Bispecific antigen-binding molecules and uses thereof

Abstract

The present invention provides a bispecific antigen-binding molecule configuration, and further provides a bispecific antigen-binding molecule directed against DLL3 constructed based on this configuration, a pharmaceutical composition comprising the bispecific antigen-binding molecule, and related uses thereof in tumor treatment.

Description

[Technical field]

[0001] The present invention claims priority to Chinese Patent Application No. 2022101238168, filed on February 10, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention is in the field of immunology and relates to bispecific antigen-binding molecules, and also to associated encoding nucleic acids, vectors, host cells, drugs, and uses in the treatment of cancer. [Background technology]

[0003] Monoclonal antibodies are widely used to treat various diseases, including tumors, but the long-term efficacy of monoclonal antibody therapy is limited by mechanisms such as tumor immune evasion. One solution is to develop bispecific antibodies, which can bind to two different antigens or two different epitopes to block different signaling pathways during tumor development or directly target immune cells to tumor cells, potentially producing higher cytotoxicity and better circumventing immune evasion. Although various bispecific antibodies have been developed so far, there is still a need to develop novel forms of bispecific antibodies.

[0004] Small cell lung cancer (SCLC) accounts for approximately 15%-20% of lung cancers and is characterized by high vascularity, rapid tumor growth, genomic instability, and early metastatic spread. It is estimated that more than 234,000 cases of SCLC are diagnosed annually, with approximately 250,000 deaths worldwide each year. Localized treatment approaches such as surgery and / or radiation therapy are virtually impossible to cure SCLC. Platinum-based combination chemotherapy remains the basis of treatment, and although response rates to first-line chemotherapy are high, recurrence rates are high, with median survival times of only 14-20 months for patients with limited-stage disease and 9-11 months for those with extensive-stage disease, and for those who relapse, survival times are even shorter and there are virtually no treatment options. Topotecan, the only FDA-recommended agent, is limited by its hematological toxicity, and its treatment response rates are unsatisfactory at only 5%-24%, with a median survival time of less than 25 weeks. As there is no specific recommendation for a third-line treatment, new effective therapeutic agents are urgently needed.

[0005] Delta-like protein 3 (DLL3), also called DLL3, is a protein encoded by the DLL3 gene and is one of the ligands of the Notch family. This protein affects the signaling pathway regulated by Notch, and as a result, signals emanating from the Notch pathway promote the uncontrolled proliferation of tumor cells. Studies have shown that DLL3 is expressed on the surface of tumor cells in approximately 85% of patients with small cell lung cancer and large cell neuroendocrine carcinoma, and is also highly expressed in glioblastoma multiforme, melanoma, pancreatic cancer, and rectal cancer. However, it is not expressed in healthy tissues or non-neuroendocrine tumors, making DLL3 a desirable target for small cell lung cancer.

[0006] Bispecific antibodies targeting CD3 antigens and tumor-associated antigens can bring T cells into spatial proximity with tumor cells and utilize T cells to achieve a specific killing effect on tumor cells, and these antibodies are called T cell-engagers (TCE bispecific antibodies). Nearly half of the bispecific antibody candidates in the clinical stage target the CD3 antigen, and the safety and efficacy of TCE bispecific antibodies are being studied. Although some progress has been made in related research, many challenges remain, such as the problem of how to balance antitumor activity and safety. In this case, the development of novel T cell bispecific antibodies with high efficiency and low toxicity is particularly important.

[0007] Therefore, it has become necessary to develop novel forms of bispecific antibodies and further to utilize them to develop bispecific antibodies with high efficiency and low toxicity. Summary of the Invention

[0008] The first object of the present invention is to provide a bispecific antigen-binding molecule having a novel morphology, in which the second antigen-binding portion of the bispecific antigen-binding molecule is encapsulated between the first antigen-binding portion and the Fc structure, thereby reducing the exposure of the second antigen-binding portion and reducing the risk of non-specific cytokine release. (A) a first polypeptide comprising (i) an antigen-binding fragment (Fab) heavy chain domain specifically directed to a first antigen, (ii) a single chain antibody (scFv) domain capable of specifically binding a second antigen, and (iii) a first Fc domain; (B) a second polypeptide comprising a second Fc domain; and (C) a third polypeptide comprising a light chain domain of an antigen-binding fragment (Fab) specifically directed to the first antigen; The antigen-binding fragment (Fab) heavy chain domain and the antigen-binding fragment (Fab) light chain domain form a first binding site for a first antigen, the single chain antibody (scFv) domain forms a second binding site for a second antigen, and the first Fc domain and the second Fc domain are associated with each other.

[0009] In one embodiment, the single chain antibody (scFv) domain comprises a second heavy chain variable region and a second light chain variable region, Preferably, the second heavy chain variable region is linked to the second light chain variable region via a first linker or directly, wherein: the C-terminus of the second heavy chain variable region is fused to the N-terminus of the first linker and the C-terminus of the first linker is fused to the N-terminus of the second light chain variable region; or the C-terminus of the second light chain variable region is fused to the N-terminus of the first linker and the C-terminus of the first linker is fused to the N-terminus of the second heavy chain variable region; or the C-terminus of the second heavy chain variable region is fused to the N-terminus of the second light chain variable region; or the C-terminus of the second light chain variable region is fused to the N-terminus of the second heavy chain variable region; More preferably, the first linker comprises the amino acid sequence (G4S) n where n is any integer from 1 to 10.

[0010] In one embodiment, the first Fc domain comprises a first CH2 domain and a first CH3 domain of an immunoglobulin, and the C-terminus of the first CH2 domain is fused to the N-terminus of the first CH3 domain; and the second Fc domain comprises a second CH2 domain and a second CH3 domain of an immunoglobulin, and the C-terminus of the second CH2 domain is fused to the N-terminus of the second CH3 domain; Preferably, said first CH3 domain comprises a "knob" structure and said second CH3 domain comprises a "hole" structure, more preferably said "knob" structure comprises the amino acid substitutions S354C and T366W and said "hole" structure comprises the amino acid substitutions Y349C, T366S, L368A and Y407V; Preferably, the Fc domain is from IgG1, Preferably, the single chain antibody (scFv) domain is linked to the first Fc domain via a second linker or directly, wherein: the C-terminus of the single chain antibody (scFv) domain is fused to the N-terminus of a second linker, and the C-terminus of the second linker is fused to the N-terminus of a first Fc domain; or the C-terminus of a single chain antibody (scFv) domain is fused to the N-terminus of a first Fc domain, More preferably, said second linker comprises the amino acid sequence EPKSS (SEQ ID NO:34).

[0011] In one embodiment, the heavy chain domain of the antigen-binding fragment (Fab) comprises a first heavy chain variable region and a CH1 domain of an immunoglobulin, and the C-terminus of the first heavy chain variable region is fused to the N-terminus of the CH1 domain; the light chain domain of the antigen-binding fragment (Fab) comprises a first light chain variable region and a light chain constant region of an immunoglobulin, and the C-terminus of the first light chain variable region is fused to the N-terminus of the light chain constant region; Preferably, the heavy chain domain of the antigen-binding fragment (Fab) is linked to the single chain antibody (scFv) domain via a third linker or directly, wherein: the C-terminus of the heavy chain domain of the antigen-binding fragment (Fab) is fused to the N-terminus of a third linker, and the C-terminus of the third linker is fused to the N-terminus of a single chain antibody (scFv) domain; or the C-terminus of the single chain antibody (scFv) domain is fused to the N-terminus of a third linker, and the C-terminus of the third linker is fused to the N-terminus of a heavy chain domain of an antigen-binding fragment (Fab); or the C-terminus of the heavy chain domain of an antigen-binding fragment (Fab) is fused to the N-terminus of a single chain antibody (scFv) domain; or The C-terminus of a single chain antibody (scFv) domain is fused to the N-terminus of the heavy chain domain of an antigen-binding fragment (Fab), More preferably, the third linker comprises the amino acid sequence (G4S) n where n is any integer from 1 to 10.

[0012] In one embodiment, the first polypeptide comprises the structure Fab heavy chain domain-third linker-scFv domain-second linker-first Fc domain, or scFv domain-third linker-Fab heavy chain domain-second linker-first Fc domain, Preferably, the first polypeptide has the structure: first heavy chain variable region-CH1-third linker-second heavy chain variable region-first linker-second light chain variable region-second linker-first CH2-first CH3, or first heavy chain variable region-CH1-third linker-second light chain variable region-first linker-second heavy chain variable region-second linker-first CH2-first CH3, or second heavy chain variable region-first linker-second light chain variable region-second linker-first CH2-first CH3. The second polypeptide comprises a structure of second CH2-second CH3, and the third polypeptide comprises a structure of first light chain variable region-light chain constant region.

[0013] In one embodiment, the bispecific antigen-binding molecule comprises one or more amino acid substitutions selected from the group consisting of: (i) L234A and L235A; (ii) H435R, preferably, the H435R substitution is on the second polypeptide.

[0014] In one embodiment, the second antigen is CD3, preferably CD3ε, and preferably, the single chain antibody (scFv) domain comprises an HCDR1 having the sequence shown in SEQ ID NO:24, an HCDR2 having the sequence shown in SEQ ID NO:25, an HCDR3 having the sequence shown in SEQ ID NO:26, an LCDR1 having the sequence shown in SEQ ID NO:27, an LCDR2 having the sequence shown in SEQ ID NO:28, and an LCDR3 having the sequence shown in SEQ ID NO:29; More preferably, the single chain antibody (scFv) domain comprises a second heavy chain variable region whose sequence is shown in SEQ ID NO:21 and a second light chain variable region whose sequence is shown in SEQ ID NO:22, More preferably, the single chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO:23.

[0015] In one embodiment, the first Fc domain comprises the amino acid sequence set forth in SEQ ID NO:31 and the second Fc domain comprises the amino acid sequence set forth in SEQ ID NO:3.

[0016] In one embodiment, the first antigen is DLL3, and preferably, the heavy chain domain of the antigen-binding fragment (Fab) comprises an HCDR1 having the sequence shown in SEQ ID NO: 13, an HCDR2 having the sequence shown in SEQ ID NO: 14, and an HCDR3 having the sequence shown in SEQ ID NO: 15, and the light chain domain of the antigen-binding fragment (Fab) comprises an LCDR1 having the sequence shown in SEQ ID NO: 16, an LCDR2 having the sequence shown in SEQ ID NO: 17, and an LCDR3 having the sequence shown in SEQ ID NO: 18; More preferably, the heavy chain domain of said antigen-binding fragment (Fab) comprises a first heavy chain variable region having a sequence as shown in SEQ ID NO: 11, and the light chain domain of said antigen-binding fragment (Fab) comprises a first light chain variable region having a sequence as shown in SEQ ID NO: 12; More preferably, the heavy chain domain of the antigen-binding fragment (Fab) comprises the amino acid sequence set forth in SEQ ID NO:33, and the light chain domain of the antigen-binding fragment (Fab) comprises the amino acid sequence set forth in SEQ ID NO:4.

