was performed using the complementary region-determining region (CDR) transplantation method (US Patent 5,225,539), and the specific method is detailed below.

[0145] To select humanized receptor frameworks for mouse antibodies E44G4C10 and 47G1B2, the light and heavy chain variable region sequences of E44G4C10 and 47G1B2 were compared with the human immunoglobulin gene database (igblast) on the NCBI website. Human germline IGVH and IGVK, which showed the highest homology with E44G4C10 and 47G1B2, were selected as humanized frameworks. For E44G4C10, the selected heavy chain germline receptor sequence was human IGHV1-46*01, and the selected light chain germline receptor sequence was human IGKV1-16*01; for 47G1B2, the selected heavy chain germline receptor sequence was human IGHV1-3*01, and the selected light chain germline receptor sequence was human IGKV1-29*02. Three-dimensional structural simulations were performed on the variable domains of E44G4C10 and 47G1B2 to identify key framework amino acid residues that may play an important role in maintaining the CDR ring structure, thereby designing reversion mutations for humanized antibodies.

[0146] Table 4. Summary of reversion mutant amino acids for humanized E44G4C10 and 47G1B2

[0147] Based on the structural modeling described above, nine potential reversion mutations were identified in the E44G4C10 heavy chain (R72V, T74K, T28I, R98E, M48I, V68A, M70L, V79A, Y27F); seven potential reversion mutations were identified in the 47G1B2 heavy chain (R72V, T74K, M48I, V68A, I70L, V2A, W47F); and one potential reversion mutation (I2V) was identified in the light chain. As shown in Tables 1 and 4, three humanized heavy chain variable regions and one humanized light chain variable region were designed from E44G4C10, resulting in three humanized antibodies; similarly, three humanized heavy chain variable regions and one humanized light chain variable region were designed from 47G1B2, also resulting in three humanized antibodies. All sequence information is summarized in Tables 1 and 4.

[0148] Sequences encoding the addition of the humanized heavy chain variable region to the IgG1 constant region and sequences encoding the addition of the light chain variable region to the κ constant region were synthesized. The amino acid sequences of the heavy chain constant region and the light chain constant region are listed in SEQ ID NO:46 and 48, respectively, and were cloned into the GS expression vector (Invitrogen, USA) using EcoRI / XhoI and ClaI / HindIII restriction enzyme sites, respectively. All expression constructions were confirmed by sequencing. The expression vectors were transfected into the EXPiCHO expression system (Invitrogen, USA) to transiently express 6 humanized DLL3 antibodies, which were then purified, as described in Example 1.

[0149] Then, the nanobodies #18, #43, and #59 were humanized. The V... H The H sequence was compared with the human immunoglobulin gene database on the NCBI website. The human germline IGVH with the highest homology was selected as the framework for humanization. The selected heavy chain germline receptor sequences were human IGHV3-66*01, human IGHV3-23*01, and human IGHV3-23*01. Three-dimensional structural simulations were performed on the variable domains of nanobodies #18, #43, and #59 to identify key framework amino acid residues that may play an important role in maintaining the CDR ring structure, thereby designing reversion mutations for the humanized antibodies.

[0150] Table 5. Summary of reverting mutant amino acids in humanized nanobodies

[0151] Based on the above structures, nine potential reversion mutations were identified in nanobody 18 (F27Y, T28R, V29Y, L45R, S49A, R71L, L78V, A96G, R97A); nine potential reversion mutations were identified in nanobody 43 (A24T, F27Y, F29D, V37F, L45R, W47G, S49A, R71Q, K97A); and nine potential reversion mutations were identified in nanobody 59 (W47L, R71Q, N73K, K97A, F28Y, S30N, T29I, V37Y, L45R). As shown in Table 5, three humanized variable regions were designed for nanobody 18; four for nanobody 43; and four for nanobody 59. All sequence information is summarized in Tables 2 and 5.

[0152] The sequence encoding the humanized heavy chain variable region added to the IgG1 constant region (CH2-CH3, SEQ ID NO:47) was synthesized and cloned into the GS expression vector (Invitrogen, USA) using EcoRI / XhoI and ClaI / HindIII restriction enzyme sites, respectively. All expression constructions were confirmed by sequencing. Eleven humanized DLL3 nanobodies were transiently expressed and purified using the expression vector transfected into the EXPiCHO expression system (Invitrogen, USA), following the procedures described in Example 1.

