Anti-DLL3 antibodies and uses thereof

Abstract

The present invention provides an antibody, or an antigen-binding fragment thereof, which can specifically bind to a DLL3 protein, such as a human DLL3 protein. The invention also provides multispecific binding molecules, such as multispecific antibodies, that specifically bind to DLL3 and one or more tumor-associated antigens. The invention further provides a nucleic acid molecule for coding the antibody, an expression vector for expressing the antibody, a host cell and a preparation method thereof. The invention also provides diagnostic and therapeutic methods using the antibodies of the invention.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202410297394.5 and application date March 15, 2024, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby introduced into this application as a reference.

[0003] The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to a DLL3 protein, such as a human DLL3 protein. The present invention also provides multispecific binding molecules, such as multispecific antibodies, that specifically bind to DLL3 and one or more tumor-associated antigens. The present invention further provides nucleic acid molecules encoding the antibodies, expression vectors and host cells for expressing the antibodies, and methods for preparing the same. The present invention also provides diagnostic and therapeutic methods using the antibodies of the present invention. Background of the Invention

[0004] DLL3 (i.e., delta-like ligand 3 or delta-like protein 3) is a type I single-pass transmembrane protein and a Notch ligand. It contains one DSL domain, six epidermal growth factor-like domains, and one transmembrane domain. Under normal circumstances, DLL3 expression is limited in normal tissues and localizes to the Golgi apparatus, not the cell membrane. However, in some neuroendocrine tumor types (including SCLC and LCNEC), such as small cell lung cancer, DLL3 is abnormally highly expressed (70%-80%). This abnormally expressed DLL3 is not only localized to the Golgi apparatus but can also be detected on the cell membrane.

[0005] Therefore, DLL3 is a promising target for the treatment of neuroendocrine tumors. In recent years, therapeutic agents targeting DLL3 have been developed for the treatment of tumors or cancers expressing DLL3.

[0006] For DLL3, an antibody-toxin conjugate (ADC) consisting of an antibody and a toxin can be developed. However, this strategy faces significant challenges in clinical practice, likely due to the low abundance of DLL3 on the cell surface. The Rova-T drug uses a highly active PBD as the toxin payload, resulting in low clinical doses and limited efficacy.

[0007] Attempts have also been made to combine targeting of DLL3 with targeting of other proteins. For example, WO2019234220A1, US10294300 or Susanne Hipp et al., A Bispecific DLL3 / CD3 IgG-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer, Clin Cancer Res 2020; 26: 5258–68 or Giffin MJ et al., AMG 757, a Half-Life Extended, DLL3-Targeted Bispecific T-Cell Engager, Shows High Potency and Sensitivity in Preclinical Models of Small-Cell Lung Cancer. Clin Cancer Res. 2021 Mar 1; 27(5): 1526-1537 all describe bispecific antibody constructs that bind to human DLL3 on the surface of target cells and to human CD3 on the surface of T cells.

[0008] CD3 is a surface molecule specifically expressed on T cells. It serves as a co-receptor for the TCR. The intracellular domain of the TCR lacks signaling domains. Therefore, upon receiving a signal from the MHC / HLA complex, the different CD3 subunits can aggregate, transmitting the T cell activation signal downstream. Functional CD3 is formed by dimers of two of four different chains: ε, ζ, δ, and γ. CD3 dimer arrangements include γ / ε, δ / ε, and ζ / ζ. Antibodies targeting CD3 have been shown to aggregate CD3 on T cells, leading to T cell activation in a manner similar to engagement of the TCR by peptide-loaded MHC molecules. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation. Furthermore, bispecific antibodies that bind to CD3 and target tumor surface antigens have been proposed to bridge tumor cells and T cells, crosslinking CD3 on the T cell surface, and thereby directly activating T cells, releasing granzymes, perforins, and cytokines to kill tumors, thereby achieving tumor suppression therapeutic goals.

[0009] However, the field still needs to develop safe anti-DLL3 antibodies suitable for use as drugs, as well as multispecific antigen-binding molecules constructed based on them that specifically bind to DLL3 and other target antigens, especially CD3, for use in treating tumors. Summary of the Invention

[0010] One aspect of the present invention relates to a binding molecule that specifically binds to DLL3, such as an anti-DLL3 antibody or antigen-binding fragment thereof, including multispecific binding molecules that specifically bind to DLL3 and other targets, such as multispecific antibodies, such as bispecific antibodies.

[0011] One aspect of the present invention relates to an anti-DLL3 antibody or an antigen-binding fragment thereof.

[0012] In some embodiments, the anti-DLL3 antibodies or antigen-binding fragments thereof of the invention specifically bind to DLL3 (e.g., human DLL3). In some embodiments, the binding affinity of the anti-DLL3 antibodies or antigen-binding fragments thereof of the invention to DLL3 (e.g., human DLL3) is K. D In some embodiments, the binding affinity of an anti-DLL3 antibody or antigen-binding fragment thereof of the invention to DLL3 (e.g., human DLL3) is less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, or 0.6 nM, or between the values ​​recited. D Values ​​greater than or equal to approximately 0.5 nM.

[0013] In some embodiments, the anti-DLL3 antibodies or antigen-binding fragments thereof of the present invention can effectively bind to DLL3 and transmit downstream signaling pathways by binding to FcR through the Fc region. For example, the Fc end of the anti-DLL3 antibody binds to macrophages to transmit CD64 downstream pathways and induce macrophage phagocytosis.

[0014] Another aspect of the present invention relates to a multispecific binding molecule (e.g., a multispecific antibody, such as a bispecific antibody) constructed based on the DLL3 antibody of the present invention, which comprises a target binding region from an anti-DLL3 antibody of the present invention, and optionally one or more other target binding regions (antigen binding regions).

[0015] In one embodiment, the invention relates to a multispecific antibody, such as a bispecific antibody, that specifically binds DLL3 and one or more other antigens. In some embodiments, the other antigen is CD3.

[0016] In one embodiment, the invention relates to a bispecific antibody that specifically binds DLL3 (eg, human DLL3) and CD3 (eg, human CD3).

[0017] In some embodiments, the bispecific antibodies of the present invention can activate the CD3 signaling pathway in a DLL3-dependent manner. In some embodiments, the bispecific antibodies of the present invention can activate T cells, such as CD4 or CD8 T cells, in DLL3-positive tumors, for example, activating them to release cytokines, such as TNF, such as TNFα, or interleukins, such as IL2.

[0018] In some embodiments, the bispecific antibodies of the present invention can activate human T cells to kill tumor cells, for example, activate human T cells to kill DLL3-positive tumor cells.

[0019] In some embodiments, the bispecific antibodies of the invention are capable of specifically binding to DLL3 (eg, human DLL3) and CD3 (eg, human CD3).

[0020] In some embodiments, a bispecific antibody of the invention, eg, a bispecific antibody that specifically binds to DLL3 and CD3, has anti-tumor (eg, DLL3-positive tumor) activity, eg, has in vivo anti-tumor activity.

[0021] In some embodiments, the bispecific antibodies of the present invention can be used in combination with other therapeutic agents to treat tumors (eg, DLL3-positive tumors).

[0022] In some embodiments, the half-life of the bispecific antibodies of the invention is long, greater than about 100 hours, 110 hours, 120 hours, 130 hours, 140 hours, 145 hours, 150 hours, or 155 hours, or between them, such as between 100-200 hours, such as between 140-160 hours, such as between about 148-159 hours.

[0023] In some embodiments, the bispecific antibodies of the present invention have better drugability, for example, they have low drugability risk, for example, they have better colloidal stability, lower non-specific binding, lower hydrophobicity, and higher thermal stability (for example, Tagg is above about 60°C, for example, above about 61°C, 62°C, 63°C or 64°C). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Binding of the test antibodies to DLL3-overexpressing CHO cells is shown;

[0025] Figure 2 The results showed that the tested antibodies activated the CD64 downstream signaling pathway in a dose-dependent manner;

[0026] Figure 3 The preparation route (A) and structure (B) of the bispecific antibody are shown;

[0027] Figure 4 The affinities of the tested bispecific antibodies DLL3 / CD3 and human DLL3 (A) and human CD3 (B) are shown;

[0028] Figure 5 Binding of the tested bispecific antibody DLL3 / CD3 to DLL3-overexpressing CHO cells is shown;

[0029] Figure 6 The tested bispecific antibody DLL3 / CD3 and human CD4 T cells ( Figure 6 B) and CD8 T cells ( Figure 6 A) Surface CD3 binding;

[0030] Figure 7 The activity of the tested bispecific antibody DLL3 / CD3 in activating NFAT signaling in cells is shown;

[0031] Figure 8 The results showed that the tested bispecific antibody DLL3 / CD3 induced the killing effect of human CD8+ T cells on SHP77 target cells in a dose-dependent manner ( Figure 8 A) and the effects on non-target cells H460 ( Figure 8 B);

[0032] Figure 9 The binding activity and expression yield of the monoclonal antibody to DLL3 after the hotspot mutation site was removed are shown;

[0033] Figure 10 The binding of the mAb to DLL3-overexpressing CHO cells after removal of the hotspot mutation site is shown;

[0034] Figure 11 Binding of the tested bispecific antibody DLL3 / CD3 to DLL3-overexpressing CHO cells is shown;

[0035] Figure 12 The test bispecific antibody DLL3 / CD3 activates NFAT signaling in cells;

[0036] Figure 13 and 14 The results showed that the tested bispecific antibody DLL3 / CD3 dose-dependently induced the killing effect of human CD8+ T cells on SHP77 target cells;

[0037] FIG15 shows the binding of the tested bispecific antibodies to human DLL3;

[0038] Figure 16 Binding of the tested bispecific antibodies to CHO-hDLL1 and 293T-hDLL4 cells is shown;

[0039] Figure 17 Binding of the tested bispecific antibodies to DLL3-overexpressing cell lines and T cells is shown;

[0040] Figure 18 The results show that the tested bispecific antibodies activate NFAT signaling in cells in vitro;

[0041] Figure 19 The test bispecific antibody showed activity against tumor cells in vitro;

[0042] Figure 20 The results show the effect of the tested bispecific antibodies on cytokine production by immune cells in PBMCs without DLL3 cross-linking.

[0043] Figure 21 The test bispecific antibody demonstrated activity in inhibiting tumor growth in vivo;

[0044] Figure 22 Shown is the group IgG1 0.1mpk( Figure 22 A), BI-D3C1analog 0.01mpk( Figure 22 B) and D3-107 / CD3-C1 0.01mpk( Figure 22 C) Infiltration of CD3-positive cells in tumors after drug administration;

[0045] Figure 23 The number of CD3-positive cells per unit area in different drug administration groups is shown;

[0046] Figure 24 The test bispecific antibody demonstrated activity in inhibiting tumor growth in vivo;

[0047] Figure 25 The test bispecific antibody demonstrated activity in inhibiting tumor growth in vivo;

[0048] Figure 26 Demonstrated in vivo tumor growth inhibition activity of the tested bispecific antibody in combination with standard chemotherapy;

[0049] Figure 27 Demonstrated in vivo tumor growth inhibition activity of the tested bispecific antibody in combination with an immune checkpoint inhibitor;

[0050] Figure 28 The results of in vivo PK testing of the tested bispecific antibodies are shown. Detailed Description of the Invention

[0051] It should be understood that the present invention is not limited to the specific methods, protocols, examples and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which will only be limited by the appended claims.

[0052] I. Definition

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0054] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0055] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5%, 4%, 3%, 2% or 1% less than the specified numerical value and an upper limit that is 5%, 4%, 3%, 2% or 1% greater than the specified numerical value.

[0056] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0057] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, but does not indicate the positions of the two domains in any way.

[0058] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0059] As used herein, the term "DLL3" refers to a type I transmembrane protein belonging to the Notch ligand family. Human DLL3 includes, for example, an amino acid sequence as shown in UniProtKB / Swiss-Prot: Q9NYJ7. In some embodiments, DLL3 is human DLL3.

[0060] As used herein, an "antibody that binds to DLL3" or an "anti-DLL3 antibody" or an "antibody that specifically binds to DLL3" refers to an antibody that is capable of binding to DLL3 with appropriate affinity.

[0061] In some aspects, the anti-DLL3 antibodies described herein also encompass multispecific antibodies, such as bispecific antibodies, that simultaneously specifically bind DLL3 and other target antigens.

[0062] As used herein, the term "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR), i.e., the T cell engaging antigen, the T cell surface glycoprotein CD3, which is composed of a homodimer or heterodimer formed by two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-εn (hCD3ε) comprises the amino acid sequence set forth in UniProtKB / Swiss-Prot: P07766. Human CD3-δ (hCD3δ) comprises the amino acid sequence set forth in UniProtKB / Swiss-Prot: P04234. In some embodiments, the CD3 described herein refers to CD3 from humans or monkeys (e.g., cynomolgus monkeys).

[0063] As used herein, the term "antibody that binds to CD3" or "anti-CD3 antibody" includes antibodies that specifically recognize or bind to a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies that specifically recognize or bind to a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3, bound CD3, and / or cell-surface expressed CD3. Soluble CD3 includes native CD3 protein and recombinant CD3 protein variants, for example, monomeric and dimeric CD3 structures that lack a transmembrane region or are otherwise not bound to the cell membrane. The binding affinity of the antibody to CD3 can be detected by flow cytometry or optical interferometry of biological membranes.

[0064] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells can also include natural killer cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes).

[0065] General information concerning the amino acid and nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0066] The multispecific antibodies of the present invention may comprise a linker. As used herein, the term "linker" refers to any molecule that enables the direct connection of the different parts of a multispecific antibody. Examples of linkers for establishing covalent linkages between different parts of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the term "peptide linker" according to the present invention refers to an amino acid sequence, wherein the sequence connects the amino acid sequences of the various parts of the multispecific antibody together. Preferably, the peptide linker has a length that is sufficient to connect the two entities in a manner that allows them to maintain their conformations relative to each other so as not to interfere with the desired activity. The peptide linker may or may not primarily include the following amino acid residues: Gly, Ser, Ala or Thr.

[0067] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the amino acid residue numbering in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Two Fc regions can achieve dimerization to form a dimeric Fc, and two different Fc heterodimerizations form heterodimeric Fc. In this article, the terms "Fc region", "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulin and heavy chain constant region CH1 and light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In one embodiment, a human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231, to the carboxyl-terminus of the heavy chain.

[0068] In one embodiment, the human IgG1 Fc region polypeptide (including part of the hinge region) comprises or consists of the following amino acid sequence:

[0069] DKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:26); or

[0070] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:27).

[0071] In one embodiment, the Fc region is derived from a human Fc region. In one embodiment, the Fc region comprises all or part of a human constant region. The antibody Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is of the human IgG4 subclass. In one embodiment, the Fc region is of the human IgG1 subclass. In one embodiment, the Fc region is of a human IgG1, IgG2, IgG3, or IgG4 Fc region.

[0072] Herein, a "heterodimeric Fc scaffold" refers to a scaffold comprising two different Fc regions or formed by dimerization of two different Fc regions, which can be connected at its N-terminus or C-terminus to an antigen-binding domain (e.g., an antibody heavy chain and / or light chain variable region or an antibody antigen-binding fragment that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule) to form a multispecific antibody such as a bispecific antibody.

[0073] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide that extends from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises or consists of the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 28).

[0074] In some embodiments, CH1 may comprise a portion of the hinge region. In some embodiments, the Fc region may comprise a portion of the hinge region.

[0075] References to specific amino acid residues in the constant region of an antibody IgG are made herein according to the EU numbering system. For example, "S364" refers to the serine at EU position 364. Amino acid mutations at specific positions in the constant region are indicated by (original amino acid, amino acid position, mutated amino acid). For example, "S364R" refers to the substitution of the serine (S) at EU position 364 with an arginine (R). When referring to combinations of mutations, the combined mutations are connected by a plus sign (+), a "-" sign, or an "and" sign. "S364R+D399K," "S364R-D399K," or "S364R and D399K" indicate that the Fc region contains both the S364R and D399K mutations. When multiple mutation possibilities exist at a specific position, this is indicated herein by the symbol " / ." For example, the mutation "K370T / S" indicates that the K residue at position 370 can be replaced with either a T or S residue.

[0076] For polypeptide sequences, "conservative changes" include replacements, deletions or additions to the polypeptide sequence, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative replacements: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein molecule.

[0077] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0078] The term "binding molecule" refers to any molecule capable of specifically binding to a target, such as an antibody or antigen-binding fragment or fusion protein thereof.

[0079] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor. The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including essentially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule. When the binding molecule of the present invention refers to a target binding region derived from an antibody, "target" and "antigen" can be used interchangeably. In some embodiments, the antigen is CD3.

[0080] As used herein, the term "target binding region" refers to the portion of a binding molecule, such as a multispecific binding molecule or a bispecific binding molecule, that binds a specific target or antigen. The target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the rest of the binding molecule and may or may not bind to its target as a separate entity. The target binding region can also be a receptor or a ligand, or a domain of a receptor that is capable of binding a ligand. In the case of multispecific antibodies or bispecific antibodies, the "target binding region" is also referred to as an "antigen binding region."

[0081] As used herein, the term "antigen binding region" refers to any portion of an antibody or antigen binding fragment thereof, such as a multispecific antibody or bispecific antibody, that binds a specific target or antigen. The antigen binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such an antigen binding region may or may not have a tertiary structure independent of the remainder of the multispecific antibody or bispecific antibody and may or may not bind to its antigen / epitope as a separate entity. Where the binding molecules of the present invention relate to a target binding region derived from an antibody, "target binding region" and "antigen binding region" can be used interchangeably.

[0082] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, e.g., a bispecific binding molecule, i.e., the molecule comprises at least a first target binding region and a second target binding region, wherein the first target binding region binds one target or antigen and the second target binding region binds another antigen or target. Multispecific binding molecules according to the present invention also encompass multispecific binding molecules comprising multiple target binding regions / binding sites. In some embodiments, the multispecific binding molecules of the present invention are multispecific antibodies. In some embodiments, the bispecific binding molecules of the present invention are bispecific antibodies.

[0083] As used herein, the term "monospecific" refers to a polypeptide / protein molecule having one or more target binding regions, each of which binds to the same site or structure of the same target or the same epitope of the same antigen.

[0084] As used herein, the term "multispecific" binding molecule, such as an antibody, refers to a molecule having at least two target binding regions, each of which binds to a different site / structure of the same target or to a different target. A multispecific binding molecule is a binding molecule that has binding specificity for at least two different targets or sites / structures. In one embodiment, provided herein is a bispecific binding molecule that has binding specificity for a first target and a second target.

[0085] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. A multispecific antibody is an antibody that has binding specificity for at least two different antigenic epitopes. In one embodiment, provided herein is a bispecific antibody that has binding specificity for a first antigen and a second antigen.

[0086] When referring to a "first antigen-binding region" in a multispecific antibody or bispecific antibody, it refers to the binding region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific antibody or bispecific antibody may contain one or more first antigen-binding regions. For example, a bispecific antibody contains a first antigen-binding region and a second antigen-binding region, but may contain one or more first antigen-binding regions and one or more second antigen-binding regions.