[0017] In one embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

[0018] Another embodiment of the bispecific antigen-binding molecule comprises two homologous polypeptides, each of which comprises (i) a nanobody (VHH) domain capable of specifically binding to a first antigen, (ii) a single-chain antibody (scFv) domain capable of specifically binding to a second antigen, and (iii) an Fc domain, wherein the Fc domains of the two polypeptides are associated with each other.

[0019] In one embodiment, the single chain antibody (scFv) domain comprises a heavy chain variable region and a light chain variable region, Preferably, the heavy chain variable region is linked to the light chain variable region via a first linker or directly, wherein: the C-terminus of the heavy chain variable region is fused to the N-terminus of a first linker and the C-terminus of the first linker is fused to the N-terminus of the light chain variable region; or the C-terminus of the light chain variable region is fused to the N-terminus of the first linker and the C-terminus of the first linker is fused to the N-terminus of the heavy chain variable region; or the C-terminus of the heavy chain variable region is fused to the N-terminus of the light chain variable region; or the C-terminus of the light chain variable region is fused to the N-terminus of the heavy chain variable region, More preferably, the first linker comprises the amino acid sequence (G4S) n where n is any integer from 1 to 10.

[0020] In one embodiment, the Fc domain comprises an immunoglobulin CH2 domain and a CH3 domain, the C-terminus of the CH2 domain fused to the N-terminus of the CH3 domain, Preferably, the Fc domain is from IgG1, Preferably, the Fc domain is linked to the single chain antibody (scFv) domain via a second linker or directly, wherein: the C-terminus of the single chain antibody (scFv) domain is fused to the N-terminus of a second linker, and the C-terminus of the second linker is fused to the N-terminus of the Fc domain; or The C-terminus of a single chain antibody (scFv) domain is fused to the N-terminus of an Fc domain, More preferably, said second linker comprises the amino acid sequence EPKSS (SEQ ID NO:34).

[0021] In one embodiment, said Nanobody (VHH) domain is linked to said single chain antibody (scFv) domain via a third linker or directly, wherein: the C-terminus of the nanobody (VHH) domain is fused to the N-terminus of a third linker, and the C-terminus of the third linker is fused to the N-terminus of a single chain antibody (scFv) domain; or the C-terminus of the single chain antibody (scFv) domain is fused to the N-terminus of a third linker, and the C-terminus of the third linker is fused to the N-terminus of a nanobody (VHH) domain; or the C-terminus of a nanobody (VHH) domain is fused to the N-terminus of a single chain antibody (scFv) domain; or The C-terminus of a single chain antibody (scFv) domain is fused to the N-terminus of a nanobody (VHH) domain, More preferably, the third linker comprises the amino acid sequence (G4S) n where n is any integer from 1 to 10.

[0022] In one embodiment, each of the polypeptides comprises the structure VHH domain-third linker-scFv domain-second linker-Fc domain, or scFv domain-third linker-VHH domain-second linker-Fc domain, Preferably, each of the polypeptides has the structure: VHH domain-third linker-heavy chain variable region-first linker-light chain variable region-second linker-CH2-CH3; or VHH domain-third linker-light chain variable region-first linker-heavy chain variable region-second linker-CH2-CH3; or heavy chain variable region-first linker-light chain variable region-third linker-VHH domain-second linker-CH2-CH3; or light chain variable region-first linker-heavy chain variable region-third linker-VHH domain-second linker-CH2-CH3.

[0023] In one embodiment, the bispecific antigen binding molecule comprises the amino acid substitutions L234A and L235A.

[0024] In one embodiment, the second antigen is CD3, preferably CD3ε, and preferably, the single chain antibody (scFv) domain comprises an HCDR1 having the sequence shown in SEQ ID NO:24, an HCDR2 having the sequence shown in SEQ ID NO:25, an HCDR3 having the sequence shown in SEQ ID NO:26, an LCDR1 having the sequence shown in SEQ ID NO:27, an LCDR2 having the sequence shown in SEQ ID NO:28, and an LCDR3 having the sequence shown in SEQ ID NO:29; More preferably, said single chain antibody (scFv) domain comprises a heavy chain variable region the sequence of which is shown in SEQ ID NO:21 and a light chain variable region the sequence of which is shown in SEQ ID NO:22, More preferably, the single chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO:23.

[0025] In one embodiment, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO:30.

[0026] In one embodiment, said first antigen is DLL3, preferably said Nanobody (VHH) domain comprises an HCDR1 whose sequence is shown in SEQ ID NO:6, an HCDR2 whose sequence is shown in SEQ ID NO:7, and an HCDR3 whose sequence is shown in SEQ ID NO:8, More preferably, said Nanobody (VHH) domain comprises the amino acid sequence shown in SEQ ID NO:32.

[0027] In one embodiment, each of the polypeptides in the bispecific antigen binding molecule comprises the amino acid sequence shown in SEQ ID NO:1.

[0028] A second object of the present invention is to provide an anti-DLL3 (preferably anti-DLL3 and CD3) bispecific antigen-binding molecule, which can specifically kill tumor cells with high DLL3 expression by targeting immune cell surface antigens (preferably CD3 antigens) and recruiting T cells to tumor sites, and has low ex vivo cytokine release levels and good safety.

[0029] Based on the above, the present invention provides a bispecific antigen-binding molecule that binds a specific epitope, and the epitope to which the bispecific antigen-binding molecule can bind is, (i) is identical to or overlaps with the epitope of interest of a Nanobody comprising an HCDR1 having the sequence shown in SEQ ID NO:6, an HCDR2 having the sequence shown in SEQ ID NO:7, and an HCDR3 having the sequence shown in SEQ ID NO:8, or (ii) is the same as or overlaps with the epitope of interest of an antibody comprising an HCDR1 whose sequence is set forth in SEQ ID NO:13, an HCDR2 whose sequence is set forth in SEQ ID NO:14, an HCDR3 whose sequence is set forth in SEQ ID NO:15, an LCDR1 whose sequence is set forth in SEQ ID NO:16, an LCDR2 whose sequence is set forth in SEQ ID NO:17, and an LCDR3 whose sequence is set forth in SEQ ID NO:18.

[0030] In one embodiment, the epitope to which the bispecific antigen-binding molecule can bind is (i) is identical to or overlaps with the epitope of interest of a Nanobody comprising the amino acid sequence shown in SEQ ID NO:32, or (ii) It is the same as or overlaps with the epitope of interest of an antibody comprising a first heavy chain variable region whose sequence is set forth in SEQ ID NO:11 and a first light chain variable region whose sequence is set forth in SEQ ID NO:12.

[0031] In one embodiment, the bispecific antigen-binding molecule is capable of binding to CD3, preferably CD3ε.

[0032] The present invention further provides a bispecific antigen-binding molecule, the bispecific antigen-binding molecule comprising: (A) a first binding moiety capable of specifically binding DLL3; and (B) a second binding moiety that specifically binds to a different antigen or epitope than the first binding moiety; The first binding moiety is (i) an HCDR1 whose sequence is set forth in SEQ ID NO:6, an HCDR2 whose sequence is set forth in SEQ ID NO:7, and an HCDR3 whose sequence is set forth in SEQ ID NO:8, or (ii) an HCDR1 whose sequence is shown in SEQ ID NO:13, an HCDR2 whose sequence is shown in SEQ ID NO:14, an HCDR3 whose sequence is shown in SEQ ID NO:15, an LCDR1 whose sequence is shown in SEQ ID NO:16, an LCDR2 whose sequence is shown in SEQ ID NO:17, and an LCDR3 whose sequence is shown in SEQ ID NO:18.

[0033] In one embodiment, the first binding moiety is (i) the amino acid sequence shown in SEQ ID NO:32, or (ii) comprises a first heavy chain variable region, the sequence of which is shown in SEQ ID NO:11, and a first light chain variable region, the sequence of which is shown in SEQ ID NO:12.

[0034] In one embodiment, the second binding moiety is capable of binding to CD3, preferably CD3ε.

[0035] The present invention further provides a nucleic acid molecule encoding the above-described bispecific antigen-binding molecule.

[0036] The present invention further provides an expression vector comprising the above-described nucleic acid molecule.

[0037] The present invention further provides a host cell comprising the above-mentioned nucleic acid molecule or expression vector, preferably said host cell is a prokaryotic or eukaryotic cell, said prokaryotic cell is preferably E. coli, said eukaryotic cell is preferably a mammalian cell or yeast, more preferably said mammalian cell is a CHO cell, an Expi 293 or a HEK 293 cell.

[0038] The present invention further provides a method for producing a bispecific antigen-binding molecule, said method comprising culturing the above-described host cell under suitable conditions.

[0039] The present invention further provides an antibody-drug conjugate formed by conjugating the above-mentioned bispecific antigen-binding molecule with another biologically active molecule, preferably, the other biologically active molecule is a small molecule drug, and preferably, the bispecific antigen-binding molecule is linked to the other biologically active molecule via a linker.

[0040] The present invention further provides a pharmaceutical composition comprising the above-mentioned bispecific antigen-binding molecule, nucleic acid molecule, expression vector, host cell, and / or antibody-drug conjugate.

[0041] In one embodiment, the pharmaceutical composition further comprises a pharma- ceutically acceptable vector.

[0042] In one embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0043] The present invention further provides the use of the above-mentioned bispecific antigen-binding molecule, nucleic acid molecule, expression vector, host cell and / or antibody-drug conjugate in the manufacture of a medicament for treating, alleviating and / or preventing a tumor, preferably the tumor is a DLL3-positive tumor.

[0044] The present invention further provides a method for inducing death of a DLL3-expressing cell, the method comprising contacting the cell with the above-mentioned bispecific antigen-binding molecule, nucleic acid molecule, expression vector, host cell, antibody-drug conjugate and / or drug composition, wherein the DLL3-expressing cell is a tumor cell.

[0045] The present invention further provides a method for treating a disease associated with DLL3 expression in a subject, the method comprising administering to a subject in need thereof the above-mentioned bispecific antigen-binding molecule, nucleic acid molecule, expression vector, host cell, antibody-drug conjugate and / or drug composition. Preferably, the disease is a tumor.

[0046] Preferably, the tumor / tumor cells according to the present invention are selected from small cell lung cancer, glioblastoma, neuroendocrine carcinoma, melanoma, pancreatic cancer, rectal cancer and metastases of the above tumors.