[0153] Example 7. DLL3 binding affinity / activity of humanized antibodies

[0154] Using CHO-K1 cells stably overexpressing human, monkey, or mouse DLL3, the DLL3 binding activity of humanized antibodies was tested by FACS following the procedure in Example 4. Data showed that the E44G4C10VH3VL2 antibody exhibited high binding affinity to human, monkey, and mouse DLL3 (Figure 3, AC), and the 47G1B2VH4VL2 antibody also showed high binding affinity to human, monkey, and mouse DLL3 (Figure 3, DF). The 43-VHH4 nanobody, 43-VHH5 nanobody, 59-VHH5 nanobody, and 18-VHH4 nanobody also exhibited high binding affinity to human, monkey, or mouse DLL3 (Figure 4, AI).

[0155] In addition, through BIAcore TM The binding affinity of humanized antibodies to human DLL3 was quantitatively determined using a GE Life Sciences (USA) 8K assay, following the procedure described in Example 5. The results are shown in Table 6. It can be seen that E44G4C10VH3VL2, 47G1B2VH4VL2, 18-VHH4, 43-VHH5, and 59-VHH5 exhibit high affinity for human DLL3.

[0156] Table 6. Binding affinity of humanized antibodies to human DLL3

[0157] Example 8. Identification of binding epitopes of DLL3 humanized antibodies

[0158] First, competitive SPR was performed on the humanized antibodies to conduct preliminary analysis of the binding epitopes of each antibody in this application. Briefly, 1 μg / ml of human DLL3-his (Cat#:DL3-H52H4, Bipsys, China) was coupled to a CM5 biochip (Cat#:BR-1005-30, GE Life Sciences, USA), followed by blocking unreacted groups on the chip with 1M aminoethanol. 5 μg / ml of 47G1B2VH4VL2, 43-VHH5, or E44G4C10VH3VL2 was injected into the SPR reaction solution (HBS-EP buffer, pH 7.4, Cat#:BR-1006-69, GE Life Sciences, USA) at a rate controlled at 30 μL / min. Subsequently, another DLL3 antibody from this application was injected at a concentration of 5 μg / ml at a rate controlled at 30 μL / min.

[0159] The experimental results are shown in Figure 5. 47G1B2VH4VL2 can simultaneously bind to the DLL3 antigen with 59-VHH5 and E44G4C10VH3VL2, and can also simultaneously bind to the DLL3 antigen with a portion of 43-VHH5, but cannot simultaneously bind to the DLL3 antigen with 18-VHH4 (Figure 5, A). This indicates that 47G1B2VH4VL2, 59-VHH5, and E44G4C10VH3VL2 should bind to different epitopes of human DLL3, without competition or interference, and exhibits partial binding competition with 43-VHH5. 47G1B2VH4VL2 and 18-VHH4 may bind to the same epitope, have some overlap in epitopes, or experience steric hindrance interference during epitope binding.

[0160] 43-VHH5 can bind simultaneously to the DLL3 antigen along with a portion of 59-VHH5, a portion of 18-VHH5, and a portion of E44G4C10VH3VL2 (Figure 5, B), indicating that 43-VHH5 competes with these antibodies for some epitope binding.

[0161] E44G4C10VH3VL2 and 59-VHH5 do not bind to the DLL3 antigen simultaneously (Figure 5, C), indicating that E44G4C10VH3VL2 and 59-VHH5 may bind to the same epitope, have some overlap in epitopes, or have steric interference with each other when binding to epitopes.

[0162] Figure 6 shows the degree of epitope binding competition among the humanized antibodies, where 1.0 indicates weak competition and 0 indicates strong competition.