[0087] When referring to a "first target binding region" in a binding molecule, it refers to the binding region that binds to the first target, and is not intended to limit the number of target binding regions contained in the binding molecule. For example, a multispecific binding molecule or a bispecific binding molecule can contain one or more first target binding regions. For example, a bispecific binding molecule comprising a first target binding region and a second target binding region can contain one or more first target binding regions and one or more second target binding regions.

[0088] When referring to "a target or antigen binding region is derived from an antibody", it means that the binding domain constituting the target / antigen binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, for example, the specific antigen-binding fragment of the antigen binding region, such as Fab, is or is derived from the corresponding fragment, such as Fab, of the antibody, or the heavy chain variable region and / or light chain variable region of the antigen binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the antigen binding region are CDRs of the antibody.

[0089] The term "derived from" means that the fragment in the antigen binding region is substantially identical to the fragment of the antibody from which it is derived, but has a mutation, such as a substitution, deletion or addition, at one or more sites. In a specific embodiment, the mutation is not in the CDR of the antibody.

[0090] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region at least comprises three domains CH1, CH2 and CH3 (and optionally CH4). The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region (VH) and each light chain variable region (VH) is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment.

[0091] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and that contains a portion of the full-length antibody, but is capable of binding to the antigen of the full-length antibody or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full-length antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, which is a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. An Fv fragment consists of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but can also be produced by recombinant methods using a synthetic linker peptide to link the two domains as a single protein chain in which the VL region and VH region are paired to form a single-chain Fv (scFv).

[0092] "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site. Herein, the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as the "Fab light chain."

[0093] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics The database (IMGT) (www.imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways or in combination. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0094] In some embodiments, the CDRs of the heavy chain variable region of the antibodies of the present invention are determined according to Kabat or Chothia, or a combination of Kabat and Chothia (hereinafter referred to as "Kabat & Chothia," corresponding to H26-H32 under the Kabat numbering system). In some embodiments, the CDRs of the light chain variable region of the antibodies of the present invention are determined according to Kabat.

[0095] In one embodiment, HCDR1 in the anti-DLL3 antibody of the invention is defined according to the Kabat & Chothia rules, HCDR2 and HCDR3 are defined according to the Kabat rules, and LCDRs are defined according to the Kabat rules.

[0096]

[0097] Calculation of sequence identity between sequences is performed as follows.

[0098] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0099] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing portions of the variable region that are not involved in binding with corresponding portions of a human antibody).

[0100] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0101] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (K dis and K on ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0102] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom.

[0103] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0104] The term "treat," ..."

[0105] The term "preventing" includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.

[0106] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies (such as immune checkpoint molecule antibodies), small molecule drugs, or immunomodulators (such as immunosuppressants or agonists).

[0107] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (such as tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0108] The term "drug combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents with a preventive or therapeutically effective level in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0109] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0110] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0111] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be a solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; a body fluid, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid.

[0112] Examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples, or frozen tumor samples.

[0113] II. Anti-DLL3 Antibodies

[0114] In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0115] In some aspects, the anti-DLL3 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-DLL3 antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VL). In some aspects, the anti-DLL3 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0116] In some embodiments, the heavy chain variable region VH of the anti-DLL3 antibody of the present invention is

[0117] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 12, 13, 14 or 15; or

[0118] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, 13, 14 or 15; or

[0119] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 12, 13, 14, or 15, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0120] In some embodiments, the light chain variable region VL of the anti-DLL3 antibody of the present invention is

[0121] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 16, 17 or 18; or

[0122] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, 17 or 18; or

[0123] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 16, 17, or 18, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0124] In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the anti-DLL3 antibodies of the present invention, HCDR1, HCDR2, and HCDR3 are

[0125] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12, 13, 14 or 15, or

[0126] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of (i),

[0127] For example, the HCDR1, HCDR2, and HCDR3 can be defined according to any CDR definition rules, such as the Kabat, Chothia, IMGT, AbM, or North rules, or a combination thereof. Preferably, the HCDR1 is determined according to the Kabat & Chothia rules, and the HCDR2 and HCDR3 are each determined according to the Kabat rules.

[0128] In some embodiments, the three complementarity determining regions (LCDRs) from the light chain variable region of the anti-DLL3 antibodies of the present invention, LCDR1, LCDR2, and LCDR3 are

[0129] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16, 17 or 18, or

[0130] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of (i);

[0131] For example, the LCDR1, LCDR2, LCDR3 can be defined according to any CDR definition rules, such as according to Kabat, Chothia, IMGT, AbM or Northern rules or a combination thereof. Preferably, the LCDR1, 2 and 3 are respectively determined according to the Kabat rules.

[0132] In some embodiments, the HCDR1 described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having 1, 2, or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0133] In some embodiments, the HCDR2 described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, 3, 4 or 5, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0134] In some embodiments, the HCDR3 described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having 1, 2, or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0135] In some embodiments, the LCDR1 described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 1, 2, or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0136] In some embodiments, the LCDR2 described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having 1, 2, or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0137] In some embodiments, the LCDR3 described herein comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 1, 2, or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0138] In some embodiments, in the anti-DLL3 antibodies or antigen-binding fragments thereof of the present invention,

[0139] The HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 3, 4 or 5; the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6; the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 7; the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 8; and / or the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0140] In some embodiments of the present invention, the anti-DLL3 antibodies or antigen-binding fragments thereof comprise VH and VL, wherein

[0141] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0142] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0143] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0144] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0145] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0146] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0147] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0148] The VH comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence, and the VL comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0149] In some embodiments of the present invention, the anti-DLL3 antibody or antigen-binding fragment thereof comprises

[0150] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 13, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 18; (ii) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 12, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 16; (iii) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 13, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 16; (iv) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 14, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: The three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in NO:16;

[0151] (v) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 15, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16; (vi) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12,

[0152] and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 17; (vii) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12,

[0153] and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 18;

[0154] or

[0155] (viii) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14,

[0156] and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 18;

[0157] For example, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 can be defined according to any CDR definition rules, such as according to Kabat, Chothia, IMGT, AbM or Northern rules or a combination thereof. Preferably, the HCDR1 is determined according to the Kabat & Chothia rules, and the HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are respectively determined according to the Kabat rules.

[0158] In some embodiments of the present invention, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein

[0159] The HCDR1 consists of the amino acid sequence of SEQ ID NO: 1; the HCDR2 consists of the amino acid sequence of SEQ ID NO: 2, 3, 4 or 5; the HCDR3 consists of the amino acid sequence of SEQ ID NO: 6; the LCDR1 consists of the amino acid sequence of SEQ ID NO: 7; the LCDR2 consists of the amino acid sequence of SEQ ID NO: 8; and the LCDR3 consists of the amino acid sequence of SEQ ID NO: 9.

[0160] In some embodiments of the present invention, the anti-DLL3 antibodies or antigen-binding fragments thereof comprise VH and VL, wherein

[0161] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 18;

[0162] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0163] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0164] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0165] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 15, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0166] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0167] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; or

[0168] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 18.

[0169] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions. In some embodiments, the amino acid substitutions are conservative substitutions.

[0170] In a preferred embodiment of the present invention, the anti-DLL3 antibody or antigen-binding fragment thereof of the present invention comprises a mutation that removes a post-translational modification (PTM), such as an amino acid substitution. In one embodiment, the hotspot of the anti-DLL3 antibody or antigen-binding fragment thereof of the present invention is mutated, such as in the heavy chain variable region CDR, such as HCDR2, where any one or two of the consecutive amino acids D and G are mutated, such as D is mutated to E or Q, and / or G is mutated to V. In one embodiment, the glycosylation site of the anti-DLL3 antibody or antigen-binding fragment thereof of the present invention is mutated to remove glycosylation, such as in the light chain variable region framework region, such as FR3, where any one or more of the consecutive amino acids NFS are mutated, such as N of NFS is mutated to Q, or S of NFS is mutated to A.

[0171] In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present invention is an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4 or an antigen-binding fragment thereof, for example, an antibody in the form of IgG1 or an antigen-binding fragment thereof.

[0172] In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention further comprises an antibody heavy chain constant region (HC). In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain constant region (LC). In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region (HC) and a light chain constant region (LC). In some embodiments, the heavy chain constant region of the present invention comprises an Fc region.

[0173] In some embodiments, the heavy chain constant region of the anti-DLL3 antibody of the invention is from IgG, such as the heavy chain constant region of human IgG, for example, the heavy chain constant region of (human) IgG1, IgG2, IgG3, or IgG4, such as the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, such as the heavy chain constant region of human IgG1.

[0174] In some embodiments, the IgG1 heavy chain constant region suitable for use in the antibody molecules of the invention is

[0175] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 23;

[0176] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 23; or

[0177] (iii) comprises an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 23, or consists of said amino acid sequence.

[0178] In some embodiments, the heavy chain constant region comprises a mutation that reduces binding to an Fcγ receptor, such as a LALA mutation.

[0179] In some embodiments, the heavy chain constant region described herein comprises an Fc region, such as a wild-type Fc region or a mutant Fc region. For example, the heavy chain constant region described herein may comprise an Fc region modified in terms of the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region) or a modified Fc region having altered binding affinity to one or more Fc receptors.

[0180] In some embodiments, the light chain constant region of an anti-DLL3 antibody of the invention is a light chain constant region from a lambda or kappa light chain constant region, eg, a lambda or kappa light chain constant region, eg, a human lambda or kappa light chain constant region.

[0181] In some embodiments, the light chain constant region

[0182] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 24 or 25;

[0183] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 24 or 25; or

[0184] (iii) comprises an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 24 or 25, or consists of said amino acid sequence.

[0185] In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention comprises an antibody heavy chain. In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention comprises an antibody light chain. In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain. In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention comprises two heavy chains and two light chains. In some embodiments, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains. In some embodiments, the heavy chain comprises or consists of a heavy chain variable region and a heavy chain constant region. In some embodiments, the light chain comprises or consists of a light chain variable region and a light chain constant region.

[0186] In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof has one or more of the following properties:

[0187] (i) exhibiting the same or similar binding affinity and / or specificity for DLL3 as the antibodies of the invention;

[0188] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention to DLL3;

[0189] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0190] (iv) competing with the antibodies of the invention for binding to DLL3;

[0191] (v) possess one or more biological properties of an antibody of the invention.

[0192] In some embodiments, the anti-DLL3 antibody is a monoclonal antibody.

[0193] In some embodiments, the anti-DLL3 antibody is a full-length antibody.

[0194] In some embodiments, anti-DLL3 antibodies are humanized. Humanization can be achieved by replacing one or more amino acid residues, particularly in the framework regions, in the heavy and light chain variable regions of a non-human native antibody with residues from the corresponding positions in the variable regions of a conventional human antibody. Methods for humanizing antibodies are well known in the art. Typically, humanizing substitutions are made in a manner that maintains favorable binding properties of the antibody. Assays for determining the biological properties of humanized antibodies, such as binding affinity, are well known in the art to identify and select appropriate humanizing residue mutations or combinations of mutations.

[0195] In one embodiment, the anti-DLL3 antibodies of the present invention also encompass antibody fragments thereof (e.g., antigen-binding fragments), preferably antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), diabodies, dAbs (domain antibodies), heavy chain antibodies, or linear antibodies. In one embodiment, the anti-DLL3 antibody fragment of the present invention is a Fab comprising the VH and VL described herein, as well as CH1 and CL.

[0196] In one embodiment, the anti-DLL3 antibody of the present invention may also be a bispecific antibody or a multispecific antibody that specifically binds to DLL3, as well as one or more other antigens (eg, CD3).

[0197] III. Multispecific Binding Molecules

[0198] In one aspect, the present invention relates to multispecific binding molecules that are capable of specifically binding to DLL3 and one or more other targets or antigens. In some embodiments, the other antigen is CD3. In some embodiments, the multispecific binding molecule is a multispecific antibody, such as a bispecific antibody.

[0199] In some embodiments, the antibody of the present invention is a bispecific antibody. The term "bispecific antibody" refers to an antibody comprising a first antigen binding region and a second antigen binding region, wherein the first antigen binding region binds to an antigen or epitope and the second antigen binding region binds to another antigen or another epitope. Therefore, the bispecific antibody according to the present invention comprises specificity for two different antigens, or for two different epitopes of an antigen. The bispecific antibody format comprises IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology.8:130). The most common IgG-like antibody type comprises two Fab regions and two Fc regions, and the heavy chain and light chain of each Fab can be derived from a separate monoclonal antibody. The bispecific antibody of the present invention can be prepared using bispecific antibody formats or technologies known in the art. Specific exemplary bispecific formats that can be used in the context of the present invention are described, for example, in Labrijn, et al. Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery, 2019, 18(8): 1-24.

[0200] Therefore, one aspect of the present invention relates to a bispecific antibody comprising

[0201] a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds DLL3, and the second antigen-binding region specifically binds CD3.

[0202] The first antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise or consist of an anti-DLL3 full-length antibody of the present invention or an antigen-binding fragment thereof, as long as it can specifically bind to DLL3, including but not limited to, for example, a full-length antibody, half antibody, Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody (e.g., VHH), dAb (domain antibody), diabody, heavy chain antibody, or linear antibody that specifically binds to DLL3.

[0203] In some embodiments, the first antigen-binding region is derived from an anti-DLL3 antibody or antigen-binding fragment thereof described herein, such as a Fab of an anti-DLL3 antibody described herein. In some embodiments, the first antigen-binding region that specifically binds to DLL3 comprises one, two, three, four, five, or six CDRs of an anti-DLL3 antibody described herein. In some embodiments, the first antigen-binding region comprises one, two, or three heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of an anti-DLL3 antibody described herein. In some embodiments, the first antigen-binding region comprises one, two, or three light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of an anti-DLL3 antibody described herein. In some embodiments, the first antigen-binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of an anti-DLL3 antibody described herein. In some embodiments, the first antigen-binding region comprises a heavy chain variable region of an anti-DLL3 antibody described herein. In some embodiments, the first antigen-binding region comprises a light chain variable region of an anti-DLL3 antibody described herein. In some embodiments, the first antigen binding region comprises a heavy chain variable region and a light chain variable region of an anti-DLL3 antibody described herein. In some embodiments, the first antigen binding region comprises or consists of a Fab of an anti-DLL3 antibody described herein.

[0204] The second antigen-binding region of the bispecific antibodies suitable for use in the present invention may comprise or consist of an anti-CD3 antibody or antigen-binding fragment thereof, as long as it can specifically bind to CD3, including but not limited to, for example, full-length antibodies, half antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), diabody, heavy chain antibodies, or linear antibodies that specifically bind to CD3.

[0205] In some embodiments, the second antigen-binding region is derived from an anti-CD3 antibody or antigen-binding fragment thereof, for example, an anti-CD3 Fab. In some embodiments, the second antigen-binding region of the bispecific antibody suitable for the present invention may comprise an anti-CD3 antibody or antigen-binding fragment thereof (e.g., an anti-CD3 antibody or antigen-binding fragment thereof disclosed in WO2022068809A1, such as HzSP34.24 antibody or antigen-binding fragment thereof), or be composed thereof, as long as it can specifically bind to CD3, including but not limited to, for example, full-length antibodies, half antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), diabody, heavy chain antibodies, or linear antibodies that specifically bind to CD3.

[0206] In some embodiments, the bispecific antibodies of the present invention are IgG-like bispecific antibodies. In some embodiments, the IgG-like bispecific antibodies of the present invention refer to bispecific antibodies comprising an Fc dimer (e.g., a heterodimeric Fc scaffold). Therefore, in some embodiments, the bispecific antibodies of the present invention comprise an Fc dimer, such as a heterodimeric Fc scaffold.

[0207] In one embodiment, the bispecific antibody may comprise one or more first antigen-binding regions. In one embodiment, the bispecific antibody may comprise one or more second antigen-binding regions. In some embodiments, the bispecific antibody comprises two first antigen-binding regions and two second antigen-binding regions.

[0208] The present invention has been described in detail with respect to components of multispecific binding molecules, such as bispecific antibodies (e.g., antigen-binding regions or domains contained therein, such as VH, VL, CL, CH1, or Fc regions). Those skilled in the art will appreciate that, unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of the present invention. Furthermore, those skilled in the art will appreciate that, unless the context clearly indicates otherwise, antibodies of the present invention (including any form of antibodies) may comprise any such combination.

[0209] III-1 Second antigen binding region

[0210] In some embodiments, the second antigen binding region specifically binds CD3.

[0211] In some embodiments, the second antigen binding region is an antigen binding region that specifically binds to CD3. In some embodiments, the second antigen binding region that specifically binds to CD3 is derived from an antibody that specifically binds to CD3, such as an antibody that specifically binds to CD3 disclosed in WO2022068809A1 (incorporated herein in its entirety), such as HzSP34.24 disclosed therein.

[0212] In some embodiments, the second antigen-binding region that specifically binds to CD3 comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to CD3 (e.g., a CD3 antibody disclosed in WO2022068809A1, e.g., HzSP34.24 disclosed therein). In some embodiments, the second antigen-binding region comprises 1, 2, or 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to CD3 (e.g., a CD3 antibody disclosed in WO2022068809A1, e.g., HzSP34.24 disclosed therein). In some embodiments, the second antigen-binding region comprises 1, 2, or 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to CD3 (e.g., a CD3 antibody disclosed in WO2022068809A1, e.g., HzSP34.24 disclosed therein). In some embodiments, the second antigen-binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to CD3 (e.g., a CD3 antibody disclosed in WO2022068809A1, such as HzSP34.24 disclosed therein). In some embodiments, the second antigen-binding region comprises a heavy chain variable region of a known antibody that specifically binds to CD3 (e.g., a CD3 antibody disclosed in WO2022068809A1, such as HzSP34.24 disclosed therein). In some embodiments, the second antigen-binding region comprises a light chain variable region of a known antibody that specifically binds to CD3 (e.g., a CD3 antibody disclosed in WO2022068809A1, such as HzSP34.24 disclosed therein). In some embodiments, the second antigen-binding region comprises a heavy chain variable region and a light chain variable region of a known antibody that specifically binds to CD3 (e.g., a CD3 antibody disclosed in WO2022068809A1, such as HzSP34.24 disclosed therein). In some embodiments, the second antigen-binding region comprises a Fab of a known antibody that specifically binds to CD3 (e.g., an antibody that specifically binds to CD3 disclosed in WO2022068809A1, such as HzSP34.24 disclosed therein).

[0213] In some embodiments, the second antigen-binding region that specifically binds to CD3 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region set forth in SEQ ID NO: 31. In some embodiments, the second antigen-binding region that specifically binds to CD3 comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region set forth in SEQ ID NO: 35. In some embodiments, the HCDR1 of the second antigen-binding region that specifically binds to CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 32; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0214] In some embodiments, the second antigen binding region that specifically binds to CD3 comprises a VH, wherein the VH comprises

[0215] (a) three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 32; HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 33; and HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 34; or

[0216] (b) the amino acid sequence shown in SEQ ID NO: 31 or consisting thereof.