[0047] In one embodiment, the method further comprises administering to the subject an additional therapeutic agent.

[0048] The substitutents according to the present invention are preferably represented by the EU numbering system.

[0049] The technical solution according to the present invention can bring about the following beneficial effects: strong in vitro tumor cell killing ability is maintained, while the level of in vitro non-specific cytokine release is weak or non-existent, and safety is good. [Brief description of the drawings]

[0050] The drawings are provided to further illustrate the novel features of the invention herein, and the features and advantages of the invention herein may be better understood by reference to these drawings, but it should be understood that these drawings are merely for the purpose of illustrating specific embodiments of the principles of the invention herein, and are not intended to limit the scope of the appended claims. [Figure 1] The structures of two bispecific antibodies constructed in the Examples are shown in Figure 1A, bispecific antibody 1, and Figure 1B, bispecific antibody 2. [Diagram 2] Binding status of bispecific antibody 1 and bispecific antibody 2 to SHP-77 cells expressing hDLL3 is shown. [Diagram 3] 1 shows the binding profiles of bispecific antibody 1 and bispecific antibody 2 to Jurkat cells that naturally express hCD3. [Figure 4] 1 shows the results of a CD3+ T cell cytotoxicity assay (TDCC) mediated by Bispecific Antibody 1 and Bispecific Antibody 2 against SHP-77 cells. [Diagram 5] 1 shows the results of a CD3+ T cell cytotoxicity assay (TDCC) mediated by Bispecific Antibody 1 and Bispecific Antibody 2 against NCI-H82 cells. [Figure 6A] The results of a cytokine release test from bispecific antibody 1 and bispecific antibody 2 in the presence or absence of target cells are shown. Figures 6A to 6E show the results of cytokine release from bispecific antibody 1 molecules, which are IFNγ, TNFα, IL-10, IL-6, and IL4, respectively, and Figures 6F to 6J show the results of cytokine release from bispecific antibody 2 molecules, which are IFNγ, TNFα, IL-10, IL-6, and IL4, respectively. [Figure 6B] These are the results of the cytokine release test mentioned above. [Figure 6C] These are the results of the cytokine release test mentioned above. [Figure 6D] These are the results of the cytokine release test mentioned above. [Figure 6E] These are the results of the cytokine release test mentioned above. [Figure 6F] These are the results of the cytokine release test mentioned above. [Figure 6G] These are the results of the cytokine release test mentioned above. [Figure 6H] These are the results of the cytokine release test mentioned above. [Figure 6I] These are the results of the cytokine release test mentioned above. [Figure 6J] These are the results of the cytokine release test mentioned above. [Figure 7] FIG. 1 is a schematic diagram of mouse tumor inoculation locations for in vivo efficacy testing of bispecific antibody 2. [Figure 8] FIG. 1 shows the effect of Bispecific Antibody 2 on tumor growth weight in a SHP-77 small cell lung cancer model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Terminology All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference, and the citation level shall specifically and individually indicate that each individual publication, patent, or patent application is incorporated by reference herein.

[0052] Before the present invention is described in detail below, it should be understood that the specific methodology, solutions, and reagents used in the present invention may vary and are not limited to those described herein. 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. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs, unless otherwise defined.

[0053] Some embodiments disclosed herein have numerical ranges, and some aspects of the present invention may be described in ranges. It should be understood that numerical ranges or methods of describing ranges are merely for brevity and convenience, and do not strictly limit the scope of the present invention, unless otherwise stated. Thus, by describing a range, all possible subranges and all possible specific numerical points within the range are considered to be specifically invented, as if these subranges and numerical points were expressly described herein. The above principles shall apply equally regardless of the magnitude of the numerical values ​​described above. When a range description is used, the range shall include the end points of the range.

[0054] When referring to measurable values ​​such as amounts or temporal durations, the term "about" is meant to include variations of ±20%, in some cases ±10%, in some cases ±5%, in some cases ±1%, or in some cases ±0.1% of the specified value.

[0055] The term "antibody" as used herein includes complete antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragments (i.e., antigen-binding portions) or single chains thereof, as well as products based on complete antibodies or antigen-binding fragments or single chains thereof that have been modified (e.g., by binding to other peptide fragments, rearrangement of functional units, etc.) and have antigen-specific binding ability.

[0056] In one embodiment, an antibody typically refers to a Y-shaped tetrameric protein containing two heavy (H) and two light (L) polypeptide chains linked by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have such a structure. Each light chain is composed of one variable domain (VL) and one constant domain (CL). Each heavy chain contains one variable domain (VH) and a constant region.

[0057] In the art, antibodies are broadly classified into five types, IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains called α, δ, ε, γ, and μ, respectively, and IgG and IgA are further divided into different subclasses, e.g., IgG is divided into IgG1, IgG2, IgG3, and IgG4, and IgA is divided into IgA1 and IgA2. The light chain of an antibody from any vertebrate can be assigned to one of two clearly distinct types, called κ and λ, depending on the amino acid sequence in its constant domain.

[0058] In the case of IgG, IgA and IgD antibodies, the constant region contains three domains called CH1, CH2 and CH3 (in the case of IgM and IgE, there is a fourth domain, CH4). In the IgG, IgA and IgD types, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable length fragment rich in prolines and cysteines. Each type of antibody further contains inter- and intrachain disulfide bonds formed by ligand cysteine ​​residues.

[0059] The term "variable region" or "variable domain" refers to the region that exhibits significant variation in amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable regions of each light / heavy chain pair form the antibody binding site, such that an intact IgG antibody has two binding sites (i.e., it is bivalent). Each of the heavy chain variable region (VH) domain and the light chain variable region (VL) domain contains three regions of extreme variability, called hypervariable regions (HVRs) or, more commonly, complementarity determining regions (CDRs), and each of the VH and VL has four framework regions, FR, designated FR1, FR2, FR3, and FR4, respectively. Thus, the CDR and FR sequences usually occur in the following sequence in the heavy chain variable domain (or light chain variable domain): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0060] The term "nanobody" refers to an amino acid sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively. "VHH" refers to the variable antigen-binding domain of heavy chain antibodies from the Camelidae family (camel, dromedary, llama, alpaca, etc.).

[0061] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, where the light and heavy chain variable regions are contiguous (e.g., linked via a synthetic linker, such as a short flexible polypeptide) and can be expressed in the form of a single polypeptide chain, retaining the specificity of the intact antibody from which the scFv is derived. An scFv can have the VL and VH variable regions described above in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), unless otherwise specified, and can comprise a VL-linker-VH or a VH-linker-VL.

[0062] The term "Fc" is intended to define the C-terminal region of an immunoglobulin heavy chain, which region includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain can vary slightly, the Fc region of a human IgG heavy chain is usually defined as stretching from Cys 226, or Pro 230, to the carboxyl terminus of the heavy chain, e.g., the Fc region of an IgG includes the IgG CH2 and IgG CH3 as constant regions. The numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index), unless otherwise specified herein.

[0063] The term "association" refers to a functional relationship between two or more polypeptide chains and / or two or more portions of a single polypeptide chain. In particular, the term "association" means that two or more polypeptides (or portions of a single polypeptide) are associated with each other, e.g., non-covalently linked by molecular interactions and / or covalently linked by one or more dione bridges or chemical crosslinks to generate a functional antigen-binding domain. Examples of associations that may exist in antigen-binding molecules include, but are not limited to, the association between Fc regions in an Fc domain, the association between VH and VL regions in a Fab or Fv, and the association between CH1 and CL in a Fab.

[0064] The term "knob-into-hole" refers to modifications to promote the association of the two polypeptide chains of the Fc, including a "knob" modification in one of the two polypeptide chains of the Fc and a "hole" modification in the other of the two polypeptide chains of the Fc. This technique is described, for example, in US 5,731,168 and US 7,695,936. In general, the method introduces a protrusion ("knob") at the interface of a first polypeptide chain and a corresponding recess ("hole") at the interface of a second polypeptide chain, thereby positioning the protrusion within the recess to promote heterodimer formation while preventing homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide chain with a larger side chain (e.g., tyrosine or tryptophan). A compensating recess of identical or similar size to the protrusion is created at the interface of the second polypeptide chain by replacing a large amino acid side chain with a smaller one (eg, alanine or threonine).

[0065] Thus, according to one specific embodiment, the bispecific antibody according to the invention has one amino acid residue in the CH3 region of the first polypeptide chain in the Fc region substituted with an amino acid residue having a larger side chain volume, thereby forming a protrusion in the CH3 region of the first polypeptide chain that can be left in a cavity in the CH3 region of the second polypeptide chain, while one amino acid residue in the CH3 region of the second polypeptide chain in the Fc region substituted with an amino acid residue having a smaller side chain volume, thereby forming a cavity in the CH3 region of the second polypeptide chain that can be left in the protrusion in the CH3 region of the first polypeptide chain. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0066] The term "linker" refers to any tool for linking two different functional units (e.g., antigen-binding fragments). Types of linkers include, but are not limited to, chemical linkers and polypeptide linkers. The sequence of the polypeptide linker is not limited. The polypeptide linker is preferably non-immunogenic and flexible, for example, containing serine and glycine sequences. Depending on the specific structure, the linker can be long or short.

[0067] According to the invention, the linker connecting the different functional units preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker has the amino acid sequence (G4S) n or (G4S) nPreferably, the linker linking the VH and VL domains to form the VH-VL or VL-VH scFv domain comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker comprises the amino acid sequence (G4S) n or (G4S) n A, where n is any integer from 1 to 10, and preferably comprises the amino acid sequence (G4S)3 or (G4S).

[0068] The term "antibody" as used herein is used in the broadest sense and includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

[0069] The term "monoclonal antibody" (also called "monospecific antibody") refers to an antibody that is substantially homogeneous from a single cell clone and directed only to one particular antigen epitope. Monoclonal antibodies can be produced by a variety of techniques known in the art, including hybridization tumor techniques, recombinant techniques, phage display techniques, transgenic animals, synthetic techniques, or a combination of the above techniques.

[0070] It is particularly noted that the CDR and FR divisions of the variable regions of the antibodies and bispecific antigen-binding molecules according to the present invention shall be determined based on the Kabat definition. In addition, other naming and numbering systems, such as Chothia, IMGT, AHo, etc., are known to those skilled in the art. Thus, both humanized antibodies based on the sequences of the monospecific antibodies according to the present invention and comprising one or more CDRs from any naming system are expressly included within the scope of the present invention.