[0163] Next, the binding of each antibody to the DLL3 truncated variants was tested to further analyze the binding epitopes. Specifically, cDNA sequences encoding five human DLL3 truncated variants were synthesized and cloned into the pET32a vector after restriction enzyme digestion. The resulting pET32a-DLL3-EGF1 was transformed into competent Escherichia coli BL21 cells. These five DLL3 truncated variants contain 1, 2, 3, 4, and 5 extracellular EGF-like structures, respectively, and were named DLL3 EGF1, DLL3EGF1-2, DLL3 EGF1-3, DLL3 EGF1-4, and DLL3 EGF1-5, with amino acid sequences shown in SEQ ID NO:52-56. Single colonies of different recombinant bacteria were picked and added to 3 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C with shaking for 2-3 hours. When the OD600 of the bacterial culture reached approximately 0.8-1.0, IPTG was added to a final concentration of 1.0 mM, and the culture was continued with shaking for another 5 hours. After the culture was completed, the bacteria were collected by centrifugation at 8000 rpm for 3 min and washed twice with PBS solution. Finally, the bacterial cells were resuspended in 500 μL of PBS to obtain DLL3EGF1, DLL3EGF1-2, DLL3EGF1-3, DLL3EGF1-4, and DLL3EGF1-5 antigens.

[0164] DLL3 EGF1, DLL3 EGF1-2, DLL3 EGF1-3, DLL3 EGF1-4, and DLL3 EGF1-5 antigens diluted to the same concentration, and recombinant full-length human DLL3-his (containing 6 extracellular EGF-like structures, also known as DLL3 EGF1-6, Cat#:DL3-H52H4, BispeCys, China) were coated onto ELISA plates and incubated at room temperature for 1 hour. Then, the humanized antibody was transferred to the ELISA plate and incubated horizontally at 37°C for 1.5 hours. The plates were then removed from the 37°C incubator and washed according to the prescribed procedure. After washing, the plates were centrifuged at 1600 rpm for 1 minute using a Smartplate centrifuge to remove residual droplets. Anti-human Fc-antibody-HRP was diluted 1:5000 to the working concentration, and 100 μl was added to each well. The plates were sealed with a membrane and incubated at 37°C in the dark for 60 minutes. Remove the microplate and wash it according to the pre-set plate washer program. After washing, centrifuge at 1600 rpm for 1 min using a Smartplate centrifuge to remove any residual droplets. Add 100 μl of TMB chromogenic solution to each well and incubate at 37°C in the dark for 30 minutes. Add 100 μl of 2N H2SO4 stop solution to each well to terminate the substrate reaction. Measure the absorbance at 450 nm and use the microplate reader's built-in analysis software to read the values ​​and simulate a four-parameter equation to fit the curve.

[0165] The experimental results are shown in Figure 7. E44G4C10VH3VL2 and 59-VHH5 can only bind to the full-length DLL3, indicating that the binding sites of E44G4C10VH3VL2 and 59-VHH5 are approximately at the 6th EGF-like structure. 18-VHH4 and 47G1B2VH4VL2 can bind to DLL3 EGF1-3, DLL3 EGF1-4, DLL3 EGF1-5 and the full-length DLL3 antigen, but cannot bind to DLL3 EGF1 and DLL3 EGF1-2 antigens, indicating that the binding sites of 18-VHH4 and 47G1B2VH4VL2 are approximately at the 3rd EGF-like structure. 43-VHH5 can bind to the full-length DLL3 and various DLL3 truncated variants, indicating that it may bind to complex discrete or multimeric epitopes.

[0166] Example 9. Endocytosis of DLL3 humanized antibody

[0167] Following the method steps in Example 4, the binding activity of the humanized antibody against the SHP77 (CBP60151, Nanjing Kebai Biotechnology Co., Ltd.) human small cell lung cancer cell line expressing DLL3 was measured. As shown in Figure 8, 47G1B2VH4VL2, 43-VHH5, and 18-VHH4 exhibited high binding affinity to SHP77 cells, while E44G4C10VH3VL2 and 59-VHH5 showed low binding affinity to SHP77 cells.

[0168] Digest SHP77 cells, count them, centrifuge to remove the culture medium, resuspend them in RPMI complete medium (RPMI + 10% FBS), and adjust the cell density to 2 × 10⁶ cells / year. 5 / ml. Zenon at an initial concentration of 300 μg / mL was diluted with RPMI + 10% FBS. TM pHrodo TM The iFL red human IgG labeling reagent (Cat#: Z25612, Invitrogen) was diluted to 10 μg / mL, and the antibody of this application was diluted to 10 μg / mL with RPMI + 10% FBS. The two dilutions were mixed at a 1:1 volume ratio and reacted in the dark for 5 min to obtain a working solution with a final concentration of 5 μg / mL for both the labeling reagent and the antibody. 50 μL of the above working solution was added to 50 μL of SHP77 cells (10,000 cells / well), and the reaction was carried out for 0 h, 3 h, and 6 h. 0 h refers to the time when the antibody was added to the cells at 4°C, and 3 h and 6 h refer to the time when the antibody reacted with the cells at 37°C for 3 h and 6 h, respectively. The cells were digested with trypsin and transferred to a U-bottom cell plate. The cells were washed three times with 200 μL PBS + 2% FBS at 1000g. The cells were then analyzed using flow cytometry in the PE channel using FlowJo software.