[0217] In some embodiments, the second antigen binding region that specifically binds CD3 comprises a VL, wherein the VL comprises

[0218] (a) three complementarity determining regions (HCDRs) from a light chain variable region, HCDR1, HCDR2, and HCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38; or

[0219] (b) the amino acid sequence shown in SEQ ID NO: 35 or consisting thereof.

[0220] In some embodiments, the second antigen-binding region that specifically binds to CD3 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:32; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:33, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:34, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:36, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:37, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:38.

[0221] In some embodiments, the second antigen-binding region that specifically binds to CD3 comprises VH and VL, wherein VH comprises or consists of the sequence shown in SEQ ID NO: 31; and / or VL comprises or consists of the sequence shown in SEQ ID NO: 35.

[0222] In some embodiments, the second antigen binding region that specifically binds CD3 is a Fab fragment.

[0223] III-2Fab fragment

[0224] In some embodiments, the first antigen-binding region and / or the second antigen-binding region of the present invention is a Fab fragment. Suitable for use as a multispecific antibody as described herein, such as a bispecific antibody antigen-binding region, a Fab fragment is composed of two polypeptide chains comprising an antibody VH, CH1, VL, and CL domains, wherein the VH is paired with the VL and the CH1 is paired with the CL to form an antigen-binding region. In some embodiments, in Fab, one chain comprises VH and CH1 (i.e., VH-CH1) or consists of VH and CH1 from N-terminus to C-terminus, and the other chain comprises VL and CL (i.e., VL-CL) or consists of VL and CL from N-terminus to C-terminus. In some embodiments, in a multispecific antibody of the present invention, such as a bispecific antibody, the Fab can be connected to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising VH. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain and can be connected to the antibody Fc domain via the C-terminus of the CH1 of the VH-CH1 chain. In some embodiments, the connection is a direct connection or is connected via a linker. Herein, the Fab chain comprising VH-CH1 or consisting of VH-CH1 is also referred to as a Fab heavy chain, and the Fab chain comprising or consisting of VL-CL is also referred to as a Fab light chain.

[0225] In some embodiments, the CH1 is a CH1 from IgG1, IgG2, IgG3 or IgG4, preferably a CH1 from IgG1, preferably a CH1 from human IgG1, IgG2, IgG3 or IgG4, such as a CH1 from human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the CH1

[0226] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 28;

[0227] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 28; or

[0228] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 28.

[0229] In some embodiments, the CL is a light chain constant region from a lambda or kappa light chain constant region, for example, a lambda or kappa light chain constant region, for example, a human lambda or kappa light chain constant region. In some embodiments, the CL is a human kappa light chain constant region or a human lambda light chain constant region. In some embodiments, the CL

[0230] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24 or 25;

[0231] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 24 or 25; or

[0232] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 24 or 25, or consists of said amino acid sequence.

[0233] In some embodiments, the first antigen-binding region is a Fab that specifically binds to DLL3, wherein the Fab fragment is derived from an anti-DLL3 antibody of the present invention and comprises the heavy chain variable region (VH) and the light chain variable region (VL) of an anti-DLL3 antibody of the present invention. In some embodiments, the heavy chain of the Fab that specifically binds to DLL3 as the first antigen-binding region comprises or consists of VH and CH1, wherein VH is the VH of an anti-DLL3 antibody of the present invention. In some embodiments, the light chain of the Fab that specifically binds to DLL3 as the first antigen-binding region comprises or consists of VL and CL, wherein VL is the VL of an anti-DLL3 antibody of the present invention. In some embodiments, the first antigen-binding region comprises CH1 from IgG1 and / or a kappa light chain constant region.

[0234] In some embodiments, the Fab heavy chain that specifically binds to DLL3

[0235] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 19, 20, 21 or 22;

[0236] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, 20, 21 or 22; or

[0237] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 19, 20, 21 or 22, or consists of said amino acid sequence.

[0238] In some embodiments, the Fab light chain that specifically binds DLL3 comprises

[0239] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 45, 46 or 47;

[0240] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 45, 46 or 47; or

[0241] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 45, 46 or 47, or consists of said amino acid sequence.

[0242] In some embodiments, a Fab that specifically binds to DLL3 comprises or consists of a Fab heavy chain and a Fab light chain, wherein

[0243] The Fab heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 45;

[0244] The Fab heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:20, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:45;

[0245] The Fab heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:21, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:45;

[0246] The Fab heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:22, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:45;

[0247] The Fab heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 46;

[0248] The Fab heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 47;

[0249] The Fab heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 20, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 47; or

[0250] The Fab heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 21, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 47.

[0251] In some embodiments, the second antigen-binding region is a Fab that specifically binds to CD3, wherein the Fab fragment is derived from an anti-CD3 antibody and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-CD3 antibody.

[0252] In some embodiments, the Fab as the second antigen-binding region comprises the VH or VL of the second antigen-binding region described herein, or comprises the VH and VL of the second antigen-binding region described herein.

[0253] In some embodiments, the Fab heavy chain of an antibody that specifically binds to CD3 as the second antigen-binding region comprises or consists of a VH and a CH1, wherein the VH is the VH of the second antigen-binding region described herein. In some embodiments, the Fab light chain that specifically binds to CD3 as the second antigen-binding region comprises or consists of a VL and a CL, wherein the VL is the VL of the second antigen-binding region described herein. In some embodiments, the second antigen-binding region comprises a CH1 from IgG1, and / or a lambda light chain constant region.

[0254] In some embodiments, the Fab heavy chain that specifically binds to CD3 comprises

[0255] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 39;

[0256] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 39; or

[0257] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 39.

[0258] In some embodiments, the Fab light chain that specifically binds CD3 comprises

[0259] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 40;

[0260] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 40; or

[0261] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 40.

[0262] In some embodiments, the Fab heavy chain that specifically binds to CD3 comprises or consists of the amino acid sequence shown in SEQ ID NO:39, and the Fab light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:40.

[0263] In some embodiments, the light chain constant region of the Fab light chain of the first antigen-binding region is the same as or different from the light chain constant region of the Fab light chain of the second antigen-binding region. In some embodiments, the light chain constant region of the Fab light chain of the first and second antigen-binding regions is a kappa light chain constant region or a lambda light chain constant region. In some embodiments, the light chain constant region of the Fab light chain of the first antigen-binding region is a kappa light chain constant region, and the light chain constant region of the Fab light chain of the second antigen-binding region is a lambda light chain constant region; or the light chain constant region of the Fab light chain of the first antigen-binding region is a lambda light chain constant region, and the light chain constant region of the Fab light chain of the second antigen-binding region is a kappa light chain constant region.

[0264] III-3 Fc region

[0265] In some embodiments, one or more target binding regions (antigen binding regions) of a multispecific antibody (eg, a bispecific antibody) of the present invention further comprise an Fc region, wherein the Fc regions comprised may be the same or different.

[0266] In some embodiments, the first and second Fc regions are different and are capable of dimerizing to form a heterodimeric Fc scaffold.

[0267] In this article, Fc district refers to the C-terminal region of the immunoglobulin heavy chain containing the constant region of at least a portion, and can include native sequence Fc district and variant Fc district.Native sequence F district covers naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc district (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520) of its allotype.In some embodiments, Fc district of the present invention comprises antibody CH2 and CH3.In some embodiments, antibody Fc district can also carry IgG hinge region or part IgG hinge region at N end, such as, IgG1 hinge region or part IgG1 hinge region, such as according to EU numbering, the sequence of D221 to P230.In the hinge region, can contain sudden change.

[0268] Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0269] In some embodiments, the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 98%, 99% or higher identity, to SEQ ID NO: 26 or 27.

[0270] As will be appreciated by those skilled in the art, to promote the formation of heterodimers of the multispecific antibodies of the present invention, the Fc regions comprised by the multispecific antibodies of the present invention may include mutations that facilitate heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of both Fc regions.

[0271] Methods for promoting heterodimerization of the Fc region are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner so that each CH3 region (or a heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with other CH3 regions of complementary engineering (so that the CH3 regions of the first and second Fc regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions). For example, based on the Knob-into-Hole technology, corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region. This technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0272] Corresponding mutations can also be introduced into the first CH3 region of the first Fc region and the second CH3 region of the second Fc region based on the Innobody technology. For this technology, see, for example, PCT / CN2021 / 143141 (the patent is incorporated herein in its entirety).

[0273] In a specific embodiment,

[0274] The first CH3 region comprises an S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region comprises a K370S / T / A / V mutation (preferably K370S), and optionally one or more other mutations. In some embodiments, the first CH3 region comprises an S364R / K mutation, and the second CH3 region comprises a K370S / T / A / V mutation. In some embodiments, the first CH3 region comprises an S364R mutation, and the second CH3 region comprises a K370S mutation.

[0275] In some embodiments, the first CH3 region comprises S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region comprises K370S / T / A / V (preferably K370S) mutations and K409D / E (preferably K409D) mutations and / or Y349T / S / A / V mutations. In some embodiments, the first CH3 region comprises S364R / K+D399K / R, and the second CH3 region comprises K370S / T / A / V+Y349T / S / A / V. In some embodiments, the first CH3 region comprises S364R+D399K, and the second CH3 region comprises K370S+Y349T. In some embodiments, the first CH3 region further comprises E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further comprises K409D / E (preferably K409D), Q347D / E (preferably Q347D) and / or T350V / A (preferably T350V).

[0276] In some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T + K409D. In some embodiments, the first CH3 region further comprises E357N. In some embodiments, the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region further comprises E357N, and the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region and the second CH3 region further comprise T350V, or both comprise T350V.

[0277] Thus, in some embodiments, the first CH3 region comprises S364R+D399K, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N+T350V, and the second CH3 region comprises K370S+Y349T+K409D+Q347D+T350V.

[0278] In some embodiments, the first CH3 region comprises K409E / D (preferably K409E). In some embodiments, the second CH3 region comprises D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region comprises K409E / D (preferably K409E), and the second CH3 region comprises D399K / R (preferably D399K). In some embodiments, the first CH3 region further comprises T411R / K (preferably T411R). In some embodiments, the second CH3 region further comprises K370T / S / A / V (preferably K370T). In some embodiments, the CH3 region comprises K409E / D+T411R / K, and the second CH3 region comprises D399K / R+K370T / S / A / V. In some embodiments, the CH3 region comprises K409E+T411R, and the second CH3 region comprises D399K+K370T.

[0279] In some specific embodiments, the first and second CH3 regions have the following five mutation combinations:

[0280] First CH3 region Second CH3 region S364R,D399K,E357N,T350V K370S,Y349T,Q347D,K409D,T350V K409E,T411R D399K,K370T S364R,D399K,E357N K370S,Y349T,Q347D,K409D S364R,D399K K370S,Y349T,Q347D,K409D S364R,D399K K370S,Y349T,K409D

[0281] In one embodiment, the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S, and K409D mutations.

[0282] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0283] The Fc region of the binding molecules of the invention, such as antibodies, can also be mutated to obtain desired properties. Mutations in the Fc region are known in the art.

[0284] In one embodiment, the Fc region is modified with respect to the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to a wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0285] In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, such as reduced or abolished ADCC or ADCP or CDC effector function, eg, comprises a mutation that achieves the above function.

[0286] As will be appreciated by those skilled in the art, depending on the intended use of the binding molecules of the present invention, such as antibody molecules, the binding molecules of the present invention, such as antibody molecules, may also include modifications in the Fc domain that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has an L234A / L235A mutation that reduces binding to the Fcγ receptor. In another preferred embodiment, the Fc fragment may have a mutation that results in increased serum half-life, such as a mutation that improves binding of the Fc fragment to FcRn.

[0287] Thus, in a specific embodiment, an antibody of the invention, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0288] One Fc-region polypeptide comprises the mutations S364R and D399K, while the other Fc-region polypeptide comprises the mutations Y349T, K370S and K409D.

[0289] In some embodiments, the Fc region further comprises L234A / L235A mutations, respectively, and optionally a proline deletion at position P329 (P329delP).

[0290] Therefore, in a specific embodiment, an antibody of the present invention, such as a multispecific antibody, comprises two Fc regions that are heterodimerized, wherein one Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 30.

[0291] In a specific embodiment, an antibody of the present invention, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:29, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:30.

[0292] Thus, in a specific embodiment, a binding molecule of the invention, such as an antibody, such as a multispecific antibody, comprises two Fc regions that heterodimerize, wherein

[0293] a) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 29 and comprises the mutations S364R and D399K, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 30 and comprises the mutations Y349T, K370S, and K409D; or

[0294] b) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 29 and comprises mutations S364R and D399K and L234A / L235A mutations, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 30 and comprises mutations Y349T, K370S and K409D and L234A / L235A mutations.

[0295] III-4 Exemplary bispecific antibodies

[0296] In some embodiments, the bispecific antibodies of the present invention comprise a first antigen-binding region as described herein that specifically binds to a first antigen and a second antigen-binding region as described herein that specifically binds to a second antigen, wherein the first antigen-binding region comprises a first Fab and the second antigen-binding region comprises a second Fab, wherein the first Fab is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region). In a specific embodiment, the first Fab and the first Fc region constitute a first half antibody that binds to the first antigen, the second Fab and the second Fc region constitute a second half antibody that binds to the second antigen, and the first half antibody and the second half antibody constitute the bispecific antibody of the present invention.

[0297] In a specific embodiment, the bispecific antibody is an IgG-like antibody having Figure 3 In a specific embodiment, the first Fc region and the second Fc region comprise an Innobody mutation, and optionally a mutation that reduces effector function mediated by the Fc region or a mutation that reduces binding to Fcγ receptors. In one embodiment, the bispecific antibody comprises or consists of:

[0298] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0299] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0300] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region);

[0301] Light chain 2: From N-terminus to C-terminus, it comprises the following: the light chain variable region of the second Fab - the light chain constant region.

[0302] In some embodiments, the Fab heavy chain variable region and the heavy chain constant region CH1 constitute the Fab heavy chain.In some embodiments, the Fab light chain variable region and the light chain constant region constitute the Fab light chain.

[0303] In some embodiments, the heavy chain variable region-heavy chain constant region CH1 of the first Fab and the light chain variable region-light chain constant region of the first Fab constitute the first Fab; the heavy chain variable region-heavy chain constant region CH1 of the second Fab and the light chain variable region-light chain constant region of the second Fab constitute the second Fab.

[0304] In some embodiments, the first Fc region comprises S364R and D399K mutations, and the second Fc region comprises Y349T, K370S, and K409D mutations.

[0305] In a specific embodiment, the first Fab is an anti-DLL3 Fab of the invention and the second Fab is an anti-CD3 Fab, or vice versa.

[0306] In a specific embodiment, the bispecific antibody of the invention specifically binds to DLL3 and CD3 and comprises or consists of:

[0307] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a Fab heavy chain variable region that specifically binds to DLL3 - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0308] Light chain 1: From N-terminus to C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to DLL3 - a light chain constant region;

[0309] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a Fab heavy chain variable region that specifically binds to CD3 - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region); and / or

[0310] Light chain 2: From N-terminus to C-terminus, it comprises the following: a light chain variable region of a Fab that specifically binds to CD3 - a light chain constant region.

[0311] In some embodiments, the first Fc region comprises S364R and D399K mutations, and the second Fc region comprises Y349T, K370S, and K409D mutations, or vice versa; optionally, the first Fc region and the second Fc region further comprise L234A / L235A mutations, respectively. In some embodiments, the light chain constant region in light chain 1 is the same as or different from the light chain constant region in light chain 2, for example, the light chain constant region in light chain 1 is a kappa light chain constant region and the light chain constant region in light chain 2 is a lambda light chain constant region.

[0312] The various domains of the bispecific antibodies of the invention are as defined herein.

[0313] In some specific embodiments, the bispecific antibody that specifically binds to DLL3 and CD3 comprises or consists of heavy chain 1, heavy chain 2, light chain 1, and light chain 2, wherein

[0314] Heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 41, 42, 43 or 44, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0315] Light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 45, 46, or 47, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0316] Heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and / or

[0317] Light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 40, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or consists of the amino acid sequence.

[0318] In some specific embodiments, the bispecific antibody that specifically binds to DLL3 and CD3 comprises or consists of heavy chain 1, heavy chain 2, light chain 1, and light chain 2, wherein

[0319] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:41, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:45, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0320] heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:42, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:47, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0321] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:43, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:47, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0322] In some specific embodiments, the bispecific antibody that specifically binds to DLL3 and CD3 comprises or consists of heavy chain 1, heavy chain 2, light chain 1, and light chain 2, wherein

[0323] Heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO:41; light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO:45; heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO:48; and light chain 2 comprises or consists of the amino acid sequence of SEQ ID NO:40;

[0324] Heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO:42; light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO:47; heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO:48; and light chain 2 comprises or consists of the amino acid sequence of SEQ ID NO:40; or

[0325] Heavy chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO:43; light chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO:47; heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO:48; and light chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO:40.

[0326] IV. Polynucleotides, Vectors, and Host Cells

[0327] The present invention provides nucleic acids encoding any of the above antibody molecules of the present invention (eg, an anti-DLL3 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody of the present invention).

[0328] In one aspect, the invention provides nucleic acids encoding any of the above anti-DLL3 antibodies or antigen-binding fragments thereof, or multispecific antibodies, such as bispecific antibodies.

[0329] To facilitate production and purification, an anti-DLL3 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody can be fused to a secretory signal peptide at the N-terminus or C-terminus (e.g., the C-terminus), and / or a tag peptide that facilitates purification, such as a hexahistidine tag or a biotin tag.

[0330] As will be apparent to those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0331] In some embodiments, the nucleic acid of the present invention comprises a nucleic acid selected from any one of SEQ ID NO: 10 or 11, or a nucleic acid having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence selected from any one of SEQ ID NO: 10 or 11.

[0332] In some embodiments, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 12-22 and 41-47, or a nucleic acid encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 12-22 and 41-47.

[0333] Nucleic acid sequences encoding molecules of the present invention can be generated using methods well known in the art, such as de novo solid phase DNA synthesis or PCR amplification.

[0334] In one aspect, the present invention also provides a vector comprising a nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. Vectors include but are not limited to viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

[0335] In one aspect, the present invention also provides a host cell comprising the nucleic acid or the vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, e.g., HEK 293 or 293F cells or 293FT cells or Expi293 cells). In another embodiment, the host cell is prokaryotic.

[0336] Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells or 293FT cells or Expi293 cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2 / 0, etc. Mammalian host cell lines suitable for producing antibodies are known in the art. In a preferred embodiment, the host cell is a CHO or HEK293 cell or a 293FT cell or an Expi293 cell.

[0337] V. Production and Purification of the Molecules of the Invention

[0338] In another aspect, the present invention provides a method for producing an antibody molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof, or a multispecific antibody such as a bispecific antibody), the method comprising: culturing a host cell containing a protein encoding the polypeptide chain under conditions suitable for expression of the polypeptide chain of the molecule; optionally further comprising assembling the polypeptide chains to produce the molecule under conditions suitable for assembly of the polypeptide chains into the molecule.