[0071] The term "humanized antibody" refers to an antibody in which all or some of the amino acids other than the CDRs of a non-human antibody (e.g., a mouse antibody) are replaced with the corresponding amino acids from a human immunoglobulin. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as they do not remove the ability of the antibody to bind to a specific antigen. A "humanized" antibody maintains antigen specificity similar to that of the original antibody.

[0072] The term "chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a mouse antibody and whose constant region is derived from a human antibody.

[0073] The term "antibody fragment" includes at least a portion of a complete antibody. For example, as used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment that specifically binds to or reacts with a selected antigen or an immunogenic determining portion thereof in an immunoglobulin or antibody, or a fusion protein product further derived from this fragment, such as a single chain antibody, an extracellular binding region in a chimeric antigen receptor, and the like. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to, variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single domain antibodies, linear antibodies, single chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments.

[0074] The term "antigen" refers to a substance that is recognized and then specifically bound by an antibody or an antibody-binding fragment. In a broad sense, an antigen includes any immunogenic fragment or determinant in a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, a complete extracellular domain (ECD) or a protein. Any peptide, protein, glycoprotein, polysaccharide, lipid, portion thereof, and combinations thereof can constitute an antigen. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, and the like. An "antigen" may also refer to a molecule that elicits an immune response. Any form of antigen or any cell or preparation containing the antigen can generate antibodies with specificity for an antigenic determinant. Antigens include isolated full-length proteins, cell surface proteins (e.g., immunization with a cell expressing at least a portion of the antigen on its surface), soluble proteins (e.g., immunization with only the ECD portion of the protein), or protein constructs (e.g., Fc antigens). The antigens can be generated in genetically modified cells. Any of the above antigens may be used alone or in combination with one or more immunogenicity enhancing adjuvants known in the art. The DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA) and may encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector may be used to transduce cells expressing the antigen, including, but not limited to, adenoviral vectors, slow viral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0075] The terms "epitope" and "antigenic determinant" refer to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from adjacent amino acids are usually retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are usually lost after treatment with denaturing solvents. Epitopes usually exist in a unique spatial conformation and contain at least 3-15 amino acids. Methods for determining the epitope that binds to a defined antibody are well known in the art and include immunoblotting, immunoprecipitation detection analysis, and the like. Methods for determining the spatial conformation of an epitope include techniques in the art, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and the like.

[0076] The term "bispecific" means that an antigen-binding molecule can specifically bind to two different antigenic determinants. The term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds to an antigenic determinant. Exemplary antigen-binding molecules include immunoglobulins and their derivatives, such as fragments. The term "bispecific antigen-binding molecule" refers to a binding molecule (e.g., a molecule comprising an antibody or an antibody fragment) that has specificity for two different antigens (or epitopes), preferably a bispecific antibody.

[0077] The term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, including, for example, surface plasmon resonance (SPR) technology (analysis performed on a BIAcore instrument).

[0078] When preparing antibodies, binding molecules, bispecific binding molecules, or multispecific binding molecules using the variable regions of the present invention, the constant region is not particularly limited, and any constant region known to those skilled in the art or a constant region obtained by oneself can be used. Furthermore, amino acid mutations (e.g., mutations that improve or reduce binding to Fcγ receptors or FcRn) can also be introduced into the constant region portion.

[0079] The method for obtaining the binding molecule, antigen-binding fragment, antibody, bispecific binding molecule or multispecific binding molecule according to the present invention is not particularly limited and can be obtained by any method, for example, the antibody test technical manual of Reizei Port (chapters 5 to 8 and chapter 15). The binding molecule, antigen-binding fragment, antibody, bispecific binding molecule or multispecific binding molecule according to the present invention can be prepared and purified by a conventional method. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended conventional technique, a mammalian expression system will cause glycosylation of the antibody, especially at the highly conserved N-terminus of the Fc region. A stable clone is obtained by expressing an antibody that specifically binds to a human-derived antigen. The positive clone is cultured in a serum-free medium in a bioreactor to produce the antibody. The culture solution into which the antibody is secreted can be purified and collected by a conventional technique. The antibody can be filtered and concentrated by a conventional method. Soluble impurities and polymers can likewise be removed by conventional techniques such as molecular sieving, ion exchange, and the like.

[0080] The term "antibody drug conjugate" (ADC) refers to an antibody to which a therapeutically active agent or active drug component (API) is covalently attached. Furthermore, the therapeutically active agent or active drug component (API) can be targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active agent or active drug component can be a cytotoxin capable of killing ADC target cells, preferably malignant or cancer cells. Covalent attachment of the therapeutically active agent, active drug component or cytotoxin can be performed non-site-specifically by standard chemical linkers with payloads attached to lysine or cysteine ​​residues, or preferably, the attachment can be performed site-specifically, allowing full control of the binding site and the drug-antibody ratio of the resulting ADC.

[0081] The terms "amino acid substitution," "substitution," or "substitution" refer to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid.

[0082] The term "affinity" or "binding affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a particular antibody-antigen interaction. Measurement of binding affinity can be performed by various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0083] The term "biological activity" refers to the ability of an antibody to effect a measurable biological response by binding to an antigen, said biological response being measurable in vitro or in vivo.

[0084] The pharmaceutical composition according to the present invention can be formulated by mixing with an appropriate vector, medium, etc. that is inactive against the pharmaceutical composition and pharma- ceutically acceptable, as necessary. Examples of the pharmaceutical composition include physiological saline, sterilized water, excipients, stabilizers, antioxidants (e.g., ascorbic acid, etc.), buffers, preservatives, surfactants, chelating agents (e.g., EDTA, etc.), adhesives, etc. In addition, the pharmaceutical composition may contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulins, amino acids such as glycine, glutamine, asparagine, glutamic acid, asparagine, methionine, arginine, and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols such as mannitol and sorbitol. When prepared in the form of an injectable aqueous solution, examples of the solution include isotonic solutions containing physiological saline, glucose and other excipients, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and may be used in combination with suitable cosolvents, such as alcohol (ethanol, etc.), polyols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, polysorbate 20, poloxamer 188, HCO-50), etc. Also, by mixing hyaluronidase into the preparation, a larger volume of solution can be administered subcutaneously.

[0085] In the present invention, the binding molecules or antigen-binding fragments can be used in combination with other drugs, and the active ingredients can be mixed to form a single dosage unit or each can be used separately as a dosage unit.

[0086] The term "effective amount" refers to the amount of the drug formulation of the antibody or fragment according to the present invention that, after administration to the patient in a single or multiple doses, produces the desired effect in the treated patient. The effective amount can be easily determined by the attending physician, who is skilled in the art, taking into account various factors such as ethnic differences, body weight, age, health condition, the specific disease involved, the severity of the disease, the individual response of the patient, the specific antibody administered, the mode of administration, the bioavailability characteristics of the administered formulation, the selected administration scheme, and the use of any concomitant therapy.

[0087] As used herein, the term "individual" or "subject" refers to any animal, including, for example, a mammal or marsupial. Individuals according to the present invention include, but are not limited to, humans, non-human primates (e.g., cynomolgus or Ganges or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any type of poultry.

[0088] As used herein, the term "disease", "pathology" or "disturbance" refers to any change or disorder that impairs or impairs the normal function of a cell, tissue or organ. For example, the above-mentioned "disease" includes, but is not limited to, tumor, pathogen infection, autoimmune disease, T cell dysfunction disease, or defective immune tolerance (e.g., transplant rejection).

[0089] As used herein, the term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Tumor growth is usually uncontrolled and progressive, without inducing or suppressing the proliferation of normal cells.

[0090] The term "treatment" as used herein refers to clinical intervention in the process of attempting to modify an individual or treat a disease caused by a cell, and may be preventive or intervene in the clinical pathology process. Therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, relief of any direct or indirect pathological consequences of the disease, prevention of metastasis, delay in the rate of disease progression, amelioration or alleviation of the pathological condition, alleviation or improvement of prognosis, etc. EXAMPLES

[0091] The present invention will be further described below with specific examples. It should be understood that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. In the following examples, test methods for which no specific conditions are specified will usually be carried out according to normal conditions or conditions recommended by the manufacturer.

[0092] Example 1. Design of anti-CD3-DLL3 bispecific antibodies and their sequences The bispecific antigen-binding molecule (hereinafter abbreviated as bispecific antibody) constructed in the examples is formed by linking the heavy chain of the Fab fragment of the anti-DLL3 nanoantibody or the anti-DLL3 full-length antibody to the binding domain of the human T-cell receptor subunit CD3ε via a flexible linker. Here, the anti-DLL3 nanoantibody is hDLL3-3-1-NA, the sequence of which is shown in SEQ ID NO:5. The anti-DLL3 full-length antibody is H2-39E2D11-NA, the heavy chain sequence of which is shown in SEQ ID NO:9, and the light chain sequence of which is shown in SEQ ID NO:10. The CD3ε binding domain is derived from the full-length antibody h160C9AA, the heavy chain sequence of which is shown in SEQ ID NO:19, and the light chain sequence of which is shown in SEQ ID NO:20. In order to reduce the ADCC activity of the antibody, both of the Fc fragments of the finally constructed bispecific antibodies have amino acid substitutions of L234A and L235A.

[0093] The single chain antibody scFv formed by linking the heavy chain variable region and the light chain variable region of h160C9AA via a flexible linker has the structure VH-(G4S)3-VL and its sequence is shown in SEQ ID NO: 23. The above scFv is further fused to the heavy chain of the Fab fragment of the DLL3 full-length antibody or the C-terminus of the DLL3 nanobody via a flexible linker.

[0094] When the scFv was fused to the C-terminus of the DLL3 nanobody, the bispecific antibody formed was named Bispecific Antibody 1 and contained two homologous chains, the sequence of the fused chains being shown in SEQ ID NO:1, a schematic of which can be seen in Figure 1A.

[0095] When the scFv is fused to the C-terminus of the heavy chain of the Fab fragment of the full-length DLL3 antibody, the bispecific antibody formed is named bispecific antibody 2 and contains one light chain (i.e., the third polypeptide) and two heterologous heavy chains (i.e., the first polypeptide and the second polypeptide). Of the two heterologous heavy chains, the heavy chain containing the scFv is designed to have a "knob" structure (named the "knob" structure heavy chain of bispecific antibody 2) and contains amino acid substitutions at two sites, S354C and T366W. Of the two heterologous heavy chains, the heavy chain not containing the scFv is designed to have a "hole" structure (named the "hole" structure heavy chain of bispecific antibody 2) and contains amino acid substitutions at four sites, Y349C, T366S, L368A, and Y407V. In order to facilitate the purification of the bispecific antibody, it is also necessary to make a substitution of H435R in the "hole" structure heavy chain. The sequence of the engineered "knob" heavy chain is shown in SEQ ID NO:2, the sequence of the "hole" heavy chain is shown in SEQ ID NO:3, and the sequence of the light chain is shown in SEQ ID NO:4 (designated Bispecific Antibody 2 Light Chain), a schematic of which can be seen in FIG. 1B.