[0169] Figures 9(A) and 9(B) show the MFI and percentage of luminescent cells in each group, respectively. It can be seen that all detected humanized DLL3 antibodies were internalized by SHP77 cells. Antibodies E44G4C10VH3VL2 and 18-VHH4 exhibited the best internalization effect, i.e., the highest percentage of cells were internalized and the fastest internalization rate. This was followed by 47G1B2VH4VL2, 43-VHH5, and 59-VHH5.

[0170] Example 10. Drug conjugation and cytotoxic effect of humanized DLL3 antibody

[0171] Take 8 mg of the above-mentioned humanized antibody and add 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) stock solution (Cat#: C4706-10G, Sigma, USA). Mix well and place in a constant temperature mixer at 37°C for 2 h with stirring. Cool the sample to about 4°C, pre-add a certain amount of 10 mM DMSO solution, and then add 10 mM DMSO solution containing 10 equivalents (i.e., the number of adapter-drug is 10 times that of antibody) of Deruxtecan (MC-GGFG-DXD, Cat#: HY-13631E, MedChemExpress, USA). The total amount of DMSO solution added in this step is 10% of the reaction system. Mix well and react at 25°C for 1 h. After the reaction is completed, transfer the reaction solution to a 50 KD ultrafiltration tube (Cat#: UFC905008, Merck Millipore, USA), add 1×PBS and ultrafilter 5 times, concentrating 10 times each time to remove small molecules and DMSO. It was then transferred to a 5 mL sterile centrifuge tube, sterilized and filtered, and the concentration was measured by a UV spectrophotometer.

[0172] The SHP77 cell line was treated with the obtained humanized antibody-DXD conjugate to test the cytotoxicity of the antibody-drug conjugate. CellTrace was used to analyze the drug. TM The cell proliferation kit (Cat#:C34554, Invitrogen) was used to label the SHP77 cell line with the dye.

[0173] Dye-labeled SHP77 cells were resuspended in RPMI complete medium (RPMI + 10% FBS) and the cell density was adjusted to 2.5 × 10⁶ cells / year. 5Live cells / ml, 100 μl was added to a 96-well plate, along with 100 μl of a 10-fold serially diluted antibody-DXD conjugate (initial concentration of 10 μg / ml based on antibody concentration). The plate was incubated at 37°C and 5% CO2 for 48 hours. Using an ATP assay kit (Cell Counting - Lite2.0 luminescent cell viability assay, Cat#:DD1101-02, Nanjing Novizan Biotechnology Co., Ltd.), an equal volume of reagent was added directly to the cell culture. After 10 minutes, the luminescence value was read using a microplate reader to calculate the cell viability.

[0174] The experimental results are shown in Figure 10. E44G4C10VH3VL2-DXD has the strongest killing effect on SHP77, followed by 18-VHH4-DXD, 43-VHH5-DXD, 47G1B2VH4VL2-DXD and 59-VHH5-DXD.

[0175] Example 11. In vivo antitumor efficacy of DLL3 humanized antibody-DXD conjugate

[0176] The small cell lung cancer cell line SHP77, which expresses DLL3, was subcutaneously injected into immunodeficient mice B6-NDG (Biocytogen). About 2 to 3 weeks before tumor cell inoculation, the small cell lung cancer cell line SHP77 was resuscitated and cultured in RPMI medium containing 10% FBS, and passaged every 3 to 4 days.