[0339] For recombinant production, the polynucleotide encoding the polypeptide chain of the molecule of the present invention can be inserted into one or more vectors for further cloning and / or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Once an expression vector comprising one or more polynucleotides of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection or other conventional techniques.

[0340] The molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0341] The purity of the molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the antibody molecules provided herein can be identified, screened or characterized by a variety of assays known in the art.

[0342] VI. Assay

[0343] The molecules provided herein (e.g., anti-DLL3 antibodies, antigen-binding fragments thereof, or multispecific antibodies, such as bispecific antibodies) can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. The Examples illustrate methods for determining the properties of the anti-DLL3 antibodies, antigen-binding fragments thereof, or bispecific antibodies of the invention, such as thin-layer interferometry (BLI), T cell reporter gene assays, T cell activation assays, T cell-mediated killing assays, or anti-tumor activity assays.

[0344] VII. Immunoconjugates

[0345] In one aspect, the invention provides immunoconjugates comprising a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody) and one or more other active ingredients (e.g., an active ingredient from a drug or therapeutic agent for treating a disease of the invention, such as a small molecule that enhances the therapeutic effect of the molecule of the invention).

[0346] In some embodiments, the immunoconjugate is an antibody-drug-conjugate (ADC).

[0347] VIII. Drugs and Uses

[0348] In some embodiments, the invention also relates to compositions (eg, pharmaceutical compositions or pharmaceutical formulations) comprising a molecule of the invention (eg, an anti-DLL3 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody, or an immunoconjugate).

[0349] In one embodiment, a composition, such as a pharmaceutical composition, comprises a molecule of the invention in combination with one or more other therapeutic agents.

[0350] The compositions of the present invention can also include suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients as known in the art, including buffers. As used herein, "pharmaceutical carriers" include any and all solvents, dispersion media, isotonic agents or absorption delay agents that are physiologically compatible. For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago. The compositions of the present invention can be in various forms. These forms, for example, include liquid, semisolid and solid dosage forms, such as liquid solutions (for example, injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0351] Medicaments comprising the antibody molecules or immunoconjugates described herein can be prepared by mixing an antibody molecule (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody) or immunoconjugate of the invention having the desired degree of purity with one or more optional pharmaceutical excipients, for example in the form of a lyophilized formulation or an aqueous solution.

[0352] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody, or an immunoconjugate), and one or more other therapeutic agents.

[0353] Another object of the present invention is to provide a kit of parts comprising the pharmaceutical combination of the present invention, preferably in the form of pharmaceutical dosage units, whereby dosage units can be provided according to a dosing regimen or interval of drug administration.

[0354] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0355] - a first container containing a pharmaceutical composition comprising a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody, or an immunoconjugate);

[0356] - One or more containers containing a pharmaceutical composition comprising one or more additional therapeutic agents.

[0357] In some embodiments, when a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or a bispecific antibody or immunoconjugate thereof of the invention) is used to treat a tumor, the other therapeutic agent is, for example, a variety of therapeutic agents used to treat a tumor, such as a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, a small molecule drug, or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist).

[0358] In some specific embodiments, the other therapeutic agent can be other antibodies, such as antibodies that specifically bind to immune checkpoint molecules, such as antibodies that inhibit the PD-1 / PD-L1 signaling pathway, such as anti-PD-1 antibodies; or anti-PD-L1 antibodies or anti-PD-L2 antibodies.

[0359] In some specific embodiments, the pharmaceutical combination of the present invention comprises a molecule of the present invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or a bispecific antibody or immunoconjugate thereof) and an antibody that inhibits the PD-1 / PD-L1 signaling pathway, such as an anti-PD-1 antibody; or an anti-PD-L1 antibody or an anti-PD-L2 antibody.

[0360] In some embodiments, the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, cemiplizumab, toripalimab, sintilimab, tislelizumab, camrelizumab, penampalimumab, sepalimumab, putalimumab, or adebelimumab.

[0361] In some specific embodiments, the other therapeutic agent is a chemotherapeutic agent, such as a chemotherapeutic agent used in standard chemotherapy for treating tumors. In some specific embodiments, the other chemotherapeutic agent is selected from platinum chemotherapy drugs such as carboplatin; or a topoisomerase inhibitor, such as a topoisomerase II inhibitor, such as etoposide.

[0362] In some specific embodiments, the pharmaceutical combination of the invention comprises a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or a bispecific antibody or immunoconjugate thereof) and one, two or more chemotherapeutic agents, e.g., two chemotherapeutic agents, wherein one chemotherapeutic agent is a platinum chemotherapeutic agent such as carboplatin and the other chemotherapeutic agent is a topoisomerase inhibitor, e.g., a topoisomerase II inhibitor, etoposide.

[0363] In some specific embodiments, the pharmaceutical combination of the invention comprises a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or bispecific antibody or immunoconjugate thereof of the invention), and a combination of carboplatin and etoposide.

[0364] Another aspect of the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof, or a multispecific antibody such as a bispecific antibody or immunoconjugate thereof), pharmaceutical composition, pharmaceutical combination, or kit. In some embodiments, the disease is, for example, a tumor such as cancer.

[0365] In some embodiments, the invention relates to a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody or immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination or a kit of the invention for use in therapy, e.g., for treating a tumor such as a cancer.

[0366] In some embodiments, the invention relates to methods of treating a disease, such as a tumor, such as cancer, using a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody or immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination or a kit of the invention, or uses thereof, or uses thereof for the preparation of a medicament for the treatment thereof.

[0367] In some embodiments, the tumor is a solid tumor or a hematological tumor and a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in the early, middle or late stages or be a metastatic cancer. In some embodiments, the tumor has tumor immune escape.

[0368] In some embodiments, the tumor is a DLL3-positive tumor or cancer. In some embodiments, the tumor is associated with aberrant expression or aberrant activity of DLL3.

[0369] In some embodiments, a DLL3-positive tumor or cancer refers to abnormal expression or activity of DLL3 in a subject having the tumor, e.g., cancer. In some embodiments, the subject (particularly an adult subject) has DLL3 expression. In some embodiments, the subject has (e.g., elevated levels, e.g., nucleic acid or protein levels or activity) DLL3 (e.g., compared to a healthy subject). In some embodiments, the subject has (e.g., elevated levels, e.g., nucleic acid or protein levels or activity) DLL3 in a biological sample (e.g., tumor cells or tumor tissue) (e.g., compared to a biological sample of a healthy subject (e.g., a corresponding tissue or cell in a healthy subject), or compared to DLL3 in an adjacent healthy tissue or cell of the subject).

[0370] In some embodiments, a DLL3-positive tumor is a tumor containing tumor cells that aberrantly express DLL3. In some embodiments, aberrant expression of DLL3 refers to expression of DLL3 on the cell membrane of the tumor cells. In some embodiments, aberrant expression of DLL3 refers to higher expression of DLL3 on tumor cells compared to control cells (e.g., healthy cells from corresponding tissue of a healthy individual, or healthy cells adjacent to tumor cells).

[0371] In some embodiments, the tumor is a neuroendocrine tumor (eg, a DLL3-positive neuroendocrine tumor), such as a lung cancer (eg, a DLL3-positive lung cancer), such as small cell lung cancer (SCLC).

[0372] Depending on their therapeutic use, the molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies or immunoconjugates thereof) or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, e.g., treatment modalities and / or other therapeutic agents, for the methods or uses described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0373] In some embodiments, when a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof, or a bispecific antibody or immunoconjugate thereof) is used to treat a tumor, the other therapeutic agent, for example, various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). In some embodiments, the therapeutic agent is a PD-1 antibody or antigen-binding fragment thereof. In some embodiments, when a molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof, or a bispecific antibody or immunoconjugate thereof) is used to treat a tumor, the treatment modality includes surgery; radiotherapy, local irradiation or focused irradiation, etc.

[0374] In some specific embodiments, the other therapeutic agent can be other antibodies, such as antibodies that specifically bind to immune checkpoint molecules, such as antibodies that inhibit the PD-1 / PD-L1 signaling pathway, such as anti-PD-1 antibodies; or anti-PD-L1 antibodies or anti-PD-L2 antibodies.

[0375] In some specific embodiments, the molecules of the invention (e.g., the anti-DLL3 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof of the invention) are administered in combination with an antibody that inhibits the PD-1 / PD-L1 signaling pathway, such as an anti-PD-1 antibody; or an anti-PD-L1 antibody or an anti-PD-L2 antibody, for the methods or uses described herein.

[0376] In some embodiments, the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, cemiplizumab, toripalimab, sintilimab, tislelizumab, camrelizumab, penampalimumab, sepalimumab, putalimumab, or adebelimumab.

[0377] In some specific embodiments, the other therapeutic agent is a chemotherapeutic agent, such as a chemotherapeutic agent used in standard chemotherapy for treating tumors. In some specific embodiments, the other chemotherapeutic agent is selected from platinum chemotherapy drugs such as carboplatin; or a topoisomerase inhibitor, such as a topoisomerase II inhibitor, such as etoposide.

[0378] In some specific embodiments, molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof of the invention) are administered in combination with standard chemotherapy for the methods or uses described herein.

[0379] As described herein, standard chemotherapy refers to a chemotherapy method that is widely recognized and used as a conventional treatment for a specific type of cancer after clinical research and practical verification. In some specific embodiments, standard chemotherapy includes standard chemotherapy methods suitable for treating tumors described herein.

[0380] In some embodiments, standard chemotherapy for treating tumors described herein comprises administering one, two or more chemotherapeutic agents in combination, for example, two chemotherapeutic agents, for example, one of which is a platinum chemotherapy drug such as carboplatin, and the other chemotherapeutic agent is a topoisomerase inhibitor, for example, a topoisomerase II inhibitor, for example, etoposide. In some embodiments, standard chemotherapy for treating tumors described herein comprises administering carboplatin and etoposide in combination.

[0381] In some specific embodiments, the molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof) are administered in combination with one, two or more chemotherapeutic agents, e.g., two chemotherapeutic agents, for the methods or uses described herein, preferably, wherein one chemotherapeutic agent is a platinum-based chemotherapeutic drug such as carboplatin and the other chemotherapeutic agent is a topoisomerase inhibitor, e.g., a topoisomerase II inhibitor, etoposide.

[0382] In some specific embodiments, molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof of the invention) are administered in combination with carboplatin and etoposide for the methods or uses described herein.

[0383] In other aspects, the present invention provides the use of the molecules of the present invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof, etc.) or compositions or combination products comprising the same in the production or preparation of medicaments for the uses described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0384] In other aspects, the present invention also provides molecules of the present invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof, etc.), or compositions or drugs or formulations or combination products comprising the same, which are used for therapy, e.g., for treating the relevant diseases or disorders mentioned herein.

[0385] IX. Diagnosis and Testing

[0386] In certain embodiments, the molecules provided herein (eg, anti-DLL3 antibodies or antigen-binding fragments thereof or bispecific antibodies or immunoconjugates thereof, etc.) can be used to detect the presence of DLL3 in a biological sample.

[0387] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid.

[0388] In certain embodiments, the method comprises contacting a biological sample with a molecule as described herein (e.g., a bispecific antibody or immunoconjugate thereof) under conditions permissive for binding to DLL3, and detecting whether a complex forms between the molecule and DLL3, wherein the formation of a complex indicates the presence of DLL3. The method can be in vitro or in vivo. In one embodiment, the molecule of the invention is used to select a subject suitable for treatment with an anti-DLL3 antibody or antigen-binding fragment thereof, a bispecific antibody or immunoconjugate thereof, etc., of the invention, e.g., wherein DLL3 is a biomarker used to select the subject.

[0389] In certain embodiments, labeled molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof, bispecific antibodies or immunoconjugates thereof, etc.) are provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., by an enzymatic reaction or molecular interaction.

[0390] In some embodiments provided herein, the sample is obtained prior to treatment with a molecule of the invention, or a composition, medicament, formulation, or combination product comprising the same. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during treatment with another therapy, or after treatment with another therapy.

[0391] In some embodiments, DLL3 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0392] In some embodiments, a method of treating a disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of DLL3, thereby determining a DLL3 value, comparing the DLL3 value to a control value (e.g., a value in a normal individual), and if the DLL3 value is greater than the control value, administering to the subject a therapeutically effective amount of a bispecific antibody of the present invention, or a composition, medicament, or formulation comprising the same, optionally in combination with one or more other therapies, thereby treating the disease.

[0393] X. Specific Implementation Plan

[0394] 1. An anti-DLL3 antibody or antigen-binding fragment thereof, comprising three CDRs of the heavy chain variable region VH, namely HCDR1, HCDR2 and HCDR3, and three CDRs of the light chain variable region VL, namely LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from

[0395] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 13, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 18; (ii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12,

[0396] and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16; (iii) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 13,

[0397] and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16; (iv) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14,

[0398] and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16; (v) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 15,

[0399] and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16; (vi) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12,

[0400] and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 17; (vii) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12,

[0401] and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 18;

[0402] or

[0403] (viii) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14,

[0404] and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:18.

[0405] 2. An anti-DLL3 antibody or antigen-binding fragment thereof comprising a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1 consists of the amino acid sequence of SEQ ID NO: 1; the HCDR2 consists of the amino acid sequence of SEQ ID NO: 2; the HCDR3 consists of the amino acid sequence of SEQ ID NO: 6; the LCDR1 consists of the amino acid sequence of SEQ ID NO: 7; the LCDR2 consists of the amino acid sequence of SEQ ID NO: 8; and the LCDR3 consists of the amino acid sequence of SEQ ID NO: 9;

[0406] Optionally, any amino acid D or G in the consecutive amino acids DG in HCDR2 can be substituted, for example, D can be substituted with E or Q, and / or G is substituted with V, for example, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 3, 4 or 5.

[0407] 3. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain can

[0408] Variable Zone

[0409] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 12, 13, 14 or 15; or

[0410] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12, 13, 14 or 15.

[0411] 4. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, comprising a light chain variable region VL, wherein

[0412] Light chain variable region

[0413] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 16; or

[0414] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16;

[0415] Optionally, any of the consecutive amino acids NFS in the framework region FR3 of the VL may be mutated, for example, N therein is substituted with Q and / or S therein is substituted with A; for example

[0416] The light chain variable region

[0417] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 17 or 18; or

[0418] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 17 or 18.

[0419] 5. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0420] (i) The VH contains the amino acid sequence shown in SEQ ID NO: 13 or has at least 90%, 91%, 92%, 93%,

[0421] The amino acid sequence of the present invention is 94%, 95%, 96%, 97%, 98% or 99% identical to or consists of said amino acid sequence, and said VL comprises the amino acid sequence of SEQ ID NO: 18 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0422] an amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical to, or consists of, such an amino acid sequence;

[0423] (ii) The VH contains the amino acid sequence shown in SEQ ID NO: 12 or has at least 90%, 91%, 92%, 93%,

[0424] The amino acid sequence of the present invention is 94%, 95%, 96%, 97%, 98% or 99% identical to or consists of said amino acid sequence, and said VL comprises the amino acid sequence of SEQ ID NO: 16 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0425] an amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical to, or consists of, such an amino acid sequence;

[0426] (iii) The VH contains the amino acid sequence shown in SEQ ID NO: 13 or has at least 90%, 91%, 92%, 93%,

[0427] The amino acid sequence of the present invention is 94%, 95%, 96%, 97%, 98% or 99% identical to or consists of said amino acid sequence, and said VL comprises the amino acid sequence of SEQ ID NO: 16 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0428] an amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical to, or consists of, such an amino acid sequence;

[0429] (iv) The VH contains the amino acid sequence shown in SEQ ID NO: 14 or has at least 90%, 91%, 92%, 93%,

[0430] The amino acid sequence of the present invention is 94%, 95%, 96%, 97%, 98% or 99% identical to or consists of said amino acid sequence, and said VL comprises the amino acid sequence of SEQ ID NO: 16 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0431] an amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical to, or consists of, such an amino acid sequence;

[0432] (v) The VH contains the amino acid sequence shown in SEQ ID NO: 15 or has at least 90%, 91%, 92%, 93%,

[0433] The amino acid sequence of the present invention is 94%, 95%, 96%, 97%, 98% or 99% identical to or consists of said amino acid sequence, and said VL comprises the amino acid sequence of SEQ ID NO: 16 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0434] an amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical to, or consists of, such an amino acid sequence;

[0435] (vi) The VH contains the amino acid sequence shown in SEQ ID NO: 12 or has at least 90%, 91%, 92%, 93%,

[0436] The amino acid sequence of the present invention is 94%, 95%, 96%, 97%, 98% or 99% identical to or consists of said amino acid sequence, and said VL comprises the amino acid sequence of SEQ ID NO: 17 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0437] an amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical to, or consists of, such an amino acid sequence;

[0438] (vii) The VH contains the amino acid sequence shown in SEQ ID NO: 12 or has at least 90%, 91%, 92%, 93%,

[0439] The amino acid sequence of the present invention is 94%, 95%, 96%, 97%, 98% or 99% identical to or consists of said amino acid sequence, and said VL comprises the amino acid sequence of SEQ ID NO: 18 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0440] or (viii). the VH comprises an amino acid sequence as set forth in SEQ ID NO: 14 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consists of said amino acid sequence; or (viii). the VH comprises an amino acid sequence as set forth in SEQ ID NO: 14 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consists of said amino acid sequence.

[0441] The amino acid sequence of the present invention is 94%, 95%, 96%, 97%, 98% or 99% identical to or consists of said amino acid sequence, and said VL comprises the amino acid sequence of SEQ ID NO: 18 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0442] or consisting of an amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical.

[0443] 6. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0444] (i) The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 18;

[0445] (ii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0446] (iii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0447] (iv) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0448] (v) the VH comprises the amino acid sequence shown in SEQ ID NO: 15 or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO: 16 or consists of the amino acid sequence;

[0449] (vi) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0450] (vii). the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; or (viii). the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 18.

[0451] 7. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, further comprising a heavy chain constant region HC, for example, the antibody heavy chain constant region HC is from IgG, such as the heavy chain constant region of human IgG, for example, the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0452] 8. The antibody or antigen-binding fragment thereof of embodiment 7, wherein the heavy chain constant region

[0453] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 23; or

[0454] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 23.

[0455] 9. The antibody or antigen-binding fragment thereof of embodiment 7 or 8, wherein the heavy chain constant region comprises an Fc region, wherein the Fc region is an Fc region from IgG, such as a human IgG, for example, an IgG1, IgG2, IgG3 or IgG4 Fc, preferably a human IgG1, IgG2, IgG3 or IgG4 Fc, optionally, the Fc region comprises

[0456] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 26 or 27; or

[0457] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 26 or 27.