[0096] The structures and related molecular sequences of Bispecific Antibody 1 and Bispecific Antibody 2 are summarized in Tables 1 and 2, respectively.

[0097] Table 1: Structure of bispecific antibodies [Table 1]

[0098] Table 2: Amino acid sequences of bispecific antibodies [Table 2-1] [Table 2-2] [Table 2-3]

[0099] Example 2. Construction of anti-CD3-DLL3 bispecific antibody and its instantaneous transfection expression in eukaryotic cells The gene fragments of the bispecific antibody molecules described above were each cloned into a PTT5 expression vector to prepare expression plasmids at the transfection level.

[0100] Expi293F in serum-free medium TM Cells (Thermo Fisher Scientific) were cultured, and the cells were seeded in shake flasks (purchased from Corning Inc.) and cultured on a shaker table at 37°C and 8% CO2. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in an appropriate ratio and added to a shake flask for cell culture. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove the cell chip, and affinity purified by Protein A column. The column was washed with PBS until the A280 reading returned to the baseline. The target protein was eluted with an acidic eluent of pH 3.0 to pH 3.5 and neutralized with 1M Tris-HCl (pH 8.0 to 9.0). After the eluted sample was appropriately concentrated, it was further purified by gel chromatography Superdex200 (GE) equilibrated with PBS to remove aggregates, and the monomer peak was collected and exchanged with PBS for preparation. The final purified antibody was subjected to SDS-PAGE, HPLC purity analysis and A280 concentration measurement.

[0101] Example 3. Affinity measurement study of anti-CD3-DLL3 bispecific antibodies A. Affinity determination of anti-CD3-DLL3 bispecific antibodies to hDLL3 and hCD3 expressing cells The binding of the anti-CD3-DLL3 bispecific antibody to SHP-77 cells expressing hDLL3 and T lymphocytes (Jurkat) naturally expressing hCD3 was measured by FACS.

[0102] SHP-77 (ATCC, CRL-2195) and Jurkat cells (ATCC, TIB-152) were cultured, and the medium for both SHP-77 and Jurkat cells was RPMI 1640 + 10% FBS, and they were cultured in a T75 cell culture bottle at 37 °C and 5% CO2 in a culture box. When using the cells, SHP-77 cells were washed with sterile DPBS and digested with 0.25% trypsin EDTA for about 5 minutes, then terminated with complete medium and placed in a 50 mL centrifuge tube. Jurkat cells were directly placed in a 50 mL centrifuge tube without digestion.

[0103] For SHP-77 and Jurkat, the cells were centrifuged at 1000 rpm at room temperature for 5 minutes, the supernatant was discarded, and the cells were resuspended in 100 μL of 1% BSA (in PBS). The cells were counted and the cell density was adjusted to 1E6 / mL. The cells were placed in a 96-well round-bottom culture plate (Corning, catalog number 3799), centrifuged at 1500 rpm at 4°C for 5 minutes, the supernatant was discarded, and the plate was left at 4°C to prepare. The antibody to be measured and the negative control IgG1 LALA (purchased from Hyakuei Seibutsu, catalog number B109802) were diluted with 1% BSA (in PBS) to a starting concentration of 100 nM, and diluted 10-fold to the following 7 concentrations. The cells were resuspended in the diluted antibody, 100 μL / well, and cultured at 4°C for 1 hour. The cells were centrifuged at 1500 rpm at 4°C for 5 minutes, and the supernatant was discarded. The cells were washed by resuspension in 160 μL of 1% BSA in PBS, centrifuged at 1500 rpm at 4°C for 5 min, and the supernatant was discarded. The double antibody (goat anti human IgG Fc PE) was diluted 1:200 in 1% BSA in PBS according to the manufacturer's instructions, and the cells were resuspended in the diluted double antibody, 100 μL / well, and incubated at 4°C for 0.5 h. The cells were centrifuged at 1500 rpm at 4°C for 5 min, and the supernatant was discarded. The cells were washed by resuspension in 160 μL of 1% BSA in PBS, centrifuged at 1500 rpm at 4°C for 5 min, and the supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA in PBS, filtered through 300 mesh gauze, and the mean fluorescence intensity of the PE channel was measured using a flow cytometer.

[0104] The FCS file was extracted from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel for each sample was analyzed using flowjo software. The mean fluorescence intensity obtained by the analysis was imported into Graphpad to calculate the half-binding concentration (EC 50 and the results are shown in Table 3, Figure 2 (SHP-77 cells) and Figure 3 (Jurkat cells). Both of the two bispecific antibody molecules had weaker binding ability to the Jurkat cell line than to the SHP-77 cell line, and furthermore, two bispecific antibody molecules had weaker binding ability to both of these cells than one bispecific antibody molecule.

[0105] Table 3: Affinity of each bispecific antibody for hDLL3 (SHP-77 cell line) and hCD3 (Jurkat cell line) [Table 3]

[0106] B. In vitro recombinant protein binding affinity and kinetics of anti-CD3-DLL3 bispecific antibodies The affinity and kinetic properties of the anti-CD3-DLL3 bispecific antibody molecule to human / cynomolgus DLL3 and human / cynomolgus CD3 were analyzed by a Biacore 8K instrument.

[0107] [Table 3-2]

[0108] After each test, the chip was regenerated by washing with 3M MgCl2 solution at a flow rate of 30 μL / min for 30 s to remove the captured antibody together with the antigen. The raw data was analyzed by Biacore Insight Evaluation Software (3.0.12.15655) software and fitted with the (1:1) Langmuir model. The obtained test data on the affinity and kinetics of the bispecific antibodies are shown in Table 4.

[0109] Table 4: Binding affinity and kinetics of anti-CD3-DLL3 bispecific antibodies to human / cynomolgus DLL3 protein. [Table 4]

[0110] The affinity and kinetic properties of human CD3 (purchased from Acro, catalog number CDD-H52W1) / cynomolgus CD3 (purchased from Acro, catalog number CDD-C52W4) were measured by direct curing of human / cynomolgus CD3 molecules on a CM5 chip. After activating the CM5 chip with EDC and NHS, human / cynomolgus CD3 molecules were diluted to 1 μg / ml in acetate solution at pH=5, cured at a flow rate of 10 μL / min for 60 s, and then blocked with ethanolamine. Anti-CD3-DLL3 bispecific antibody molecules were diluted 2-fold to series concentrations (100 nM–0.39 nM) in HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer and bound for 90 s at a flow rate of 50 µL / min and dissociated for 360 s.

[0111] After each test, the chip was washed with 3M MgCl2 solution at a flow rate of 30 μL / min for 30 s to remove the anti-CD3-DLL3 bispecific antibody molecules and regenerate the chip. The raw data was analyzed by Biacore Insight Evaluation Software (3.0.12.15655) software and fitted with the (1:1) Langmuir model. The obtained test data on the affinity and kinetics of the bispecific antibody are shown in Table 5.

[0112] Table 5: Binding affinity and kinetics of anti-CD3-DLL3 bispecific antibodies to human / cynomolgus CD3 protein. [Table 5]

[0113] The results of the study demonstrated that both bispecific antibody molecules bound to human / cynomolgus DLL3 and human / cynomolgus CD3, with one bispecific antibody molecule having a higher or equal affinity for the CD3 end than the DLL3 end, and with two bispecific antibody molecules having a higher affinity for the DLL3 end than the CD3 end.

[0114] Example 4. In vitro functional testing of anti-CD3-DLL3 bispecific antibodies A. T cell-mediated cytotoxicity test (TDCC) using SHP-77 as target cells SHP-77 cells were cultured in RPMI 1640 + 10% FBS medium in a T75 cell culture bottle at 37℃, 5% CO2 incubator. When using the cells, the cells were washed with sterile DPBS, digested with 0.25% trypsin EDTA for about 5 minutes, and terminated with complete medium. They were placed in a 50 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were resuspended in complete medium, counted, and the cell density was adjusted to 5E4 / mL.

[0115] Target cells were plated in black, clear bottom and opaque wall 96-well plates from Greiner (cat. no. 655090) at 100 μL / well and cultured overnight at 37° C., 5% CO2. CD3 T cells were isolated from fresh PBMCs using a T cell negative selection kit (StemCell, cat. no. 17951). + T cells were sorted, cells were counted, and cell density was adjusted to 5E5 / mL with RPMI 1640+10% FBS. Effector cells were plated in 96-well plates at 100 μL / well and cultured at 37°C, 5% CO2.

[0116] Antibodies were diluted in complete medium to a starting concentration of 110 nM (11X) and diluted 5-fold or less. The diluted antibodies were added to the cell culture plate at 20 μL / well, so the starting final concentration was 10 nM. The plates were incubated at 37°C and 5% CO2 for 48 hours. The lymphocytes in the plate were aspirated with a pipette tip, and 100 μL of CTG reagent was added per well, incubated for 10 minutes in the dark with rocking at 300 rpm, and the chemiluminescence value was read on the Envision.

[0117] The cell killing rate due to the TDCC effect is calculated using the following formula: % Cell Killed = 1 - (Sample Well Reading - T Cell Well Reading) / (Maximum Signal - T Cell Well Reading) where the reading for the T cell well is the reading for the well to which only T cells and no target cells SHP-77 were added.

[0118] EC by GraphPad software 50 The maximum killing was calculated, and the results are shown in Table 6 and Figure 4. Both of the two bispecific antibody molecules could reach 100% maximum killing ability against SHP-77 cells, and the half effective concentration of one bispecific antibody molecule was lower than that of two bispecific antibody molecules.

[0119] B. T cell-mediated cytotoxicity test (TDCC) using NCI-H82 as target cells NCI-H82 cells (ATCC, HTB-175) were cultured in RPMI 1640 + 10% FBS medium in a T75 cell culture flask at 37°C in a 5% CO2 culture chamber. The cells were centrifuged at 1000 rpm for 5 minutes and resuspended in DPBS. The cells were counted, adjusted to a cell density of 1E6 / ml, and stained with 30 nM Cell Trace Far Red Cell Proliferation Kit in a 37°C, 5% CO2 culture chamber for 20 minutes. An equal volume of complete medium was added, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed once with DPBS.

[0120] Target cells were resuspended in complete medium, counted, and adjusted to a cell density of 2E5 / mL before plating in round-bottom culture plates (Corning, Cat. No. 3799) at 50 μL / well. CD3 T cells were isolated from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951). + T cells were sorted, cells were counted, and the cell density was adjusted to 2E6 / mL with RPMI 1640 + 10% FBS. Effector cells were plated in 96-well plates at 100 μL / well and cultured at 37°C, 5% CO2.