[0177] SHP77 cells were collected and the SHP77 tumor cell density was adjusted to 2.5 × 10⁻⁶ cells using a 1:1 volume mixture of Matrigel (Cat#: 356234, GIBCO) and PBS. 7 / ml, 0.2ml was injected subcutaneously into the abdomen of each mouse, which is 0.5×10 7 Cells / mouse. When the tumor grows to 50-100 mm 3 The mice were divided into 3 groups of 4 mice each. Each group of mice was intravenously injected with E44G4C10VH3VL2-DXD, 18-VHH4-DXD and PBS, respectively, at a dose of 10 mg / kg per mouse (calculated based on antibody-DXD). This day was recorded as day 0.

[0178] Tumor size was measured on days 2, 4, 6, 8, 10, 13, 16, 21, 23, and 28. Tumor volume was calculated as 0.5 × length × width. 2 .

[0179] Figure 11 shows the changes in average tumor size in each group of mice. It can be seen that, compared with the PBS group, both E44G4C10VH3VL2-DXD and 18-VHH4-DXD have significant tumor-suppressing effects, and 18-VHH4-DXD has a stronger tumor-suppressing effect.

[0180] Example 12. Pharmacokinetic Study of DLL3 Humanized Antibody-DXD Conjugate

[0181] SHP77 cells were subcutaneously injected into immunodeficient mice B6-NDG (Biocytogen). About 2 to 3 weeks before tumor cell inoculation, the small cell lung cancer cell line SHP77 was resuscitated and cultured in RPMI medium containing 10% FBS, and passaged every 3 to 4 days.

[0182] SHP77 cells were collected, and the tumor cell density was adjusted to 2.5 × 10⁻⁶ cells / mL using a 1:1 volume mixture of matrix gel (GIBCO, 356234) and PBS. 7 / ml, 0.2ml was injected subcutaneously into the abdomen of each mouse, which is 0.5×10 7 Cells / mouse. Tumors grew to 50-100 mm. 3 Mice were divided into 4 groups of 5 each. Each group of mice was intravenously injected with E44G4C10VH3VL2-DXD, 18-VHH4-DXD, HEL-DXD (prepared according to Example 10), and PBS, respectively, at a dose of 10 mg / kg per mouse (calculated based on antibody-DXD).

[0183] At 2, 24, 48, 72, 96, 144, 192, and 240 hours after injection, approximately 60-80 μL of blood was collected from the orbital venous plexus of mice in each group using sterile venous blood collection, and serum was separated.

[0184] 100 μl of goat anti-hIgG Fc (Cat#: 12136-1ML, Sigma) diluted to 1 μg / mL with PBS was coated onto an ELISA plate and incubated at 37°C for 2 h. The plate was washed three times with PBST (PBS mixed with 0.5% Tween 20), and 200 μl of 3% BSA (Cat#: 900900-1KG, Sigma) was added to each well for blocking, and the plate was incubated overnight at 4°C. 100 μl of 1500-fold diluted serum sample to be tested or 100 μl of the antibody-DXD conjugate of this application (final concentrations of 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.2 ng / mL, 153.6 ng / mL, 7.8 ng / mL, and 3.9 ng / mL) was added to each well and incubated at 37°C for 2 h. Wash the plate three times with PBST, then add 100 μl of goat anti-hIgG-total-HRP (Cat#: A8667-2ML, Sigma) diluted 5000 times with 3% BSA, and incubate at 37°C for 1 h. Wash the plate three times with PBST, then add 100 μl of TMB (Cat#: ES-002, Beijing Bio-Science & Technology Co., Ltd.) for color development. Stop the reaction with 100 μl of H2SO4, read the OD450 value using a microplate reader, and calculate the sample concentration. Calculate the serum antibody-DXD concentration based on the standard curve.

[0185] Figure 12 shows the changes in antibody-DXD concentration in the serum of mice in each group. It can be seen that, compared with the persistent high presence of the negative control in mouse serum, the concentrations of E44G4C10VH3VL2-DXD and 18-VHH4-DXD in mouse serum decreased over time.

[0186] The preliminary PK test results above show that 18-VHH4-DXD has a lower clearance rate than E44G4C10VH3VL2-DXD.

Claims

1. An antibody or its antigen-binding portion thereof, which is capable of specifically binding to the DLL3 protein, wherein, i) The antibody or its antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 respectively contain i-1)SEQ ID NO:1, 2, 3, 4, 5 and 6, or i-2) The amino acid sequence shown in SEQ ID NO:7, 8, 9, 10, 11 and 12, or ii) The antibody or its antigen-binding portion comprises a variable region, wherein the variable region comprises CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 respectively contain ii-1)SEQ ID NO:13, 14 and 15, ii-2)SEQ ID NO:16, 17 and 18, or (ii-3) The amino acid sequences shown in SEQ ID NO:19, 20 and 21.