[0458] 10. The antibody or antigen-binding fragment thereof of any one of embodiments 1-9, further comprising a light chain constant region, e.g., the light chain constant region is a light chain constant region from a lambda or kappa light chain constant region, e.g., a lambda or kappa light chain constant region, e.g., a human lambda or kappa light chain constant region.

[0459] 11. The antibody or antigen-binding fragment thereof according to embodiment 10, wherein the light chain constant region

[0460] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 24 or 25; or

[0461] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 24 or 25.

[0462] 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-11, wherein the antibody is a humanized antibody or a chimeric antibody.

[0463] 13. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-12, wherein the antibody is a monoclonal antibody.

[0464] 14. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-13, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, diabody, or linear antibody.

[0465] 15. The antibody or antigen-binding fragment thereof of any one of embodiments 1-11, wherein the antibody is a bispecific antibody or a multispecific antibody comprising a first binding specificity for DLL3 and additional binding specificities for one or more other antigens.

[0466] 16. The antibody or antigen-binding fragment thereof according to embodiment 15, wherein the other antigen is CD3.

[0467] 17. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region,

[0468] The first antigen binding region specifically binds to DLL3 and comprises VH and VL, wherein

[0469] The VH comprises HCDR1, HCDR2, HCDR3 as defined in embodiment 1 or 2 and the VL comprises LCDR1, LCDR2 and LCDR3 as defined in embodiment 1 or 2; or

[0470] The VH and VL are as defined in any one of embodiments 3-6; and

[0471] The second antigen binding region specifically binds to CD3.

[0472] 18. The bispecific antibody of embodiment 17, wherein the first antigen binding region is a Fab of an anti-DLL3 antibody as defined in any one of embodiments 1-14.

[0473] 19. The bispecific antibody of embodiment 17 or 18, wherein the second antigen binding region is a Fab that specifically binds to CD3.

[0474] 20. The bispecific antibody of embodiment 18 or 19, wherein the Fab serving as the first antigen binding region or the second antigen binding region comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from human IgG1, IgG2, IgG3 or IgG4, for example, CH1 from human IgG1, IgG2, IgG3 or IgG4.

[0475] 21. The bispecific antibody of embodiment 20, wherein the CH1

[0476] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 28; or

[0477] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 28.

[0478] 22. The bispecific antibody of any one of embodiments 18-21, wherein the Fab as the first antigen binding region or the second antigen binding region comprises a light chain constant region, wherein the light chain constant region is a light chain constant region from a lambda or kappa light chain constant region, for example, a lambda or kappa light chain constant region, for example, a human lambda or kappa light chain constant region, optionally, the first antigen binding region and the second binding region comprise the same or different light chain constant regions, for example, the first antigen binding region comprises a kappa light chain constant region and the second antigen binding region comprises a lambda light chain constant region.

[0479] 23. The bispecific antibody of embodiment 22, wherein the Kappa light chain constant region comprises

[0480] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 25; or

[0481] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 25;

[0482] and / or

[0483] The Lambda light chain constant region comprises

[0484] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 24; or

[0485] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 24.

[0486] 24. The bispecific antibody of any one of embodiments 17-23, wherein the bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer, the first Fc region and the second Fc region are the same or different, optionally, the Fc regions are respectively from IgG, such as human IgG, for example, IgG1, IgG2, IgG3 or IgG4, preferably human IgG1, IgG2, IgG3 or IgG4 Fc, optionally, the Fc region

[0487] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 26 or 27; or

[0488] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 26 or 27.

[0489] 25. The bispecific antibody of embodiment 24, wherein the two Fc regions are different, wherein based on Innobody technology, mutations are introduced into the first Fc region and the second Fc region to promote heterodimerization of the first Fc region and the second Fc region.

[0490] 26. The antibody or antigen-binding fragment thereof of embodiment 25, wherein the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S and K409D mutations.

[0491] 27. The bispecific antibody of any one of embodiments 24-26, wherein the first and / or second Fc region comprises L234A / L235A mutations.

[0492] 28. The bispecific antibody of embodiment 26 or 27, wherein

[0493] a) one Fc region comprises, or consists of, the amino acid sequence of SEQ ID NO: 29, and the other Fc region comprises, or consists of, the amino acid sequence of SEQ ID NO: 30;

[0494] b) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 29 and comprises the mutations S364R and D399K, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 30 and comprises the mutations Y349T, K370S, and K409D; or

[0495] c) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 29 and comprises mutations S364R and D399K and L234A / L235A mutations, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 30 and comprises mutations Y349T, K370S and K409D and L234A / L235A mutations.

[0496] 29. The bispecific antibody of any one of embodiments 17-28, wherein the second antigen-binding region comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein

[0497] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 32; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0498] 30. The bispecific antibody of embodiment 29, wherein the second antigen-binding region comprises VH and VL, wherein VH comprises or consists of the sequence shown in SEQ ID NO: 31; and VL comprises or consists of the sequence shown in SEQ ID NO: 35.

[0499] 31. The bispecific antibody of any one of embodiments 17-30, wherein the bispecific antibody comprises a first antigen binding region that specifically binds to DLL3 and a second antigen binding region that specifically binds to CD3, wherein the first antigen binding region comprises a first Fab and the second antigen binding region comprises a second Fab, wherein the first Fab is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region).

[0500] 32. The bispecific antibody of embodiment 31, wherein the bispecific antibody is an IgG-like antibody having Figure 3 Configuration shown.

[0501] 33. The bispecific antibody of embodiment 31 or 32, comprising a first Fab as a first antigen-binding region that specifically binds to DLL3 and a second Fab as a second antigen-binding region that specifically binds to CD3, wherein the bispecific antibody comprises

[0502] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0503] Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region;

[0504] Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region);

[0505] Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region;

[0506] The first Fab heavy chain variable region-heavy chain constant region CH1 and the first Fab light chain variable region-light chain constant region constitute the first Fab, and the second Fab heavy chain variable region-heavy chain constant region CH1 and the second Fab light chain variable region-light chain constant region constitute the second Fab;

[0507] Optionally, the light chain constant region in light chain 1 and the light chain constant region in light chain 2 are different, e.g., the light chain constant region in light chain 1 is a Kappa light chain constant region and the light chain constant region in light chain 2 is a Lambda light chain constant region.

[0508] 34. The bispecific antibody of embodiment 33, wherein

[0509] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:41, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:45, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0510] heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:42, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:47, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0511] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:43, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:47, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0512] 35. A nucleic acid molecule comprising or consisting of a polynucleotide encoding any one chain of the antibody or antigen-binding fragment thereof of any one of embodiments 1-16, or any one chain of the bispecific antibody of any one of embodiments 17-34.

[0513] 36. An expression vector comprising the nucleic acid molecule of embodiment 35, preferably, the expression vector is pCDNA, such as pCDNA3.1.

[0514] 37. A host cell comprising the nucleic acid molecule of embodiment 35 or the expression vector of embodiment 36. Preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as Expi293 cells.

[0515] 38. A method for preparing the antibody or antigen-binding fragment thereof of any one of embodiments 1-16 or the bispecific antibody of any one of embodiments 17-34, the method comprising culturing a host cell comprising the nucleic acid molecule of embodiment 35 or the expression vector of embodiment 36 under conditions suitable for expression of the polypeptide chain of the antibody or antigen-binding fragment thereof or bispecific antibody, and optionally recovering the antibody or antigen-binding fragment thereof or bispecific antibody from the host cell (or host cell culture medium).

[0516] 39. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-16 or the bispecific antibody of any one of embodiments 17-34.

[0517] 40. A pharmaceutical composition, medicament or formulation comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-16, the bispecific antibody of any one of embodiments 17-34, or the immunoconjugate of embodiment 39, and optionally a pharmaceutically acceptable excipient.

[0518] 41. A drug combination comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-16 or the bispecific antibody of any one of embodiments 17-34, or the immunoconjugate of embodiment 39, and one or more other therapeutic agents, preferably, the therapeutic agents are various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists).

[0519] 42. The drug combination of embodiment 41, comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-16 or the bispecific antibody of any one of embodiments 17-34, or the immunoconjugate of embodiment 39 and other antibodies, preferably, the other antibodies are antibodies that inhibit the PD-1 / PD-L1 signaling pathway, such as anti-PD-1 antibodies; or anti-PD-L1 antibodies or anti-PD-L2 antibodies; or

[0520] Comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-16, or the bispecific antibody of any one of embodiments 17-34, or the immunoconjugate of embodiment 39 and one or more chemotherapeutic agents, such as a platinum-based chemotherapeutic drug such as carboplatin; or a topoisomerase inhibitor, such as a topoisomerase II inhibitor, such as etoposide, or a combination thereof;

[0521] Preferably, the drug combination comprises the antibody or antigen-binding fragment thereof according to any one of embodiments 1-16, or the bispecific antibody according to any one of embodiments 17-34, or the immunoconjugate according to embodiment 39 and a combination of carboplatin and etoposide.

[0522] 43. A method for preventing or treating a tumor in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 1-16, or the bispecific antibody of any one of embodiments 17-34, or the immunoconjugate of embodiment 39, or the pharmaceutical composition or formulation of embodiment 40; or the pharmaceutical combination of embodiment 41 or 42.

[0523] 44. The method of embodiment 43, wherein the tumor is a solid tumor or a blood tumor, such as a DLL3-positive tumor or cancer, and optionally the tumor is a neuroendocrine tumor (such as a DLL3-positive neuroendocrine tumor), such as a lung cancer (such as a DLL3-positive lung cancer), such as small cell lung cancer (SCLC).

[0524] 45. The method described in embodiment 43 or 44, wherein the method further comprises administering in combination with other therapies such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents, preferably, the therapeutic agents are various therapeutic agents used to treat tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0525] 46. ​​The method of embodiment 45, wherein the method further comprises administering in combination with other antibodies, preferably, the other antibodies are antibodies that inhibit the PD-1 / PD-L1 signaling pathway, such as anti-PD-1 antibodies; or anti-PD-L1 antibodies or anti-PD-L2 antibodies; or

[0526] The method further comprises administering in combination with one or more chemotherapeutic agents, wherein the chemotherapeutic agent is selected from a platinum chemotherapy drug such as carboplatin; or a topoisomerase inhibitor, such as a topoisomerase II inhibitor, such as etoposide, or a combination thereof; or

[0527] The method also includes administration in combination with carboplatin and etoposide. Example

[0528] Example 1. Generation and screening of DLL3 monoclonal antibodies

[0529] immunity

[0530] The human DLL3 extracellular segment protein (Kaixia, DLL-HM103) was emulsified with Freund's complete adjuvant (Sigma, F5881) and then immunized with RenMab mice (purchased from Biocytogen). Two weeks later, it was emulsified with Freund's incomplete adjuvant (Sigma, F5506) and immunized three times, with subcutaneous injection once every two weeks (50 μg protein per mouse).

[0531] Single B cell fluorescence staining and sorting

[0532] When the serum titer meets the requirements, the spleen of the mouse is removed to obtain suspended B cells. Next, the cell density is adjusted to 1*10 8 cells / vial, with a volume of 0.25-2 mL, add 50 μL / mL Rat Serum and 50 μL / mL Isolation Cocktails, let it stand at room temperature for 5 minutes, then add 50 μL / mL RapidSpheres, let it stand at room temperature for 2.5 minutes, add EasySep Buffer to 2.5 mL, place the flow tube on a magnetic stand at room temperature for 2.5 minutes, and collect the cell suspension. Wash the cells with 10 mL EasySep Buffer and adjust the cell density to 1*10 8 cells / mL, add PE-Cy 7Rat Anti-Mouse CD45R / B220 (BD, 552772) and FITC (Invitrogen, A20181) to label hu-DLL3, incubate for 1 hour in the dark, wash once with 10mL pre-cooled EasySep Buffer, and adjust the cell density to 1*10 8 cells / vial, added APC-Cyanine 7 anti-mouse IgM Antibody (Biolegend, 406516), Brilliant Violet 510 anti-mouse IgD Antibody (Biolegend, 405723), R-Phycoerythrin AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG (Jackson Immunoresearch Inc, 115-116-071), incubated in the dark for 20 min; washed once with 10 mL pre-cooled EasySep Buffer, and adjusted the cell density to 10 based on the cell number. 7 cells / mL. Single B cells were sorted using an Aria III flow cytometer. The gate logic for flow cytometry sorting was: PE-Cy 7 pos / FITC pos / APC pos / APC-Cyanine 7 neg / Brilliant Violet 510 neg / R-Phycoerythrin pos , cells were sorted into 96-well PCR plates containing lysis buffer.

[0533] Antibody variable region amplification, vector construction, expression and detection

[0534] Amplification products of the antibody variable regions were obtained through a single round of RT-PCR and nested PCR. The paired amplification products were sequenced and analyzed and aligned using MEGA7 software. Simultaneously, the paired amplification products were homologously recombined into a pcDNA3.1 vector digested with BsmBI (NEB, R0739L), with the heavy chain constant region selected to be IgG1 wild-type (SEQ ID NO: 23) and the light chain constant region selected to be kappa (SEQ ID NO: 25). Five microliters of the homologous recombinant product was transformed into 30 microliters of DH5a competent culture medium. The resulting bacterial suspension was then inoculated into 2 mL of LB medium containing Amp-resistant lysine and cultured overnight at 37°C in a shaker at 220 rpm. Plasmids were extracted from the overnight culture using the 96Plus Miniprep Kit (QIAGENE, 27291), and the plasmid concentration was determined using a nanodrop. The light chain plasmid and heavy chain plasmid of the same antibody were mixed at a molar ratio of 1:1 and transfected into 293F cells using polyethyleneimine (PEI) (Polysciences, 23966). After culturing for 5-7 days, the supernatant was collected for subsequent detection.

[0535] Anti-DLL3 antibody supernatants were screened by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS). For the ELISA, 100 μL of human-DLL3 (1 μg / mL) was coated per well and blocked with 200 μL of 5% BSA. The primary antibodies were 50 μL / well of transient transfection supernatant and 5A4 (benchmark, homemade). Next, 100 μL of anti-human IgG Fc (Thermo, A24476) secondary antibody (1:10,000 dilution in antibody diluent) was added to each well. Coating, blocking, and primary and secondary antibody incubation were all performed at 37°C for 1 hour. Color development was performed with 100 μL / well of TMB (Solarbio, PR1200) in the dark for 3 minutes. Finally, 50 μL of ELISA stop buffer (Solarbio, C1058) was added to each well, and absorbance at OD 450 nm was measured using a microplate reader. The FACS experiment was performed by diluting the cells to be tested (huDLL3 / GS-CHO, constructed in-house) to 2×10 6Cells were plated at 400 cells / mL and 50 μL / well was added to a U-bottom 96-well plate. 50 μL / well of supernatant was added to the U-plate and the cells were resuspended. The cells were incubated at 4°C for 30 minutes. The supernatant was removed by centrifugation at 500 g for 5 minutes, and the cells were washed once with PBS. 100 μL of a PE-conjugated secondary antibody (1:500 dilution in PBS) against human Fab (BioLegend, 409304) was added to each well and incubated at 4°C in the dark for 30 minutes. The supernatant was removed by 500 g for 5 minutes, and the cells were washed once with PBS. The cells were resuspended in 50 μL of 1× PBS and analyzed by FACS. The supernatants of 89 positive clones were selected and purified using a Protein A affinity column to generate monoclonal antibodies. Binding of the purified antibodies to cells expressing different DLL3 truncations (created in-house) was determined by FACS to determine the binding region of the antibodies. Affinity analysis of these purified antibodies was also performed using Fortebio. Based on the results and sequence diversity, 19 matching sequences were identified for subsequent bispecific antibody construction. The amino acid sequence and DNA sequence of the obtained VH and VL of D3-107 are shown in the sequence listing.

[0536] Table 1: DLL3 clone initial screening results

[0537]

[0538] Table 2: DLL3 clone affinity results

[0539]

[0540] Example 2. DLL3 monoclonal antibody affinity experiment

[0541] The affinity (KD) of antibody D3-107 for binding to DLL3 was determined using biofilm thin-layer interferometry (BLI). Half an hour before the experiment, an appropriate number of AHC sensors (18-5060, Sartorius) were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20) based on the number of samples. The antibody and human DLL3 (DL3-H52H4, Acrobiosystems) were diluted to 100 nM, respectively.

[0542] SD buffer, antibody solution, and human DLL3 were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a Fortebio Octet Red96e plate, with the plate arranged according to the sample position and the sensor position selected.

[0543] The instrument was set up with the following parameters: run steps: baseline equilibration for 120 s, immobilized antibody loading for 100 s, baseline equilibration for 120 s, antigen binding for 100 s, and dissociation for 120 s, at a speed of 1000 rpm and a temperature of 30°C. After the experiment, KD values ​​were analyzed using ForteBio Octet analysis software. The results are shown in Table 3 below.

[0544] Table 3. Affinity testing of exemplary antibodies to human DLL3

[0545]

[0546] Example 3. In vitro binding assay of DLL3 monoclonal antibodies

[0547] CHO-hDLL3 (human DLL3 gene NCBI ID: 10683 was constructed into the pXC17.4 vector and then electroporated into CHO cells and positive single clones were sorted) was cultured and passaged according to routine procedures.

[0548] The cells were centrifuged and resuspended, and the cell density was adjusted to 4×10 6 Pour 50 μL of cells / mL into a sample reservoir and seed each well of a 96-well plate using a dispenser. Add 50 μL of serially diluted antibody sample (starting at 100 nM, 3-fold dilutions, 11 steps total) to each well of a 96-well plate. Incubate in a 4°C refrigerator for 30 minutes. Centrifuge at 400 g for 5 minutes, shake off the plate, and resuspend in 200 μL of PBS. Centrifuge again and resuspend twice. Add PE anti-human IgG Fc recombinant antibody (Biolegend, 409304, 1:200) and incubate in the dark at 4°C for 30 minutes. Wash twice with 200 μL of PBS, shake off the plate, and resuspend in 100 μL of PBS for flow cytometry reading. Fit the curve and calculate the EC50 value.

[0549] The results are as follows Figure 1 As shown, the candidate mAb and the positive control had comparable binding abilities on DLL3-overexpressing CHO cells.

[0550] Example 4. Verification of ADCP activity of DLL3 monoclonal antibody

[0551] The Fab end of the DLL3 example antibody binds to DLL3 on the surface of small cell lung cancer cells, while the Fc end binds to CD64 on the surface of FcγRI ADCPBioassay Effector Cells, activating downstream signaling pathways through DLL3-dependent CD64 cross-linking.

[0552] In this study, the luciferase reporter gene assay was used to detect FcγRI ADCP Bioassay Effector Cells. Under the conditions of co-culture with DLL3-positive cells, the expression of luciferase after overnight culture with the addition of the sample antibody was detected to reflect the strength of the antibody activation ability.

[0553] Adjust the cell density of CHO-hDLL3 and FcγRI ADCP Bioassay Effector Cells (Promega G9871) to 6.0×10 5 CHO-hDLL3-overexpressing cells and Effortor cells were mixed at a 1:1 ratio for later use. 100 μL of cell suspension and a serial dilution (starting at 30 nM, 3X dilution, 10 steps) of the test antibody (D3-107, DLL3BMK antibody (Rova-T), DLL3BMK (Amgen)) were added to each well and incubated overnight. The next day, 80 μL of Bio-Glo Luciferase Assay System (Promega, G7940) detection solution was added to each well. The assay was read on a microplate reader (Molecular Devices, SpectraMax I3).