[0121] Antibody dilution and sample addition: Antibodies were diluted in complete medium to a starting concentration of 110 nM (11X) and diluted 5-fold or less. The diluted antibodies were added to the cell culture plate at 10 μL / well, so the starting final concentration was 10 nM. The cells were incubated at 37°C, 5% CO2 for 48 hours. 10 μL of the viability dye PI was added per well to a final concentration of 4 μg / mL PI, mixed uniformly, and stained at room temperature for 10 minutes. The cells were filtered through 300 mesh gauze and the percentage of APC channel (stained target cells) and PE channel (stained live or dead cells) was measured by flow cytometer.

[0122] The cell killing rate due to the TDCC effect is calculated using the following formula: Target cell death (%) = (Far Red+PI+ cells) / [(Far Red+PI+ cells)+(Far Red+PI- cells)] Cell killing (%) = target cell death (%) - target cell spontaneous death (%) where target cell spontaneous death (%) is the number of dead cells in wells where only target cells (NCI-H82) but no T cells were added.

[0123] EC by GraphPad software 50The DLL3 expression level in NCI-H82 cells was only 1 / 3 that in SHP-77 cells according to literature reports, so the maximum killing power of the two bispecific antibody molecules to NCI-H82 cells was much weaker than that to SHP-77 cells, both of which were about 15%, and at the half effective concentration, bispecific antibody 1 was lower than bispecific antibody 2.

[0124] Table 6: Results of T cell-mediated cytotoxicity test (TDCC) of anti-CD3-DLL3 bispecific antibodies [Table 6]

[0125] C. Cytokine Release Assay SHP-77 cells were cultured in RPMI 1640 + 10% FBS medium in T75 cell culture bottles at 37°C in a 5% CO2 culture box. When using the cells, the cells were washed with sterile DPBS, digested with 0.25% trypsin EDTA for about 5 minutes, and terminated with complete medium. They were placed in a 50 mL centrifuge tube, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended and counted in complete medium, and the cell density was adjusted to 1E5 / mL. SHP-77 cells were plated in a 96-well plate (Corning, Cat. No. 3799) at 100 μL / well and cultured at 37°C in 5% CO2.

[0126] Fresh PBMCs were purchased, cells were counted, and the cell density was adjusted to 2E6 / mL with RPMI 1640 + 10% FBS. Effector cells were plated in 96-well plates at 100 μL / well and cultured at 37°C, 5% CO2.

[0127] Antibodies were diluted in complete medium to a starting concentration of 110 nM (11X) and diluted no more than 5-fold. The diluted antibodies were added to cell culture plates at 20 μL / well, so the starting final concentration was 10 nM. Incubation was carried out for 48 hours at 37°C, 5% CO2.

[0128] The culture plate was centrifuged at 400 g for 10 minutes, and 80 μL of the supernatant was removed and frozen at −80° C. for storage.

[0129] Cytokine standards were diluted according to the instructions of the CBA flow cytometry kit (BD, 551809), 2-fold dilution, with a maximum concentration of 5000 pg / mL and a minimum concentration of 20 pg / mL. The beads were homogenized by vortex mixing, and human Th1 / Th2 cytokine capture beads were mixed 1:1, and six types of cytokine beads were mixed in equal proportions and added to a 96-well plate (corning 3799) at 40 μL / well. Prepared standards or thawed samples were added to the 96-well plate at 40 μL / well. Human Th1 / Th2-II PE reagent was added to the 96-well plate, rocked at 1100 rpm in the dark for 5 minutes, and incubated at room temperature in the dark for 3 hours. The plate was washed with wash buffer at 160 μL / well and washed twice. Samples were filtered through 300 mesh gauze and fluorescence readings were measured in the APC and PE channels by flow cytometry.

[0130] The FCS files were derived from the instrument and imported into the FCAP software to analyze the amount of cytokine release for each sample. As a result, as shown in Figure 6 (Figures 6A-6E show the results of cytokine release of IFNγ, TNFα, IL-10, IL-6, and IL4 from one bispecific antibody molecule in the presence or absence of target cells, respectively, and Figures 6F-6J show the results of cytokine release of IFNγ, TNFα, IL-10, IL-6, and IL4 from two bispecific antibody molecules in the presence or absence of target cells, respectively), compared with one bispecific antibody molecule, two bispecific antibody molecules induced weaker levels of each type of cytokine release, and under the condition where only PBMCs were present, the cytokine release levels of both bispecific antibody molecules were weak or absent, indicating that there is an appropriate safety window for the co-culture conditions of PBMCs and SHP-77 tumor cells.

[0131] Example 5. Physical stability studies of anti-CD3-DLL3 bispecific antibodies The thermal stability of different antibodies was measured in PBS buffer at pH 7.4 by NanoDSF (differential fluorescence scanning technique). The sample concentration was about 1mg / ml, and the measurements were performed using Prometheus NT. Plex (nano DSF). Before the measurements, each sample was centrifuged at 10000g for 10 minutes. 40μl of sample was added per well of the sample plate (instrument loading volume was 10μl, and there was one duplicate well per sample). The scanning temperature started from 30℃ and ended at 95℃, and the scanning speed was 0.5℃ / min. The test results are shown in Table 7. Both bispecific antibody molecules exhibited good thermal stability, and the onset aggregation temperature (Tagg) of two bispecific antibody molecules was higher than that of one bispecific antibody molecule.

[0132] Table 7: NanoDSF measurement results of anti-CD3-DLL3 bispecific antibodies [Table 7]

[0133] Example 6. Pharmacokinetic studies of anti-CD3-DLL3 bispecific antibodies Two naive cynomolgus monkeys were prepared for the study and allowed to drink water ad libitum. The dose of bispecific antibody was 1 mg / kg, and the intravenous infusion was terminated at 30 min. Blood was collected at the following times: pre-dose, 5 min (during drip), 30 min (end of administration), 2 hr, 4 hr, 6 hr, 24 hr (1 d), 48 hr (2 d), 72 hr (3 d), 120 hr (5 d), 168 hr (7 d), 336 hr (14 d), 504 hr (21 d), and 672 hr (28 d). Whole blood samples were collected in polyethylene tubes without anticoagulant, left at room temperature for about 1 h, centrifuged at 6000 g and 25°C, and immediately two portions (PK sample and cytokine measurement sample) were placed separately on dry ice and transferred to a -80°C refrigerator for long-term storage.

[0134] The concentration of DLL3 / CD3 complete molecules in serum was measured by ELISA. Human DLL3 protein was used to coat a 96-well plate at a concentration of 1 μg / ml, 100 μL per well, and left at 4°C overnight. The plate was washed three times with 200 μL PBST per well, 300 μL of encapsulation reagent (5% powdered milk) was added, and incubated at 37°C for 1 hour. The plate was washed three times with 200 μL PBST per well, 100 μL of test sample was added, and incubated at 37°C for 1 hour. The plate was washed six times with 300 μL PBST per well, and 100 μL of Biotin-CD3e (1:10000), SA-HRP (1:10000), and TMB were added, and the plate was left in the dark for 10 min, and then 100 μL of stop solution was added to stop the color reaction. The concentration of DLL3 / CD3 intact molecules was quantitatively determined based on the color reaction.

[0135] The absorbance values ​​at a wavelength of 450 nm were measured by an M5 reader from MD, and the data were processed by softmax software.

[0136] The monkey serum concentration of the bispecific antibody Bispecific Antibody 2 was calculated by Phoenix Winnolin 8.2 software and the pharmacokinetic parameters were obtained, as shown in Table 8 below.

[0137] Table 8: Monkey serum concentrations and PK parameters of the bispecific antibody Bispecific Antibody 2 [Table 8]

[0138] In the above table, the concentration units are both μg / mL, and the BLQ was below the measurement limit.

[0139] The bispecific antibody 2 had a half-life of 99.9±21 h in monkeys, a Cmax of 25.3±1.2 μg / mL, and an AUC of 60.4±0.9 day*μg / mL. This bispecific antibody was stable in monkeys, had no obvious nonspecific binding, and had good pharmacokinetic properties.

[0140] Example 7. Cytokine measurements accompanying pharmacokinetic studies of anti-CD3-DLL3 bispecific antibodies The measurement kit was a Muti-Analyte Flow assay kit (Biolegend, Cat. No. 740391). Before use, 250 μL of buffer was added to the lyophilized NHP Th cytokine, mixed uniformly, and left at room temperature for 10 minutes. The standard was diluted 1:4 with the assay buffer in the kit, and the PK serum sample was diluted 1:4 with the assay buffer in the kit. 10 mL of LEGENDplex Assay Buffer was added to lyophilized Matrix B, which was dissolved at room temperature for 15 minutes.

[0141] 25 μL of Matrix B and 25 μL of standard were added to standard wells, and 25 μL of assay buffer and 25 μL of serum sample were added to sample wells. After homogenizing the microbeads by vortexing, the various assay beads were mixed 1:1 and diluted to working concentration with assay buffer, and 25 μL was added per well. The plate was sealed with parafilm and incubated for 2 hours in the dark with rocking at 800 rpm. The plate was centrifuged for 5 minutes at 250 g, the supernatant was discarded, and washed with 200 μL / well of wash buffer. 25 μL of the assay antibody was added to the plate, sealed with parafilm, and incubated for 1 hour at room temperature with rocking at 600 rpm in the dark, and 25 μL of SA-PE was added, sealed with parafilm, and incubated for 0.5 hours at room temperature with rocking at 600 rpm in the dark. The cells were centrifuged at 250 g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed with 200 μL / well of wash buffer. The samples were resuspended in 1% BSA (in PBS), filtered through 300 mesh gauze, and measured using a flow cytometer.

[0142] FCS files were derived and the samples were analyzed for cytokine release by LENEGDplex 8.0 software, and the results are shown in Table 9.

[0143] Table 9: Cytokine measurements associated with pharmacokinetic studies of anti-CD3-DLL3 bispecific antibodies [Table 9]

[0144] As is evident from the test results, various cytokine measurements accompanying the pharmacokinetic study of bispecific antibody 2 in cynomolgus monkeys showed low overall release levels, all below 300 pg / ml, indicating good safety.

[0145] Example 8. In vivo efficacy testing of anti-CD3-DLL3 bispecific antibodies Each test animal was kept in a separate thermo-hygrostat-ventilated box, the temperature of the breeding room was 20.0°C to 26.0°C, the humidity was 40% to 70%, and the day-night (light / dark) alternating period was 12h / 12h.