2. The antibody or its antigen-binding portion as described in claim 1, wherein, i) The antibody or its antigen-binding moiety contains a heavy chain variable region and a light chain variable region, wherein, i-1) The heavy chain variable region contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO:24, 22, 23 or 25, and the light chain variable region contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO:44, or i-2) The heavy chain variable region contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO:29, 26, 27 or 28, and the light chain variable region contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO:45, or ii) The antibody or its antigen-binding moiety contains a variable region, wherein, ii-1) The variable region contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 34, 30, 31, 32 or 33. ii-2) The variable region contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 38, 35, 36 or 37, or ii-3) The variable region contains an amino acid sequence that has at least 95% sequence identity with SEQ ID NO:43, 39, 40, 41 or 42.

3. An antibody or its antigen-binding portion thereof, which is capable of specifically binding to the DLL3 protein, wherein, i) The antibody or its antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3, wherein... i-1) The VH-CDR1, VH-CDR2, and VH-CDR3 are identical to the VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:24, 22, 23, or 25, respectively; and the VL-CDR1, VL-CDR2, and VL-CDR3 are identical to the VL-CDR1, VL-CDR2, and VL-CDR3 of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:44, respectively. i-2) The VH-CDR1, VH-CDR2, and VH-CDR3 are identical to the VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:29, 26, 27, or 28, respectively; and the VL-CDR1, VL-CDR2, and VL-CDR3 are identical to the VL-CDR1, VL-CDR2, and VL-CDR3 of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:45, respectively. ii) The antibody or its antigen-binding moiety contains a variable region, wherein, ii-1) The CDR1, CDR2, and CDR3 are identical to the CDR1, CDR2, and CDR3 containing the variable regions of the amino acid sequences shown in SEQ ID NO: 34, 30, 31, 32, or 33, respectively. ii-2) The CDR1, CDR2, and CDR3 are identical to the CDR1, CDR2, and CDR3 containing the variable regions of the amino acid sequences shown in SEQ ID NO: 38, 35, 36, or 37, respectively. ii-3) The CDR1, CDR2 and CDR3 are identical to the CDR1, CDR2 and CDR3 containing the variable regions of the amino acid sequences shown in SEQ ID NO:43, 39, 40, 41 or 42, respectively.

4. The antibody or its antigen-binding portion as described in claim 1 or 3, wherein, i) The antibody or its antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the antibody or its antigen-binding moiety further comprises a heavy chain constant region linked to the heavy chain variable region and / or a light chain constant region linked to the light chain variable region, or ii) The antibody or its antigen-binding portion contains a variable region, wherein the antibody or its antigen-binding portion further contains a heavy chain constant region connected to the variable region.

5. The antibody or its antigen-binding portion as claimed in claim 4, wherein the heavy chain constant region is the human IgG1 heavy chain constant region or its CH2-CH3 region, and / or the light chain constant region is the human κ constant region.

6. The antibody or its antigen-binding portion as claimed in claim 5, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:46 or 47, and / or the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

48.

7. An immunoconjugate comprising an antibody or antigen-binding portion thereof as described in any one of claims 1-6, and an effector molecule.

8. The immunoconjugate of claim 7, wherein the effector molecule is a toxin.

9. A nucleic acid molecule encoding an antibody or antigen-binding portion thereof as described in any one of claims 1-6.

10. An expression vector comprising the nucleic acid molecule of claim 9.

11. An isolated host cell comprising the nucleic acid molecule of claim 9 or the expression vector of claim 10.

12. A composition comprising an antibody or antigen-binding portion thereof as described in any one of claims 1-6, an immunoconjugate as described in claim 7 or 8, a nucleic acid molecule as described in claim 9, an expression vector as described in claim 10, or a host cell as described in claim 11.

13. Use of the composition of claim 12 in the preparation of a treatment for tumors or cancers associated with DLL3 overexpression.

14. The use as described in claim 13, wherein the tumor or cancer is small cell lung cancer.

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