[0554] In experiments performed as described in the above assays, the exemplary antibodies were able to dose-dependently activate CD64 downstream signaling (see Figure 2 ), and the activation intensity was slightly stronger than that of the control antibody.

[0555] Example 5. Expression and purification of DLL3 bispecific antibodies

[0556] The bispecific antibodies in the present invention are prepared by first expressing and purifying two parent proteins separately and then recombining them in vitro through reduction and oxidation. Figure 3 A, structure see Figure 3 B.

[0557] Plasmid construction: The sequence was synthesized by GENEWIZ and loaded into the pcDNA3.1 vector. The encoding gene sequence is shown in Table 4 below:

[0558]

[0559]

[0560] Expi293F cells (purchased from Gibco) were cultured with Expi293F medium (Gibco, REF#A14351-01). The cell density was checked one day before transfection (viability should be greater than 95%) and adjusted to 3 × 10 6The cell density was adjusted to 3 × 10 cells / ml on the day of transfection. 6 cells / ml.

[0561] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as the transfection buffer, add the DNA to be transfected at a ratio of 1 mg / L, where the light and heavy chain plasmids are in a 1:1 ratio, mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 minutes, and then gently pour the mixture into the Expi293F cell suspension while shaking. The cells are cultured in a shaker under the conditions of 8% CO2, 36.5°C, and 120 rpm.

[0562] After 16-18 hours of culture, the cell suspension was supplemented with 2% (volume ratio) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution with a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) with a final concentration of 2.2 mM. The suspension was gently mixed and cultured for 7 days at 8% CO₂, 36.5°C, and 120 rpm before sampling. The cell suspension was then mixed with diatomaceous earth (Sartorius, Cat# 1000037025) (40 g of diatomaceous earth per 1 L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0563] Affinity chromatography to purify the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing and column were washed with 10-20 column volumes of distilled water. The packed column was equilibrated with 5 column volumes of 1× PBS (Gibco); the filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins; the packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), the eluate was collected, the pH was adjusted to 6.0 with 2 M Tris, and the column was filtered and sterilized for further ion exchange chromatography.

[0564] In vitro reduction and oxidation: Mix the protein fractions from affinity chromatography at a 1:1 molar ratio, add an appropriate amount of GSH, and adjust the reaction pH to 8.0 with 2M Tris. Incubate at room temperature overnight. Exchange the reaction mixture into PBS and store at 4°C until needed.

[0565] Ion exchange chromatography purification of bispecific antibodies: A Mono S 5 / 50GL (GE Healthcare) ion exchange chromatography column was selected and placed in an AKTApure system (GE healthcare). The AKTApure system equipped with a Mono S 5 / 50GL ion exchange chromatography column was detoxified with 0.5 M NaOH for 2 hours, and then the system and column were washed with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (20 mM NaPO4, pH 6.6) until the conductivity and pH were stable; the protein obtained by affinity chromatography was diluted 10-fold with loading buffer and then loaded; the column was re-equilibrated with 5 column volumes of loading buffer; linear elution was performed with a gradient of 0-40% elution buffer (20 mM NaPO4, 1 M NaCl, pH 6.6) for a total of 30 column volumes, and samples were collected based on UV absorbance.

[0566] The purity of the collected samples in each fraction was tested by size exclusion chromatography (SEC), and the samples in the fractions with a purity greater than 95% were combined based on the SEC results.

[0567] The purified bispecific antibody solution was centrifuged at 4500 rpm for 30 minutes in a 15 ml ultrafiltration centrifuge tube. The protein was diluted with PBS and centrifuged again at 4500 rpm for 30 minutes. This process was repeated several times to replace the buffer. The antibodies after buffer exchange were combined and the antibody concentration was measured. The composition and content of the bispecific antibody DLL3 / CD3 were further qualitatively and quantitatively determined using capillary electrophoresis (CE-SDS) combined with liquid chromatography-mass spectrometry (LC-MS).

[0568] Similar expression and purification positive control antibody AMGEN757 (AMG757, INN Prop. List 123 tarlatamab, referred to herein as AMG757 or AMG757 analog) and BI's DLL3 / CD3 dual antibody BI-D3C1 (WO2019 / 234220A1; SEQ ID NO: 75 DLL3#3 chain; SEQ ID NO: 79 CD3#1 chain, referred to herein as BI-D3C1 or BI-D3C1 analog). Negative control IgG1 was from BioXCell BP0297.

[0569] The antibody Anti-mPD-1 (heavy chain is SEQ ID NO: 49, light chain is SEQ ID NO: 50) was similarly expressed and purified.

[0570] Example 6. DLL3 / CD3 dual antibody affinity

[0571] The affinity (KD) of the bispecific antibody binding to DLL3 was determined using biofilm thin-layer interferometry (BLI). Half an hour before the experiment, an appropriate number of AHC sensors (18-5060, Sartorius) and HIS1K sensors (18-5120, Sartorius) were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20), depending on the sample quantity. The bispecific antibody, human DLL3 (DL3-H52H4, Acro biosystems), and human CD3 (CDD-H52W1, Acrobiosystems) were diluted to 100 nM.

[0572] SD buffer, antibody solution, human DLL3, and human CD3 were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a ForteBio Octet Red96e, with the plate arranged according to the sample position and the sensor position selected. Instrument parameters were set as follows: Run steps: 120 s baseline equilibration, 100 s addition of immobilized antibody, 120 s baseline equilibration, 100 s antigen binding, and 120 s dissociation. The speed was 1000 rpm and the temperature was 30°C. After completion of the experiment, KD values ​​were analyzed using ForteBioOctet analysis software. The results are shown in the table below.

[0573] In experiments performed as described in the above assays, the affinities of DLL3 / CD3 and human DLL3-His are shown in Table 5.

[0574]

[0575] The affinity of DLL3 / CD3 and human CD3 is shown in Table 6.

[0576]

[0577] The obtained binding affinities are shown in Table 7.

[0578] Name Antigen Ka(1 / Ms) kd(1 / s) KD(M) D3-107 / CD3 Human DLL3 4.07E+05 2.00E-04 4.91E-10 D3-107 / CD3 human CD3 2.93E+05 2.90E-03 9.92E-09

[0579] Example 7. DLL3 / CD3 dual antibody cell binding experiment

[0580] The binding ability of DLL3 / CD3 bispecific antibody to DLL3-overexpressing cell lines was detected by flow cytometry.

[0581] CHO-hDLL3 cells were cultured and passaged according to routine procedures. Cells were centrifuged, resuspended, and counted, and the cell density was adjusted to 4×10 6 Pour 50 μL of cell / mL into a sample reservoir and seed each well of a 96-well plate using a dispenser. Add 50 μL of serially diluted antibody sample (starting at 300 nM, 3-fold dilution, 12 steps total) to each 50 μL cell well and incubate in a 4°C refrigerator for 30 minutes. Centrifuge at 400 g for 5 minutes, shake off the plate, and resuspend in 200 μL of PBS. Centrifuge again and resuspend twice. Add PE anti-human IgG Fc recombinant antibody (1:200) and incubate at 4°C in the dark for 30 minutes. Wash twice with 200 μL of PBS, shake off the plate, and resuspend in 100 μL of PBS for flow cytometry reading. Fit the curve and calculate the EC50 value.

[0582] In experiments performed as described in the above assays, the exemplary antibodies bound to DLL3 on the surface of cells overexpressing DLL3. Figure 5 .like Figure 5 It can be seen that the upper plateau of the binding of the example antibody to the DLL3-overexpressing cell line is slightly stronger than that of the control antibody.

[0583] PBMC (Oricells, Catalog No. FPB005-C) were revived according to routine procedures. After resuscitation, the cells were centrifuged, resuspended, and counted. The cell density was adjusted to 4×10 6 cells / ml, add Human TruStain FcX TM (Biolegend, 422302) and mix well, then block at 4°C for 10 minutes. After blocking, add FITC anti-human CD4 Antibody (Biolegend, 300506) and APC anti-human CD8a Antibody (Biolegend, 301014) and mix well. Pour the solution into a sample reservoir and plate 50 μl per well of a 96-well plate using a pipette. Add 50 μl of serially diluted antibody sample (starting at 300 nM, 3-fold dilutions, 12 steps total) to each well of the 50 μl cell suspension and incubate in a refrigerator at 4°C for 30 minutes. Centrifuge at 400g for 5 minutes and shake off the plate. Add 200 μl of PBS and centrifuge at 400g for 5 minutes and shake off the plate. Repeat three times. Centrifuge at 400g for 5 minutes and shake off the plate. Add 200 μl of PBS and resuspend the plate. Centrifuge again and resuspend the plate. Repeat two more times. Add PE anti-human IgG Fc recombinant antibody (1:200) and incubate at 4°C in the dark for 30 minutes. Wash twice with 200 μl of PBS, shake the plate, and resuspend in 100 μl of PBS. Read the plate on a flow cytometer. Fit the curve and calculate the EC50 value.

[0584] In experiments performed as described in the assay above, the exemplified antibodies were stained with human CD4 T cells ( Figure 6 B) and CD8 T cells ( Figure 6 A) Surface CD3 binding Figure 6 .like Figure 6 It can be seen that the example antibody and the control antibody have comparable abilities to bind to CD3 on the surface of T cells.

[0585] Example 8. In vitro activation of Jurkat-NFAT-Luc by DLL3 / CD3 double antibody

[0586] The DLL3 / CD3 dual antibody simultaneously binds to DLL3 on the surface of small cell lung cancer cells and CD3 on the surface of Jurkat-NFAT-Luc cells, activating the NFAT-Luc downstream signaling pathway through DLL3-dependent CD3 cross-linking. This study used a luciferase reporter gene assay to detect luciferase expression in Jurkat-NFAT-Luc cells co-cultured with DLL3-positive cells after overnight incubation with the example antibodies, reflecting the antibody's activation ability.

[0587] Resuspend SHP77, H82 (ATCC, HTB-175), DMS53 (Nanjing Kebai, CBP60172), H460 (ATCC, HTB-177, DLL3-negative cells) and Jurkat-NFAT-Luc cells in Assay medium and adjust the cell density to 6.0 × 10 5 Tumor cells and Jurkat-NFAT-Luc cells were mixed at a 1:1 ratio (cell / ml). 100 μL of cell suspension and a serial dilution of the test antibody (50 nM starting point, 3-fold dilution, 12 steps) were added to each well and incubated overnight. The next day, 80 μL of the Bio-Glo Luciferase Assay System (Promega, G7940) detection solution was added to each well. The results were read on a microplate reader (Molecular Devices, SpectraMax I3).

[0588] In experiments performed as described in the above assays, the exemplified antibodies were able to dose-dependently activate NFAT signaling in DLL3-positive cell (SHP77, H82, DMS53) assay systems (see Figure 7 A, 7B and Figure 7 C), the activation intensity is comparable to that of the control antibody. In SHP77, where DLL3 expression is the highest, the activation intensity is the strongest, while in H982 and DMS53, where expression is slightly lower, the activation intensity is slightly lower; in the DLL3-negative cell H460 experimental system, the example antibody does not show an activation effect (see Figure 7 D) Therefore, the activation intensity of NFAT signaling by the DLL3 / CD3 dual antibody is correlated with the expression level.

[0589] Example 9. In vitro DLL3 / CD3 double antibody activated T cells to kill small cell lung cancer

[0590] The DLL3 / CD3 dual antibody simultaneously binds to DLL3 on the surface of small cell lung cancer cells and CD3 on primary T cells. Through DLL3-dependent CD3 cross-linking, it activates T cells and mediates T cell cytotoxicity against DLL3-positive tumor cells. This study evaluated the cytotoxicity of human CD8+ T cells against DLL3-positive tumor cells by using Propidium Iodide (PI) staining to measure the ratio of PI-positive tumor cells after 24 hours of co-culture of human PBMCs with DLL3-positive cells and addition of the example antibody.

[0591] Remove PBMCs from liquid nitrogen and rapidly thaw at 37°C. Add dropwise to preheated 1640 medium (containing 0.1% DNase) with 10% FBS to yield 10 ml of the mixture. Centrifuge at 400 g for 5 minutes, resuspend in 10 ml of 1640 medium containing 10% FBS, add 10 μl of DNase, and allow to adhere overnight in a 37°C, 5% CO2 incubator.

[0592] DLL3-positive SHP77 cells and DLL3-negative H460 cells (negative control) were used as target cells and centrifuged at 400g for 5 minutes. The supernatant was removed and the cells were washed twice with 30ml of PBS and centrifuged. Cells were labeled with CellTrace Far Red Cell stain (THERMO FISHER, C34564) and incubated in a 37°C incubator in the dark for 20 minutes. After incubation, the cells were added with 1640 complete medium and centrifuged at 400g for 5 minutes. The supernatant was removed, the cells were resuspended in 1640 complete medium, and the cells were transferred to a CO2 incubator and cultured overnight.

[0593] Adjust the target cell density to 2x105 / ml and the PBMC density to 4x106 / ml. Add 50ul of serially diluted antibody (starting at 10nM, 3-fold dilution, 11 steps), 50ul of target cells, and 50ul of PBMCs, mix thoroughly, and incubate in a CO2 incubator. After 24 hours of killing, remove the 96-well plate, add 100ul of PI detection solution to each well, mix thoroughly with the cells, and analyze by upflow cytometry.

[0594] In experiments performed as described in the above assays, the exemplified antibodies were able to dose-dependently induce killing of SHP77 target cells by human CD8+ T cells (see Figure 8 A), the example antibody showed no killing effect on non-target cell H460 (see Figure 8 B).

[0595] Example 10. DLL3 / CD3 dual antibody PTM removal

[0596] The D3-107 / CD3 bispecific antibody molecule contains a DG hotspot in the heavy chain variable region (HCDR2) of the DLL3 arm (D3-107) and an NFS glycosylation site in the light chain framework region (FR3). To reduce the difficulty of CMC analysis and potential drugability risks, we designed corresponding mutations for the above hotspots (Table 8). The binding activity and expression yield of the mutated molecules were verified based on the methods of Examples 2 and 3 (antibody dilution starting concentration of 300nM, 3-fold dilution, a total of 12 gradients), demonstrating that the mutated molecules can maintain good DLL3 binding activity and expression yield (Tables 8 and 10; Figure 9 ).

[0597] Based on the expression levels and cell-level binding ability results, we selected heavy chains D3-107-VH-P1 and D3-107-VH-P2 and combined them with light chain D3-107-VL-P2 to obtain D3-107-C1 and D3-107-C2, respectively. We again verified the binding activity of the obtained new mAb sequences by flow cytometry (see Example 2). The PTM-removed molecules all had better binding activity than the parent molecules (Tables 9, 10, and Figure 10 ).

[0598] Next, D3-107-C1 and D3-107-C2 were spliced ​​with CD3 arms to obtain D3-107 / CD3-C1 and D3-107 / CD3-C2 bispecific antibodies. We tested the cell-level binding ability of these two bispecific antibodies (see Examples 3 and 7 for methods and results). Figure 11 ), reporter activation (method see Example 8, results see Figure 12 ) and in vitro killing function (method see Example 9, results see Figure 13 and 14 ), we found that the in vitro activities of these two candidate molecules were comparable and consistent with the parental molecules, and we chose D3-107 / CD3-C1.

[0599] Table 8: DLL3 candidate molecule D3-107PTM removal mutation

[0600]

[0601] The above light chain variable region and heavy chain variable region were combined to prepare corresponding monoclonal antibodies as described in Example 1. The obtained antibodies are shown in Table 9 below.

[0602] Table 9: Names and variable regions of monoclonal antibodies

[0603]

[0604] Table 10: DLL3 binding activity and expression level after removal of a single hotspot in DLL3-S4-107

[0605]

[0606]

[0607] The specific sequence of the obtained D3-107 / CD3-C1 bispecific antibody is as follows:

[0608] DLL3 arm that specifically binds to DLL3 Anti-DLL3 Antibody D3-107VH (D3-107-P1-VH) SEQ ID NO:13 Anti-DLL3 Antibody D3-107VL (D3-107-P2-VL) SEQ ID NO: 18 Fc region (LALA+S364R / D399K) SEQ ID NO:29 Light chain constant region SEQ ID NO:25 Heavy chain 1 SEQ ID NO:42 Light chain 1 SEQ ID NO:47 CD3 arm that specifically binds to CD3 Anti-CD3 antibody VH SEQ ID NO:31 Anti-CD3 antibody VL SEQ ID NO:35 Fc region (LALA+K370S, K409D, Y349T) SEQ ID NO:30 Light chain constant region SEQ ID NO:24 Heavy chain 2 SEQ ID NO:48 Light chain 2 SEQ ID NO:40

[0609] The specific sequence of the obtained D3-107 / CD3-C2 bispecific antibody is as follows:

[0610] DLL3 arm that specifically binds to DLL3 Anti-DLL3 Antibody D3-107VH (D3-107-P2-VH) SEQ ID NO:14 Anti-DLL3 Antibody D3-107VL (D3-107-P2-VL) SEQ ID NO: 18 Fc region (LALA+S364R / D399K) SEQ ID NO:29 Light chain constant region SEQ ID NO:25 Heavy chain 1 SEQ ID NO:43 Light chain 1 SEQ ID NO:47 CD3 arm that specifically binds to CD3 Anti-CD3 antibody VH SEQ ID NO:31 Anti-CD3 antibody VL SEQ ID NO:35 Fc region (LALA+K370S, K409D, Y349T) SEQ ID NO:30 Light chain constant region SEQ ID NO:24 Heavy chain 2 SEQ ID NO:48 Light chain 2 SEQ ID NO:40

[0611] Example 11. PTM-removed DLL3 / CD3 bispecific antibody affinity

[0612] The affinity (KD) of the bispecific antibody binding to DLL3 was determined using biofilm thin-layer interferometry (BLI). Half an hour before the experiment, an appropriate number of AHC sensors (18-5060, Sartorius) were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20) based on the number of samples. The bispecific antibody and human DLL3 (DL3-H52H4, Acro biosystems) were diluted to 100 nM.

[0613] SD buffer, antibody solution, and human DLL3 were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a ForteBio Octet Red96e, with the plate arranged and sensor positions selected according to sample placement. Instrument parameters were set as follows: Run steps: 120 s baseline equilibration, 100 s addition of immobilized antibody, 120 s baseline equilibration, 100 s antigen binding, and 120 s dissociation. The speed was 1000 rpm and the temperature was 30°C. After the experiment, KD values ​​were analyzed using ForteBioOctet analysis software. The results are shown in Table 11 and Figure 15.