[0146] Human lung cancer cells SHP-77 were resuscitated, and SHP-77 cells in the logarithmic growth phase were harvested, the culture medium was removed, and the cells were washed twice with PBS before inoculation (the viability of SHP-77 cells before and after tumor formation was 98.8% and 96.6%, respectively). The inoculation volume was 5 × 10 6 The mixture was mixed with Matrigel at a ratio of 1:1 at 100 μL / animal and then subcutaneously inoculated into NCG mice at the right forelimb of the mouse (site number 3 in FIG. 7).

[0147] PBMCs were derived from peripheral blood of normal subjects. 48 hours after tumor cell inoculation, the cryopreservation solution was removed from PBMCs (donor number 5039), which were washed twice with PBS and then diluted with 0.8 × 10 7 A mouse model of human-derived immune cell reconstitution was established by transplanting the cells into the tail vein at 100μL / animal per day. The cell survival rates before and after inoculation were 97.7% and 95.8%, respectively.

[0148] On the third day after tumor inoculation, the mean tumor volume was 87.91 mm 3When the tumor volume reached 100 μg / kg, 35 mice were randomly divided into 5 groups based on the tumor volume: a human IgG1 (AA) negative control group (purchased from Hyakuei Seibutsu, catalog number B109802), a treatment group with 0.5 mg / kg of bispecific antibody 2, a treatment group with 0.1 mg / kg of bispecific antibody 2, a treatment group with 0.02 mg / kg of bispecific antibody 2, and a treatment group with 0.004 mg / kg of bispecific antibody 2, with 7 mice per group. The day of grouping was defined as D0, and intraperitoneal injection was started on D0, once every 3 days, for a total of 6 doses (see Table 10).

[0149] Table 10: Study Groups and Administration Methods [Table 10]

[0150] After administration, the animals' daily behavior was monitored daily for a total of 17 days. Throughout the study, tumor length and width were measured twice a week using calipers, and tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 ) was calculated. The relative tumor inhibition rate TGI (%) satisfies TGI% = (1-T / C) × 100%. T / C% was the relative tumor growth rate, i.e., the percentage value of tumor volume or tumor weight in the treatment group and the PBS control group at a certain time point. T and C were the tumor volume (TV) or tumor weight (TW) of the treatment group and the PBS control group at a certain time point, respectively. The test results of tumor volume, mouse body weight, tumor weight, etc. of animals in each group were expressed as mean ± standard error (Mean ± SEM). The significant difference between different treatment groups and the control group was detected by single sample T. Data were analyzed by SPSS. P less than 0.05 was considered to be significant. The plotting software was Graphpad prism.

[0151] Statistical analysis of the overall data on tumor volume and tumor weight at the efficacy endpoint (see Table 11, Figure 8) showed that, compared to the control group G1 (hIgG1 (AA)), the tumor inhibition rates for tumor volume and tumor weight in the three high dose groups of test drug bispecific antibody 2: G2, G3, and G4 (0.5 mg / kg, 0.1 mg / kg, and 0.02 mg / kg) all reached 90% or more, demonstrating a highly significant tumor growth inhibition effect (***P<0.001). Note that only in the lowest dose group G5, bispecific antibody 2 (0.004 mg / kg) did not show a significant tumor growth inhibition effect (TGI=17.31%, P>0.05).

[0152] In the current measurement system, bispecific antibody 2 (0.5mg / kg) in group G2, bispecific antibody 2 (0.1mg / kg) in group G3, and bispecific antibody 2 (0.02mg / kg) in group G4 all showed obvious growth inhibitory effects on mouse tumor volume and tumor weight compared to the hIgG1(AA) control group in group G1. Such tumor inhibitory effects were due to tumor killing by T cells.

[0153] Table 11: Tumor volume and tumor inhibition rate for each treatment group [Table 11]

[0154] The foregoing is merely illustrative of embodiments of the invention, and those of ordinary skill in the art will be able to recognize or ascertain, without more than ordinary experimentation, numerous equivalents to the specific compounds, materials and procedures, all of which are intended to be within the scope of the invention and covered by the claims.

Claims

1. (A) a first polypeptide comprising (i) an antigen-binding fragment (Fab) heavy chain domain specifically directed to a first antigen, (ii) a single chain antibody (scFv) domain capable of specifically binding a second antigen, and (iii) a first Fc domain; (B) a second polypeptide comprising a second Fc domain; and (C) a third polypeptide comprising a light chain domain of an antigen-binding fragment (Fab) specifically directed to the first antigen; a first binding site for a first antigen formed by the antigen-binding fragment (Fab) heavy chain domain and the antigen-binding fragment (Fab) light chain domain, a second binding site for a second antigen formed by the single chain antibody (scFv) domain, and the first Fc domain and the second Fc domain associated with each other.

2. Characterized by one or more of the following: (1) the first antigen is DLL3; Preferably, the heavy chain domain of the antigen-binding fragment (Fab) comprises an HCDR1 having the sequence shown in SEQ ID NO: 13, an HCDR2 having the sequence shown in SEQ ID NO: 14, and an HCDR3 having the sequence shown in SEQ ID NO: 15; and the light chain domain of the antigen-binding fragment (Fab) comprises an LCDR1 having the sequence shown in SEQ ID NO: 16, an LCDR2 having the sequence shown in SEQ ID NO: 17, and an LCDR3 having the sequence shown in SEQ ID NO: 18; More preferably, the heavy chain domain of said antigen-binding fragment (Fab) comprises a first heavy chain variable region having the sequence shown in SEQ ID NO: 11, and the light chain domain of said antigen-binding fragment (Fab) comprises a first light chain variable region having the sequence shown in SEQ ID NO: 12; More preferably, the heavy chain domain of the antigen-binding fragment (Fab) comprises the amino acid sequence shown in SEQ ID NO: 33, and the light chain domain of the antigen-binding fragment (Fab) comprises the amino acid sequence shown in SEQ ID NO: 4; (2) the single chain antibody (scFv) domain comprises a second heavy chain variable region and a second light chain variable region; Preferably, the second heavy chain variable region is linked to the second light chain variable region via a first linker or directly, wherein: the C-terminus of the second heavy chain variable region is fused to the N-terminus of the first linker and the C-terminus of the first linker is fused to the N-terminus of the second light chain variable region; or the C-terminus of the second light chain variable region is fused to the N-terminus of the first linker and the C-terminus of the first linker is fused to the N-terminus of the second heavy chain variable region; or the C-terminus of the second heavy chain variable region is fused to the N-terminus of the second light chain variable region; or the C-terminus of the second light chain variable region is fused to the N-terminus of the second heavy chain variable region; More preferably, the first linker has the amino acid sequence (G 4 S) n where n is any integer from 1 to 10; (3) the second antigen is CD3; Preferably it is CD3ε, Preferably, the single chain antibody (scFv) domain comprises an HCDR1 whose sequence is shown in SEQ ID NO: 24, an HCDR2 whose sequence is shown in SEQ ID NO: 25, an HCDR3 whose sequence is shown in SEQ ID NO: 26, an LCDR1 whose sequence is shown in SEQ ID NO: 27, an LCDR2 whose sequence is shown in SEQ ID NO: 28, and an LCDR3 whose sequence is shown in SEQ ID NO: 29; More preferably, the single chain antibody (scFv) domain comprises a second heavy chain variable region whose sequence is shown in SEQ ID NO: 21 and a second light chain variable region whose sequence is shown in SEQ ID NO: 22, More preferably, the single chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO: 23, (4) the first Fc domain comprises a first CH2 domain and a first CH3 domain of an immunoglobulin, the C-terminus of the first CH2 domain being fused to the N-terminus of the first CH3 domain; and the second Fc domain comprises a second CH2 domain and a second CH3 domain of an immunoglobulin, the C-terminus of the second CH2 domain being fused to the N-terminus of the second CH3 domain; Preferably, the first CH3 domain comprises a "knob" structure and the second CH3 domain comprises a "hole" structure; More preferably, said "knob" structure comprises the amino acid substitutions S354C and T366W, and said "hole" structure comprises the amino acid substitutions Y349C, T366S, L368A and Y407V; Preferably, the F C domain is derived from IgG1; Preferably, the single chain antibody (scFv) domain is linked to the first Fc domain via a second linker or directly, wherein: the C-terminus of the single chain antibody (scFv) domain is fused to the N-terminus of a second linker, and the C-terminus of the second linker is fused to the N-terminus of the first Fc domain; or the C-terminus of a single chain antibody (scFv) domain is fused to the N-terminus of a first Fc domain, More preferably, the second linker comprises the amino acid sequence EPKSS (SEQ ID NO:34), More preferably, the first Fc domain comprises the amino acid sequence shown in SEQ ID NO: 31 and the second Fc domain comprises the amino acid sequence shown in SEQ ID NO: 3; (5) The heavy chain domain of the antigen-binding fragment (Fab) comprises a first heavy chain variable region and a CH1 domain of an immunoglobulin, and the C-terminus of the first heavy chain variable region is fused to the N-terminus of the CH1 domain; and the light chain domain of the antigen-binding fragment (Fab) comprises a first light chain variable region and a light chain constant region of an immunoglobulin, and the C-terminus of the first light chain variable region is fused to the N-terminus of the light chain constant region; Preferably, the heavy chain domain of the antigen-binding fragment (Fab) is linked to the single chain antibody (scFv) domain via a third linker or directly, wherein: the C-terminus of the heavy chain domain of the antigen-binding fragment (Fab) is fused to the N-terminus of a third linker, and the C-terminus of the third linker is fused to the N-terminus of a single chain antibody (scFv) domain; or the C-terminus of the single chain antibody (scFv) domain is fused to the N-terminus of a third linker, and the C-terminus of the third linker is fused to the N-terminus of a heavy chain domain of an antigen-binding fragment (Fab); or the C-terminus of the heavy chain domain of an antigen-binding fragment (Fab) is fused to the N-terminus of a single chain antibody (scFv) domain; or The C-terminus of a single chain antibody (scFv) domain is fused to the N-terminus of the heavy chain domain of an antigen-binding fragment (Fab), More preferably, the third linker comprises the amino acid sequence (G 4 S) n where n is any integer from 1 to 10; (6) The first polypeptide has the structure a Fab heavy chain domain-third linker-scFv domain-second linker-first Fc domain, or a scFv domain-third linker-Fab heavy chain domain-second linker-first Fc domain, Preferably, the first polypeptide has the structure first heavy chain variable region-CH1-third linker-second heavy chain variable region-first linker-second light chain variable region-second linker-first CH2-first CH3; or first heavy chain variable region-CH1-third linker-second light chain variable region-first linker-second heavy chain variable region-second linker-first CH2-first CH3; or second heavy chain variable region-first linker-second light chain variable region-CH1-third linker-first heavy chain variable region-second linker-first CH2-first CH3; or a second light chain variable region-a first linker-a second heavy chain variable region-CH1-a third linker-a first heavy chain variable region-a second linker-a first CH2-a first CH3; The second polypeptide comprises a second CH2-second CH3 structure, and the third polypeptide comprises a first light chain variable region-light chain constant region structure; More preferably, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:2, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:3, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:4; (7) The bispecific antigen-binding molecule comprises one or more amino acid substitutions selected from the group consisting of: (i) L234A and L235A; (ii) H435R, preferably, the H435R substitution is on the second polypeptide; and / or (8) The epitope to which the bispecific antigen-binding molecule binds is (i) is the same as or overlaps with the epitope of interest of an antibody comprising an HCDR1 having the sequence shown in SEQ ID NO: 13, an HCDR2 having the sequence shown in SEQ ID NO: 14, an HCDR3 having the sequence shown in SEQ ID NO: 15, an LCDR1 having the sequence shown in SEQ ID NO: 16, an LCDR2 having the sequence shown in SEQ ID NO: 17, and an LCDR3 having the sequence shown in SEQ ID NO: 18, or (ii) the epitope of interest of an antibody comprising a first heavy chain variable region whose sequence is set forth in SEQ ID NO: 11 and a first light chain variable region whose sequence is set forth in SEQ ID NO: 12 is the same as or overlaps with the epitope of interest of an antibody comprising the first heavy chain variable region whose sequence is set forth in SEQ ID NO:

12.