[0614]

[0615] Example 12. Validation of Binding of PTM-Removed DLL3 / CD3 Dual Antibody to DLL1 and DLL4

[0616] In vitro affinity determination of example antibodies for DLL1 and DLL4

[0617] The affinity (KD) of the bispecific antibody binding to DLL3 was determined using biofilm thin-layer interferometry (BLI). Half an hour before the experiment, an appropriate number of AHC sensors (18-5120, Sartorius) were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20) based on the number of samples. The bispecific antibody and human DLL1 (DL1-H52H8, Acro biosystems) and human DLL4 (DL4-H5227, Acro biosystems) were diluted to 100 nM.

[0618] SD buffer, antibody solution, human DLL1, and human DLL4 were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a ForteBio Octet Red96e, with the plate arranged according to the sample position and the sensor position selected. Instrument parameters were set as follows: Run steps: 120 s baseline equilibration, 100 s addition of immobilized antibody, 120 s baseline equilibration, 100 s antigen binding, and 120 s dissociation. The speed was 1000 rpm and the temperature was 30°C. After the experiment, KD values ​​were analyzed using ForteBioOctet analysis software. The results are shown in Table 12 below.

[0619] Table 12: Example antibodies binding to DLL1 and DLL4 cells in vitro

[0620]

[0621] The binding ability of DLL3 / CD3 bispecific antibody to DLL1 and DLL4 overexpressing cell lines was detected by flow cytometry.

[0622] CHO-hDLL1 (human DLL1 gene NCBI ID: 28514 was constructed into the pXC17.4 vector, electroporated into CHO cells, and positive cell populations were sorted) and 293T-hDLL4 (human DLL4 gene NCBI ID: 54567 was constructed into the pLenti-C-MYC-DDK-IRES-puro vector, packaged into 293T cells, infected with 293T cells, and positive cell populations were sorted)

[0623] The cells were cultured and passaged according to routine procedures. The cells were centrifuged, resuspended, and counted, and the cell density was adjusted to 4×10 6Pour 50 μL of cells / mL into a sample reservoir and seed each well of a 96-well plate using a dispenser. Add 50 μL of serially diluted antibody sample (starting at 100 nM, with 10 3-fold dilutions). Positive controls include commercially available DLL1 antibodies (PE anti-human Delta-like protein 1 (DLL1); Biolegend; 346404) and DLL4 antibodies (PE anti-human Delta-like protein 4 (DLL4) Antibody; Biolegend; 346506). Incubate at 4°C for 30 minutes. Centrifuge at 400 g for 5 minutes, shake off, and resuspend in 200 μL of PBS. Centrifuge again and resuspend twice. Add PE anti-human IgG Fc recombinant antibody (1:200) and incubate at 4°C in the dark for 30 minutes. Wash twice with 200 μL of PBS, shake off, and resuspend in 100 μL of PBS for flow cytometry reading. Fit the curve and calculate the EC50 value.

[0624] In experiments performed as described in the above assays, binding of the exemplary antibodies to CHO-hDLL1 and 293T-hDLL4 cells is shown in Table 1. Figure 16 The example antibodies did not bind to cell lines overexpressing DLL3 homologous proteins (DLL1 / DLL4).

[0625] Example 13. Binding experiment of PTM-removed DLL3 / CD3 dual antibody against small cell lung cancer and T cells

[0626] Flow cytometry was used to detect the binding ability of the DLL3 / CD3 bispecific antibody to DLL3-overexpressing cell lines and T cells after PTM removal. For detailed experimental details, see Example 7.

[0627] See the results Figure 17 ,from Figure 17 It can be seen that the binding ability of the DLL3 / CD3 bispecific antibody after PTM removal is comparable to that of the positive control AMG757analog, and is better than that of the BI-D3C1analog.

[0628] Example 14. In vitro activation of NFAT-reporter cells by PTM-removed DLL3 / CD3 diabodies

[0629] The activation effect of the example antibodies on NFAT reporter cells in in vitro experiments was described in Example 8, except that IgG1 was used as a negative control and BI-D3C1 analog was used as a positive control.

[0630] The experimental results are shown in Figure 18 ,from Figure 18 As can be seen, the D3-107 / CD3-C1 antibody exhibited significant dose-dependent stimulatory activity on DLL3-positive cells, with activation levels similar to those of BI-positive drugs. However, on the DLL3-negative cells of interest, the antibody showed no stimulatory effect, suggesting a good safety profile.

[0631] Example 15. PTM-removed DLL3 / CD3 diabody stimulates T cells to kill tumor cells in vitro

[0632] We evaluated the cytotoxicity of the example antibodies against tumor cells in vitro using a co-culture system of primary PBMC and small cell lung cancer. The specific experimental method is similar to Example 9. The antibody to be tested was diluted 3-fold starting at 10 nM, with 10 steps.

[0633] Specific results such as Figure 19 The example antibody D3-107 / CD3-C1 was shown to effectively kill DLL3-positive tumor cells (SHP-77 (ATCC CRL-2195), DMS-79 (ATCC CRL-2049), and H82 (ATCC HTB-175)), with a clear dose-response. Furthermore, no nonspecific killing was observed against DLL3-negative tumor cells (293T-hDLL4 (constructed as described above) and U87 (ATCC HTB-14)), even at a dose as high as 900 nM, demonstrating the safety and reliability of the example antibody.

[0634] Example 16. Cytokine release assay of DLL3-negative cells by PTM-removed DLL3 / CD3 dual antibody

[0635] To further assess the safety of the example antibodies, we monitored cytokine production by immune cells in PBMCs without DLL3 cross-linking. The specific experimental method is similar to Example 9. However, only PBMCs and antibody diluent were added, without target cells. The antibody was initially diluted at a concentration of 10 nM, with a 3-fold dilution gradient for a total of 12 steps. After treatment, the supernatant was removed and cytokine levels were measured using the Human Th1 / Th2 / Th17 CBA Kit and analyzed using FCAP.

[0636] Specific results such as Figure 20 The results showed that the example antibody D3-107 / CD3-C1 had almost no stimulatory effect on T cells or macrophages in the absence of DLL3 cross-linking, and the detected cytokines (TNFa, IL-6, etc.) were basically the same as the baseline, indicating that this example antibody has a high safety profile.

[0637] Example 17. Study on the anti-tumor effect of PTM-removed DLL3 / CD3 diabody in vivo

[0638] This study used the PBMC model of NOG mice inoculated with human small cell lung cancer cell lines DMS79 and SHP77 cells to determine the anti-tumor effect of the exemplary antibodies.

[0639] 17.1 Antitumor Efficacy of Example Antibodies in a DMS-79 Tumor-Bearing Humanized Mouse Model

[0640] Experimental procedures

[0641] Female NOG mice (16-19 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. They were SPF grade. The mice were acclimated and quarantined for 3 days upon arrival before the study began.

[0642] PBMC cells were injected intravenously into mice, 4x10 6 DMS-79 cells were inoculated on the third day after PBMC injection. DMS-79 cells were routinely subcultured, collected by centrifugation, and dispersed with PBS. DMS-79 cells were inoculated on the right dorsal abdomen of NOG mice after shaving. 6 Each cell was inoculated with 200ul of inoculation volume.

[0643] Dosing: On day 7 after DMS-79 cell inoculation, mice were divided into groups (6 per group) based on tumor volume and PBMC proliferation. The initial tumor size of the control group was ~129 mm3.

[0644] The drug was administered once every 7 days for a total of 3 times. The dosages were 0.1 mpk and 0.01 mpk, respectively, and the administration groups were as follows:

[0645] hIgG: human IgG control (Equitech-Bio SLH56-0001);

[0646] D3-107 / CD3-C1: Example antibody D3-107 / CD3-C1

[0647] BI-D3C1analog: positive control BIDLL3 / CD3.

[0648] The tumor volume and body weight of the mice were monitored twice a week.

[0649] Tumor volume measurement: Vernier calipers were used to measure the maximum long axis (L) and maximum wide axis (W) of the tumor. Tumor volume was calculated using the following formula: V = L × W² / 2. Body weight was determined using an electronic balance. Throughout the study, mice were euthanized when tumors reached endpoint or when they experienced >20% weight loss. Tumor size was counted and tumor inhibition rate (TGI%) was calculated.

[0650] The relative tumor inhibition rate (TGI%) was calculated on the 24th day after inoculation using the following formula:

[0651] TGI%=100%×(tumor volume of hIgG control group−tumor volume of treatment group) / (tumor volume of hIgG control group−initial tumor volume of hIgG control group).

[0652] Experimental results

[0653] Tumor growth curves are shown in Figure 21 The example antibody can significantly inhibit the growth of DMS79 cells. The tumor size was counted on the 24th day, and the tumor inhibition rate and the proportion of complete tumor regression were calculated.

[0654] Compared to hIgG, the tumor inhibition rates of the exemplary antibody D3-107 / CD3-C1 and the BI positive control BIDLL3 / CD3 at a high dose of 0.1 mg / kg were 115% and 103%, respectively. Furthermore, complete tumor regression was observed in 5 / 6 mice treated with the exemplary antibody D3-107 / CD3-C1 and 2 / 6 mice treated with the BI positive control BIDLL3 / CD3, respectively. Therefore, the exemplary antibody D3-107 / CD3-C1 exhibited superior anti-tumor efficacy compared to the positive control BIDLL3 / CD3, with no significant weight loss observed in either group.

[0655] At a low dose of 0.01 mg / kg, the tumor inhibition rates of the exemplary antibody D3-107 / CD3-C1 and the BI positive control BIDLL3 / CD3 were 75% and 53%, respectively. The exemplary antibody D3-107 / CD3-C1 also exhibited a better anti-tumor effect.

[0656] DMS-79 model data

[0657] In the low-dose group, after the experiment, tumor tissue was taken, placed in formalin, and finally made into wax blocks for IHC staining of CD3 to evaluate the CD3 status in the tumor after administration.

[0658] Figure 22 A, B, and C in the figure correspond to the groups IgG1 0.1mpk, BI-D3C1analog 0.01mpk, and D3-107 / CD3-C1 0.01mpk, respectively. As can be seen from the figure, the D3-107 / CD3-C1 0.01mpk group can be observed to have obvious CD3+ cell infiltration, and the infiltration is higher than that of the hIgG group.

[0659] The number of CD3+ cells per unit area was counted using HALO software (Indica Labs). Figure 23The results showed that the D3-107 / CD3-C1 group had more positive cell infiltration in the tumor compared to the BI-D3C1analog group, consistent with a stronger tumor inhibitory effect.

[0660] 17.2 Antitumor Efficacy of Example Antibodies in SHP-77 Tumor-Bearing Humanized Mouse Model

[0661] Experimental procedures

[0662] Female NOG mice (16-19 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The grade was SPF, and the mice were acclimated and quarantined for 3 days after arrival before the study began.

[0663] PBMC cells were revived with RPMI-1640 medium preheated with 0.1% DNase, and then dispersed with PBS to prepare a cell concentration of 20*10 6 PBMC cell suspension was injected intravenously into mice, 0.2 mL / mouse, i.e. the inoculation volume was 4*10 6 cells / mouse.

[0664] SHP-77 cells were routinely subcultured for subsequent in vivo experiments. Six days after PBMC cell inoculation, SHP-77 cells were dispersed in PBS and Matrigel at a ratio of 1:1 to prepare a cell concentration of 25*10 6 NOG mice were shaved on the right side of their backs and injected subcutaneously with SHP-77 cell suspension at 0.2 mL / mouse, i.e., the inoculation volume was 5*10 6 cells / mouse.

[0665] Four days after tumor cell inoculation, mice were divided into groups (6 per group) based on tumor volume and administered once every seven days for three consecutive doses. Administration was by intraperitoneal injection. Doses were 0.6 mpk and 0.06 mpk, respectively, and the dosing groups were as follows:

[0666] h-IgG: human IgG control (Equitech-Bio SLH56-0001);

[0667] D3-107 / CD3-C1: Example antibody D3-107 / CD3-C1

[0668] BI-D3C1analog: positive control BIDLL3 / CD3.

[0669] The tumor volume and body weight of the mice were monitored twice a week for 24 days.

[0670] The relative tumor inhibition rate (TGI%) was calculated on the 24th day after inoculation using the following formula:

[0671] TGI%=100%×(tumor volume of hIgG control group−tumor volume of treatment group) / (tumor volume of hIgG control group−tumor volume of hIgG control group before administration).

[0672] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = L × W2 / 2. Body weight was measured using an electronic balance.

[0673] Experimental results

[0674] Tumor growth curves are shown in Figure 24 The example antibody D3-107 / CD3-C1 significantly inhibited SHP77 cell growth. Tumor size was measured on day 24, and the tumor inhibition rate was calculated. At a high dose of 0.6 mg / kg, the tumor inhibition rates of antibody D3-107 / CD3-C1 and the BI positive control BIDLL3 / CD3 were 108% and 111%, respectively. At a low dose of 0.06 mg / kg, the tumor inhibition rates of the example antibody D3-107 / CD3-C1 and the BI positive control BIDLL3 / CD3 were 75% and 34%, respectively. Furthermore, no significant weight loss was observed in the mouse groups receiving either antibody D3-107 / CD3-C1 or the BI positive control BIDLL3 / CD3.

[0675] 17.3 Antitumor Efficacy of Example Antibodies in SHP-77 Tumor-Bearing Humanized Mouse Model

[0676] Experimental procedures

[0677] Female NOG mice (16-19 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The grade was SPF, and the mice were acclimated and quarantined for 3 days after arrival before the study began.

[0678] PBMC cells were revived with RPMI-1640 medium preheated with 0.1% DNase, and then dispersed with PBS to prepare a cell concentration of 20*10 6 PBMC cell suspension was injected intravenously into mice, 0.2 mL / mouse, i.e. the inoculation volume was 4*10 6 cells / mouse.

[0679] SHP-77 cells were routinely subcultured for subsequent in vivo experiments. Four days after PBMC inoculation, SHP-77 cells were dispersed in a 1:1 ratio of PBS to Matrigel to prepare a cell suspension at a concentration of 25 x 106 cells / mL. NOG mice were shaved on the right side of their backs and subcutaneously injected with 0.2 mL of the SHP-77 cell suspension per mouse, yielding an inoculum of 5 x 106 cells per mouse.

[0680] Four days after tumor cell inoculation, mice were divided into groups (6 mice per group) according to tumor volume and given the drug once every 7 days for 3 consecutive times. The administration method was intraperitoneal injection. The dosage is shown in Figure 25 Legend (to ensure the same molar dosage). Dosing groups are as follows:

[0681] h-IgG: human IgG control (Equitech-Bio SLH56-0001);

[0682] D3-107 / CD3-C1: Example antibody D3-107 / CD3-C1

[0683] AMG757analog: positive control AMG757.

[0684] The tumor volume and body weight of the mice were monitored twice a week for up to day 25. The doses were matched according to equimolar ratios.

[0685] The relative tumor inhibition rate (TGI%) was calculated on the 21st day after inoculation using the following formula:

[0686] TGI% = 100% × (hIgG control group tumor volume – treatment group tumor volume) / (hIgG control group tumor volume – hIgG control group pre-dose tumor volume). Tumor volume measurement: Vernier calipers were used to measure the maximum longitudinal axis (L) and maximum width axis (W). Tumor volume was calculated using the following formula: V = L × W² / 2. Body weight was measured using an electronic balance.

[0687] Experimental results

[0688] Tumor growth curves are shown in Figure 25 , the example antibody D3-107 / CD3-C1 can significantly inhibit the growth of SHP77. The tumor size was counted on the 20th day, and the tumor inhibition rate was calculated.

[0689] The high-dose D3-107 / CD3-C1 antibody and the Amgen positive control AMG757analog achieved tumor inhibition rates of 112% and 110%, respectively. At the low dose, the D3-107 / CD3-C1 antibody and the Amgen positive control AMG757analog achieved tumor inhibition rates of 75% and 40%, respectively. Furthermore, no significant weight loss was observed in the mice treated with either D3-107 / CD3-C1 or the Amgen positive control AMG757analog.

[0690] 17.4 Antitumor Effect of Combination of Example Antibodies and Standard Chemotherapy in H82 Tumor-Bearing Humanized Mouse Model

[0691] Experimental procedures

[0692] Female NOG mice (16-19 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The grade was SPF, and the mice were acclimated and quarantined for 3 days after arrival before the study began.

[0693] PBMC cells were revived with RPMI-1640 medium preheated with 0.1% DNase, and then dispersed with PBS to prepare a cell concentration of 20*10 6 PBMC cell suspension was injected intravenously into mice, 0.2 mL / mouse, i.e. the inoculation volume was 4*10 6 cells / mouse.

[0694] H82 cells were routinely subcultured for subsequent in vivo experiments. Six days after PBMC cell inoculation, H82 cells were dispersed in PBS and Matrigel at a ratio of 1:1 to prepare a cell concentration of 12.5*10 6 NOG mice were shaved on the right side of their backs and injected subcutaneously with H82 cell suspension at 0.2 mL / mouse, i.e., the inoculation volume was 2.5*10 6 cells / mouse.

[0695] Five days after tumor cell inoculation, mice were divided into groups (6 per group) according to tumor volume and given medication. The sample antibody was given twice a week for 7 doses; the standard chemotherapy drug was given once a week for 4 doses. The administration method was intraperitoneal injection with a volume of 10 ml / kg. The dosage is shown in Figure 26 Legend. The drug administration groups are as follows:

[0696] h-IgG: IgG control (Equitech-Bio SLH56-0001)

[0697] D3-107 / CD3-C1: Example antibody D3-107 / CD3-C1

[0698] Chemotherapy(Chemo):carboplatin+etoposide

[0699] Combination therapy:carboplatin+etoposide+D3-107 / CD3-C1

[0700] The tumor volume and body weight of the mice were monitored twice a week until 31 days.

[0701] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0702] Experimental results

[0703] Tumor growth curves are shown in Figure 26 The relative tumor inhibition rate (TGI%) on day 29 after inoculation was calculated using the following formula: TGI% = 100% × (tumor volume of the hIgG control group – tumor volume of the treatment group) / (tumor volume of the hIgG control group – tumor volume of the hIgG control group before administration).

[0704] The tumor inhibition rates of the antibody D3-107 / CD3-C1 and the standard chemotherapy drug were 23.0% and 65.9%, respectively. The combined therapy achieved an inhibition rate of 83.5%, demonstrating a stronger tumor suppression effect. Furthermore, no significant weight loss was observed during the experiment.

[0705] 17.5 Anti-tumor effect of combined use of an example antibody and an immune checkpoint inhibitor in the MC38-hDLL3 tumor-bearing hCD3KI mouse model

[0706] Experimental procedures

[0707] Female hCD3EDG mice (6-8 weeks) were purchased from Shanghai Model Organisms Science Co., Ltd. The grade was SPF, and the mice were acclimated and quarantined for 3 days after arrival before the study began.