3. two homologous polypeptides, each polypeptide comprising: (i) a nanobody (VHH) domain capable of specifically binding to a first antigen; (ii) a single chain antibody (scFv) domain capable of specifically binding to a second antigen; and (iii) an Fc domain; A bispecific antigen-binding molecule, wherein the Fc domains of the two polypeptides are associated with each other.

4. Characterized by one or more of the following: (1) the first antigen is DLL3; Preferably, said Nanobody (VHH) domain comprises an HCDR1 whose sequence is shown in SEQ ID NO:6, an HCDR2 whose sequence is shown in SEQ ID NO:7, and an HCDR3 whose sequence is shown in SEQ ID NO:8, More preferably, said Nanobody (VHH) domain comprises the amino acid sequence shown in SEQ ID NO: 32, (2) the single chain antibody (scFv) domain comprises a heavy chain variable region and a light chain variable region; Preferably, the heavy chain variable region is linked to the light chain variable region via a first linker or directly, wherein: the C-terminus of the heavy chain variable region is fused to the N-terminus of a first linker and the C-terminus of the first linker is fused to the N-terminus of the light chain variable region; or the C-terminus of the light chain variable region is fused to the N-terminus of the first linker and the C-terminus of the first linker is fused to the N-terminus of the heavy chain variable region; or the C-terminus of the heavy chain variable region is fused to the N-terminus of the light chain variable region; or the C-terminus of the light chain variable region is fused to the N-terminus of the heavy chain variable region, More preferably, the first linker has the amino acid sequence (G 4 S) n where n is any integer from 1 to 10; (3) the second antigen is CD3, preferably CD3ε; Preferably, the single chain antibody (scFv) domain comprises an HCDR1 whose sequence is shown in SEQ ID NO: 24, an HCDR2 whose sequence is shown in SEQ ID NO: 25, an HCDR3 whose sequence is shown in SEQ ID NO: 26, an LCDR1 whose sequence is shown in SEQ ID NO: 27, an LCDR2 whose sequence is shown in SEQ ID NO: 28, and an LCDR3 whose sequence is shown in SEQ ID NO: 29; More preferably, said single chain antibody (scFv) domain comprises a heavy chain variable region having the sequence shown in SEQ ID NO: 21 and a light chain variable region having the sequence shown in SEQ ID NO: 22, More preferably, the single chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO: 23, (4) the Fc domain comprises an immunoglobulin CH2 domain and a CH3 domain, the C-terminus of the CH2 domain being fused to the N-terminus of the CH3 domain; Preferably, the Fc domain is from IgG1, Preferably, the Fc domain is linked to the single chain antibody (scFv) domain via a second linker or directly, wherein: the C-terminus of the single chain antibody (scFv) domain is fused to the N-terminus of a second linker, and the C-terminus of the second linker is fused to the N-terminus of the Fc domain; or The C-terminus of a single-chain antibody (scFv) domain is fused to the N-terminus of an Fc domain, More preferably, the second linker comprises the amino acid sequence EPKSS (SEQ ID NO: 34), More preferably, the Fc domain comprises the amino acid sequence shown in SEQ ID NO: 30, (5) The nanobody (VHH) domain is linked to the single chain antibody (scFv) domain via a third linker or directly, wherein: the C-terminus of the nanobody (VHH) domain is fused to the N-terminus of a third linker, and the C-terminus of the third linker is fused to the N-terminus of a single chain antibody (scFv) domain; or the C-terminus of the single chain antibody (scFv) domain is fused to the N-terminus of a third linker, and the C-terminus of the third linker is fused to the N-terminus of a nanobody (VHH) domain; or the C-terminus of a nanobody (VHH) domain is fused to the N-terminus of a single chain antibody (scFv) domain; or The C-terminus of a single chain antibody (scFv) domain is fused to the N-terminus of a nanobody (VHH) domain, More preferably, the third linker comprises the amino acid sequence (G 4 S) n where n is any integer from 1 to 10; (6) Each of the polypeptides comprises a structure of a VHH domain - a third linker - an scFv domain - a second linker - an Fc domain, or an scFv domain - a third linker - a VHH domain - a second linker - an Fc domain; Preferably, each of said polypeptides has the structure VHH domain-third linker-heavy chain variable region-first linker-light chain variable region-second linker-CH2-CH3, or VHH domain-third linker-light chain variable region-first linker-heavy chain variable region-second linker-CH2-CH3, or heavy chain variable region-first linker-light chain variable region-third linker-VHH domain-second linker-CH2-CH3; or a light chain variable region-first linker-heavy chain variable region-third linker-VHH domain-second linker-CH2-CH3; More preferably, each of said polypeptides comprises the amino acid sequence shown in SEQ ID NO:1, (7) The bispecific antigen-binding molecule comprises the amino acid substitutions L234A and L235A; and / or (8) The epitope to which the bispecific antigen-binding molecule binds is (i) is identical to or overlaps with the epitope of interest of a Nanobody comprising an HCDR1 having the sequence shown in SEQ ID NO:6, an HCDR2 having the sequence shown in SEQ ID NO:7, and an HCDR3 having the sequence shown in SEQ ID NO:8, or (ii) a bispecific antigen-binding molecule as described in claim 3, which is identical to or overlaps with the epitope of interest of a Nanobody comprising the amino acid sequence shown in SEQ ID NO:

32.

5. (A) a first binding moiety capable of specifically binding DLL3; and (B) a second binding moiety that specifically binds to a different antigen or epitope than the first binding moiety; The first binding moiety is (i) an HCDR1 whose sequence is set forth in SEQ ID NO:6, an HCDR2 whose sequence is set forth in SEQ ID NO:7, and an HCDR3 whose sequence is set forth in SEQ ID NO:8; or (ii) an HCDR1 having the sequence shown in SEQ ID NO: 13, an HCDR2 having the sequence shown in SEQ ID NO: 14, an HCDR3 having the sequence shown in SEQ ID NO: 15, an LCDR1 having the sequence shown in SEQ ID NO: 16, an LCDR2 having the sequence shown in SEQ ID NO: 17, and an LCDR3 having the sequence shown in SEQ ID NO: 18; or (iii) the amino acid sequence set forth in SEQ ID NO: 32; or (iv) A bispecific antigen-binding molecule comprising a first heavy chain variable region, the sequence of which is shown in SEQ ID NO: 11, and a first light chain variable region, the sequence of which is shown in SEQ ID NO:

12.

6. A nucleic acid encoding a bispecific antigen-binding molecule according to any one of the preceding claims, an expression vector comprising said nucleic acid or a host cell comprising said nucleic acid or said expression vector, Preferably, said host cell is a prokaryotic cell (preferably E. coli), or a eukaryotic cell (preferably a mammalian cell or a yeast, more preferably, said mammalian cell is a CHO cell, an Expi 293 or a HEK 293 cell).

7. A method for producing a bispecific antigen-binding molecule according to any one of claims 1 to 5, comprising the steps of: A method comprising culturing the host cell of claim 6 under suitable conditions.

8. An antibody-drug conjugate formed by conjugating the bispecific antigen-binding molecule according to any one of claims 1 to 5 with another biologically active molecule, Preferably, said other biologically active molecule is a small molecule drug, and preferably, said bispecific antigen-binding molecule is linked to the other biologically active molecule via a linker.

9. A drug composition comprising the bispecific antigen-binding molecule of any one of claims 1 to 5, the nucleic acid, expression vector or host cell of claim 6, or the antibody-drug conjugate of claim 8, Preferably, the pharmaceutical composition further comprises a pharma- ceutically acceptable vector, Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents.

10. Use of a bispecific antigen-binding molecule according to any one of claims 1 to 5, a nucleic acid, an expression vector or a host cell according to claim 6, or an antibody-drug conjugate according to claim 8 in the manufacture of a medicament for treating, alleviating and / or preventing a tumor, comprising: Preferably, the tumor is a DLL3-positive tumor, more preferably, the tumor is selected from the group consisting of small cell lung cancer, glioblastoma, neuroendocrine carcinoma, melanoma, pancreatic cancer, rectal cancer, and metastases of the above tumors.

11. 1. A method for inducing death of a DLL3-expressing cell, comprising: contacting the cell with the bispecific antigen-binding molecule of any one of claims 1 to 5, the nucleic acid, expression vector or host cell of claim 6, the antibody-drug conjugate of claim 8, or the drug composition of claim 9, wherein the DLL3-expressing cell is a tumor cell; Preferably, said tumor cells are cells of a tumor selected from the group consisting of small cell lung carcinoma, glioblastoma, neuroendocrine carcinoma, melanoma, pancreatic carcinoma, rectal carcinoma, and metastases of the above tumors.

12. 1. A method for treating a disease associated with DLL3 expression in a subject, comprising: Administering to a subject in need thereof a bispecific antigen-binding molecule according to any one of claims 1 to 5, a nucleic acid, an expression vector or a host cell according to claim 6, an antibody-drug conjugate according to claim 8, or a drug composition according to claim 9, Preferably, the disease is a tumor, more preferably, the tumor is small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer, and metastases of the above tumors; Preferably, the method further comprises administering to the subject an additional therapeutic agent.

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