[0708] MC38-hDLL3 cells were constructed in-house: The human DLL3 gene (NCBI ID: 10683) was constructed into the pLenti-C-MYC-DDK-IRES-puro vector, which was then packaged into 293T cells for infection with MC38 cells (Shanghai Heyuan Biotechnology, HYC0116). Positive cell populations were isolated. MC38-hDLL3 cells were routinely subcultured for subsequent in vivo experiments. MC38-hDLL3 cells were prepared at a concentration of 15 × 10 6The right side of the mouse back was shaved and the MC38-hDLL3 cell suspension was injected subcutaneously at 0.2 mL / mouse, i.e., the inoculation volume was 3*10 6 cells / mouse.

[0709] Eight days after tumor cell inoculation, mice were divided into groups (n=6 per group) based on tumor volume and administered the following: the sample antibody was administered twice weekly for five doses; the immune checkpoint inhibitor was administered once. Administration was by intraperitoneal injection at a volume of 10 ml / kg.

[0710] Dosage see Figure 27 Legend. The drug administration groups are as follows:

[0711] h-IgG: IgG control (Equitech-Bio SLH56-0001), 5 mg / kg

[0712] D3-107 / CD3-C1: Example antibody D3-107 / CD3-C1, 3 mg / kg

[0713] Anti-mPD-1: mouse PD-1 inhibitor (prepared as described in Example 1), 2 mg / kg

[0714] Combination therapy:D3-107 / CD3-C1,3mg / kg+Anti-mPD-1,2mg / kg

[0715] The tumor volume and body weight of the mice were monitored twice a week until the 29th day.

[0716] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0717] Experimental results

[0718] Tumor growth curves are shown in Figure 27 The relative tumor inhibition rate (TGI%) was calculated on the 29th day after inoculation using the following formula: TGI% = 100% × (tumor volume of the hIgG control group – tumor volume of the treatment group) / (tumor volume of the hIgG control group – tumor volume of the hIgG control group before administration).

[0719] The tumor inhibition rates of the antibody D3-107 / CD3-C1 and the mouse PD-1 inhibitor were 21.1% and 64.5%, respectively. The combined therapy achieved a tumor inhibition rate of 96.9%, demonstrating a stronger tumor suppression effect. Furthermore, no significant weight loss was observed during the experiment.

[0720] Example 18. In vivo PK study of PTM-removed DLL3 / CD3 dual antibody in mice

[0721] The in vivo stability of D3-107 / CD3-C1 was investigated in a mouse PK study. Nine BALB / c mice (Beijing Weitong Lihua) were injected with 10 mpk of D3-107 / CD3-C1 via the tail vein. Serum samples were collected for ELISA analysis at 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours, 168 hours, 336 hours, and 504 hours.

[0722] ELISA uses two methods for detection:

[0723] Method 1: Coat the plate with DLL3-his antigen (kactusbio, DLL-HM103), then add the serum to be tested, and then detect with anti-human Fc secondary antibody (BETHYL, A80-104P), and finally develop color with TMB.

[0724] Method 2: Coat the plate with DLL3-his antigen (kactusbio, DLL-HM103), then add the serum to be tested, then add biotinylated CD3 (Acrobiosystem, CDD-H82W6), then add the secondary antibody with streptavidin (BIOLEGEND, 405210), and finally develop the color with TMB. Figure 28 As shown, both arms of D3-107 / CD3-C1 were relatively stable in mice, with PK profiles similar to those of IgG, a half-life of 148-159 hours, and a clearance of 0.39-0.42 ml / kg / h. Detailed PK parameters are shown in Table 13.

[0725] Table 13: PK parameters of example bispecific antibodies

[0726]

[0727] Example 19. Study on the drugability of PTM-removed DLL3 / CD3

[0728] 1.1SMAC column detection of antibody colloidal stability

[0729] 1.1.1 Experimental steps

[0730] The colloidal stability of the antibody was tested by recording its retention time on a Zenix HPLC column. A 100 μl sample (D3-107 / CD3-C1) was centrifuged at 13,000 rpm for 5 minutes, and the supernatant was loaded onto the HPLC column. Chromatographic conditions: Zenix SEC-300 column; mobile phase: PBS; wavelength: 214 nm; column temperature: 25°C; flow rate: 0.35 ml / min; injection volume: 10 μl.

[0731] 1.2HIC detection of antibody hydrophobicity

[0732] 1.2.1 Experimental steps

[0733] The hydrophobicity of the antibody was determined by recording its retention time on the HIC-HPLC column. The sample (D3-107 / CD3-C1) was diluted to 1 mg / ml, and 10 μl was injected onto a MAbPac HIC-10 column. Chromatographic conditions were as follows: detection wavelength at 280 nm; column temperature at 25°C; flow rate at 1 ml / min. Mobile phase gradient: 100% A–100% B (0–20 min); 100% B (20–25 min); and 100% A (25–30 min). (A: 1.8 M (NH₄)₂SO₄, 100 mM NaH₂PO₄, pH 6.5; B: 100 mM NaH₂PO₄, 10% IPA, pH 6.5)

[0734] 1.3 Non-specificity of CIC detection antibodies

[0735] IgG from human serum was coated onto an NHS-activated column, and the retention time of the antibody on the column was examined by HPLC. Retention time showed a significant negative correlation with solubility, thus enabling the screening of antibodies with better solubility.

[0736] Centrifuge 100 μl of the sample (D3-107 / CD3-C1) diluted to 1 mg / ml at 13,000 rpm for 5 minutes. Apply to a chromatographic column pre-coupled with polyclonal human IgG and set up the detection method (wavelength: 280 nm; flow rate: 0.35 ml / min; injection volume: 10 μl). Run the experiment at room temperature.

[0737] 1.4 DLS detection of thermal stability of antibodies

[0738] Dynamic light scattering (DLS) uses laser to illuminate small particles doing Brownian motion in a solution and detects changes in the intensity of the scattered light. DLS can be used to detect the particle size of proteins in solution, as well as their stability at different temperatures or concentrations. Centrifuge at 13000G / min for 5 minutes and add it to the sample plate, then use a plate centrifuge to centrifuge for 1 minute to remove bubbles. After selecting the continuous temperature rise experiment in the detection Tagg, select the reading well position, and set the DLS acquisition time to 5s, the number of acquisitions to 5 times, and the experimental temperature to 25-85°C. After the experiment, analyze the changes in sample particle size with temperature.

[0739] 1.5DSC detection of the thermal stability of antibodies

[0740] Differential Scanning Calorimetry (DSC) is a thermal analysis method that measures the energy difference between a sample and a reference substance as it changes with temperature under programmed temperature conditions. As the temperature rises, the protein structure changes, and the accompanying heat changes are recorded by the differential scanning calorimeter as a DSC curve. The Tm value of the protein is determined by analyzing the DSC curve.

[0741] Dilute the samples (D3-107 / CD3-C1) to 0.5-1 mg / ml in PBS. After degassing the diluted samples and PBS buffer, add the samples to the left sample plate and add PBS buffer to the corresponding positions on the right reference plate. Set the starting temperature to 30°C and equilibrate for 10 minutes. Heat at a rate of 1°C / min to a final temperature of 90°C.

[0742] The titer of the bispecific anti-DLL3 half molecule 107-C1-innoA.LALA (heavy chain SEQ ID NO: 42; light chain: SEQ ID NO: 47) transiently transfected in CHO cells was 109 mg / L, and the titer of the anti-CD3 half molecule hzsp34.24-innoB.LALA (heavy chain SEQ ID NO: 48; light chain: SEQ ID NO: 40) was 244 mg / L.

[0743] The obtained drugability parameters are shown in Table 14. The recovery rate of the bispecific antibody molecule D3-107 / CD3-C1 after assembly was 69%, and the purity after CEX purification was 97% as determined by SEC. Early drugability HPLC chromatographic retention time detection showed colloidal stability (SMAC 8.3 min), nonspecific binding (CIC 9.8 min), and hydrophobicity (HIC 13 min), indicating a low risk of drugability indicators.

[0744] Table 14: Druggability parameters of example bispecific antibodies

[0745]

[0746] Sequence Summary

[0747]

[0748]

[0749]

[0750]

[0751]

Claims

1. An anti-DLL3 antibody or antigen-binding fragment thereof, comprising three CDRs of the heavy chain variable region VH, HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region VL, LCDR1, LCDR2, and LCDR3, wherein: The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from (i) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 13, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 18; (ii) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16; (iii) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 13, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16; (iv) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16; (v) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 15, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 16; (vi) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 17; (vii) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 18; or (viii) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:

18.

2. An anti-DLL3 antibody or antigen-binding fragment thereof comprising a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1 consists of the amino acid sequence of SEQ ID NO: 1; the HCDR2 consists of the amino acid sequence of SEQ ID NO: 2; the HCDR3 consists of the amino acid sequence of SEQ ID NO: 6; the LCDR1 consists of the amino acid sequence of SEQ ID NO: 7; the LCDR2 consists of the amino acid sequence of SEQ ID NO: 8; and the LCDR3 consists of the amino acid sequence of SEQ ID NO: 9; Optionally, any amino acid D or G in the consecutive amino acids DG in HCDR2 can be substituted, for example, D can be substituted with E or Q, and / or G is substituted with V, for example, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 3, 4 or 5.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 12, 13, 14 or 15; or (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12, 13, 14 or 15. And / or, it comprises a light chain variable region VL, wherein the light chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 16; or (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16; Optionally, any of the consecutive amino acids NFS in the framework region FR3 of the VL may be substituted, for example, N therein is substituted with Q and / or S therein is substituted with A; for example The light chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 17 or 18; or (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 17 or 18.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (i) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and The VL comprises or consists of the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and The VL comprises or consists of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iii) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and The VL comprises or consists of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iv) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and The VL comprises or consists of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (v) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and The VL comprises or consists of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (vi) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and The VL comprises or consists of the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (vii) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (viii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and The VL comprises the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (i) The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; (ii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16; (iii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16; (iv) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 16; (v) the VH comprises the amino acid sequence shown in SEQ ID NO: 15 or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO: 16 or consists of the amino acid sequence; (vi) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; (vii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; or (viii). The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO:

18.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, further comprising a heavy chain constant region HC, for example, the antibody heavy chain constant region HC is derived from IgG, for example, the heavy chain constant region of human IgG, for example, the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4; Optionally, the heavy chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 23; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 23; Optionally, the heavy chain constant region comprises an Fc region from IgG, such as human IgG, such as an Fc region of IgG1, IgG2, IgG3 or IgG4, preferably a human IgG1, IgG2, IgG3 or IgG4 Fc, and optionally, the Fc region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 26 or 27; or (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 26 or 27.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising a light chain constant region, for example, a light chain constant region from a lambda or kappa light chain constant region, for example, a lambda or kappa light chain constant region, for example, a human lambda or kappa light chain constant region; Optionally, the light chain constant region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 24 or 25; or (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 24 or 25.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody is a humanized antibody or a chimeric antibody; or, the antibody is a monoclonal antibody.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (eg, scFv), (Fab')2 diabody, or linear antibody.

10. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the antibody is a bispecific antibody or a multispecific antibody comprising a first binding specificity for DLL3 and binding specificities for one or more additional antigens; optionally, the additional antigen is CD3.

11. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, The first antigen binding region specifically binds to DLL3 and comprises VH and VL, wherein The VH comprises HCDR1, HCDR2, HCDR3 as defined in claim 1 or 2, and the VL comprises LCDR1, LCDR2 and LCDR3 as defined in claim 1 or 2; or The VH and VL are the VH and VL defined in any one of claims 3 to 6; and The second antigen-binding region specifically binds to CD3; Optionally, the first antigen binding region is a Fab of an anti-DLL3 antibody as defined in any one of claims 1 to 14; and / or the second antigen binding region is a Fab that specifically binds to CD3.

12. The bispecific antibody of claim 11, wherein the Fab as the first antigen-binding region or the second antigen-binding region comprises a CH1, wherein the CH1 is a CH1 derived from IgG1, IgG2, IgG3 or IgG4, preferably a CH1 derived from human IgG1, IgG2, IgG3 or IgG4, for example, a CH1 of human IgG1, IgG2, IgG3 or IgG4; Optionally, the CH1 (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 28; or (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO:

28.

13. The bispecific antibody of claim 11 or 12, wherein the Fab as the first or second antigen-binding region comprises a light chain constant region, wherein the light chain constant region is a light chain constant region from a lambda or kappa light chain constant region, for example, a lambda or kappa light chain constant region, for example, a human lambda or kappa light chain constant region, optionally, the first and second antigen-binding regions comprise the same or different light chain constant regions, for example, the first antigen-binding region comprises a kappa light chain constant region and the second antigen-binding region comprises a lambda light chain constant region; Optionally, the Kappa light chain constant region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 25; or (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 25; and / or The Lambda light chain constant region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 24; or (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO:

24.

14. The bispecific antibody of any one of claims 11 to 13, wherein the bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer, the first Fc region and the second Fc region are identical or different, optionally, the Fc regions are respectively Fc regions from IgG, such as human IgG, for example, IgG1, IgG2, IgG3 or IgG4, preferably human IgG1, IgG2, IgG3 or IgG4 Fc, optionally, the Fc regions (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 26 or 27; or (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 26 or 27; Optionally, the two Fc regions are different, wherein mutations are introduced into the first Fc region and the second Fc region based on the Innobody technology to promote heterodimerization of the first Fc region and the second Fc region; for example, the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S and K409D mutations; Optionally, wherein the first and / or second Fc region comprises L234A / L235A mutation; For example, a) one Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, and the other Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 30; b) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 29 and comprises the mutations S364R and D399K, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 30 and comprises the mutations Y349T, K370S, and K409D; or c) one Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 29 and comprises mutations S364R and D399K and L234A / L235A mutations, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 30 and comprises mutations Y349T, K370S and K409D and L234A / L235A mutations.

15. The bispecific antibody of any one of claims 11 to 14, wherein the second antigen-binding region comprises: three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 32; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36; LCD R2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

38.

16. The bispecific antibody of claim 15, wherein the second antigen-binding region comprises VH and VL, wherein VH comprises or consists of the sequence shown in SEQ ID NO: 31; and VL comprises or consists of the sequence shown in SEQ ID NO:

35.

17. The bispecific antibody of any one of claims 11 to 16, wherein the bispecific antibody comprises a first antigen-binding region that specifically binds to DLL3 and a second antigen-binding region that specifically binds to CD3, wherein the first antigen-binding region comprises a first Fab and the second antigen-binding region comprises a second Fab, wherein the first Fab is linked at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), and the second Fab is linked at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region); Optionally, the bispecific antibody is an IgG-like antibody having the configuration shown in FIG3 .

18. The bispecific antibody of claim 16 or 17, comprising a first Fab as a first antigen-binding region that specifically binds to DLL3 and a second Fab as a second antigen-binding region that specifically binds to CD3, wherein the bispecific antibody comprises Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a first Fab heavy chain variable region-a heavy chain constant region CH1-a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region); Light chain 1: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the first Fab - the light chain constant region; Heavy chain 2: comprises or consists of the following from N-terminus to C-terminus: a second Fab heavy chain variable region - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region); Light chain 2: from N-terminus to C-terminus, comprises or consists of: the light chain variable region of the second Fab - the light chain constant region; The first Fab heavy chain variable region-heavy chain constant region CH1 and the first Fab light chain variable region-light chain constant region constitute the first Fab, and the second Fab heavy chain variable region-heavy chain constant region CH1 and the second Fab light chain variable region-light chain constant region constitute the second Fab; Optionally, the light chain constant region in light chain 1 and the light chain constant region in light chain 2 are different, e.g., the light chain constant region in light chain 1 is a Kappa light chain constant region and the light chain constant region in light chain 2 is a Lambda light chain constant region.

19. The bispecific antibody of claim 18, wherein (i) heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:41, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:45, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; (ii) heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:42, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:47, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; or (iii) heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:43, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:47, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

20. A nucleic acid molecule comprising or consisting of a polynucleotide encoding any one chain of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or any one chain of the bispecific antibody according to any one of claims 11 to 19.

21. An expression vector comprising the nucleic acid molecule according to claim 20, preferably, the expression vector is pCDNA, such as pCDNA3.

1.

22. A host cell comprising the nucleic acid molecule of claim 20 or the expression vector of claim 21, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as Expi293 cells.

23. A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1 to 10 or the bispecific antibody of any one of claims 11 to 19, the method comprising culturing a host cell comprising the nucleic acid molecule of claim 20 or the expression vector of claim 21 under conditions suitable for expression of the polypeptide chain of the antibody or antigen-binding fragment thereof or bispecific antibody, and optionally recovering the antibody or antigen-binding fragment thereof or bispecific antibody from the host cell (or host cell culture medium).

24. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the bispecific antibody according to any one of claims 11 to 19.

25. A pharmaceutical composition, medicament or formulation comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the bispecific antibody according to any one of claims 11 to 19, or the immunoconjugate according to claim 24, and optionally a pharmaceutically acceptable excipient.

26. A pharmaceutical combination comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the bispecific antibody of any one of claims 11 to 19, or the immunoconjugate of claim 24, and one or more other therapeutic agents, preferably, the therapeutic agents are various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immune checkpoint inhibitors or agonists); Optionally, the drug combination Comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the bispecific antibody according to any one of claims 11 to 19, or the immunoconjugate according to claim 39 and other antibodies, preferably, the other antibodies are antibodies that inhibit the PD-1 / PD-L1 signaling pathway, such as anti-PD-1 antibodies; or anti-PD-L1 antibodies or anti-PD-L2 antibodies; or A method comprising: comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the bispecific antibody according to any one of claims 11 to 19, or the immunoconjugate according to claim 39 and one or more chemotherapeutic agents, such as a platinum-based chemotherapeutic drug such as carboplatin; or a topoisomerase inhibitor, such as a topoisomerase II inhibitor, such as etoposide, or a combination thereof; Preferably, the pharmaceutical combination comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the bispecific antibody according to any one of claims 11 to 19, or the immunoconjugate according to claim 39 and a combination of carboplatin and etoposide.

27. A method for preventing or treating a tumor in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the bispecific antibody according to any one of claims 11 to 19, or the immunoconjugate according to claim 24, or the pharmaceutical composition or formulation according to claim 25, or the pharmaceutical combination according to claim 26; Optionally, the tumor is a solid tumor or a blood tumor, such as a DLL3-positive tumor or cancer, optionally the tumor is a neuroendocrine tumor (such as a DLL3-positive neuroendocrine tumor), such as a lung cancer (such as a DLL3-positive lung cancer), such as small cell lung cancer (SCLC).

28. The method of claim 27, wherein the method further comprises administering in combination with other therapies, such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents, preferably, the therapeutic agents are various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists); Optionally, the method further comprises administering in combination with other antibodies, preferably, the other antibodies are antibodies that inhibit the PD-1 / PD-L1 signaling pathway, such as anti-PD-1 antibodies; or anti-PD-L1 antibodies or anti-PD-L2 antibodies; or The method further comprises administering in combination with one or more chemotherapeutic agents, wherein the chemotherapeutic agent is selected from a platinum chemotherapy drug such as carboplatin; or a topoisomerase inhibitor, such as a topoisomerase II inhibitor, such as etoposide, or a combination thereof; or The method also includes administration in combination with carboplatin and etoposide.

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