Monoclonal antibody which specifically recognizes delta-like ligand 3, and application thereof

Abstract

The present invention relates to a DLL3 antibody, and an application thereof. Specifically, the present invention relates to an antibody or an antigen-binding fragment thereof which specifically binds to DLL3, an encoding nucleic acid thereof, a recombinant vector and a host cell, a preparation method, a pharmaceutical composition, and a use for treating diseases, for example, a use for treating a tumor. The present invention has important significance with respect to the development of DLL3-targeted treatment drugs and detection reagents.

Description

Monoclonal antibodies specifically recognizing Delta-like ligand 3 and their applications

[0001] The present invention claims priority to Chinese patent application No. 202311254160.4 filed on September 27, 2023, entitled “Monoclonal antibodies specifically recognizing Delta-like ligand 3 and their applications” and Chinese patent application No. 202311604853.1 filed on November 28, 2023, entitled “Monoclonal antibodies specifically recognizing Delta-like ligand 3 and their applications”, the entire contents of which including the appendix are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of tumor immunotherapy or diagnosis, and in particular to a monoclonal antibody that specifically recognizes Delta-like ligand 3 (DLL3) and applications thereof. Background Art

[0003] Delta-like ligand 3 (DLL3) is a member of the Notch ligand family. Two splice variants exist, with the primary splice variant sharing 91% human-monkey extracellular domain sequence identity and 84% human-mouse extracellular domain sequence identity. DLL3 is primarily distributed in the Golgi apparatus, with some localization to the cell membrane. Under physiological conditions, it primarily participates in somite formation during embryonic development. DLL3 knockout mice display defects in cranial or neuronal development, while DLL3 mutants exhibit defects such as vertebral rib hypoplasia. Unlike other Notch family ligands that activate signaling pathways, DLL3 can interact with DLL1 or Notch1 in cis or trans, inhibiting signaling pathway activation.

[0004] Neuroendocrine lung cancer accounts for 20% of all lung cancers, with small cell lung cancer accounting for approximately 14%. Most patients also develop hematogenous metastases, while only approximately one-third of patients with limited-stage disease have disease confined to the chest. Current standard first-line treatments for patients with extensive-stage small cell lung cancer include radiotherapy, carboplatin + etoposide + atezolizumab, carboplatin + etoposide + durvalumab, and cisplatin + etoposide + durvalumab. Untreated patients are highly sensitive to chemotherapy and radiotherapy, but due to a small number of residual cancer cells and insensitivity to treatment of cancer stem cells, most patients will relapse within a short period of time. The median progression-free survival (PFS) for patients treated with first-line therapy is 2-3 months, and the median overall survival (OS) is 8-13 months, with a 5-year survival rate of less than 5%. For patients with relapsed or refractory disease, there are currently few effective treatment options. Recommended second-line therapies for patients who relapse within 6 months include topotecan and rubicin, with other recommended therapies including pembrolizumab. For patients who relapse more than 6 months after relapse, maintenance therapy is recommended. Given the current treatment status, there is an urgent need to provide patients with new and effective treatments.

[0005] DLL3 is essentially absent in normal tissues. While mRNA transcription levels are high in the nervous system, pancreas, and testes, protein expression is low. Furthermore, in normal tissues, DLL3 is primarily localized to the cellular matrix. DLL3 expression is high in small cell lung cancer samples, distributed across the cell membrane and cellular matrix, with a trend of correlation with disease grade, though not significant. It is essentially absent in normal lung tissue and squamous cell lung carcinoma. DLL3 is also expressed in other neuroendocrine tumors, such as melanoma, multiforme glioma, and small cell bladder cancer. StemCentrx reports that DLL3 expression H-scores exceeding 100 are found in 65% of samples from patients with large cell neuroendocrine lung cancer, 72% of patients with untreated small cell lung cancer, and 85% of patients with relapsed or refractory small cell lung cancer. Abbvie reports that 83% of samples from patients with small cell lung cancer are DLL3-positive, with 32% of samples strongly positive (50% of cells positive). Therefore, DLL3 could be considered an effective target for small cell lung cancer or neuroendocrine cancer. ADC products, such as Rova-T, have provided strong proof-of-concept for the clinical efficacy of DLL3. However, due to their reliance on cell surface antigen expression and the choice of toxin, ADC products have failed to generate positive overall survival data in Phase III clinical trials. The present invention aims to obtain antibody sequences targeting DLL3 to generate more DLL3-targeted drug forms.

[0006] Summary of the Invention

[0007] The present invention provides a new antibody or antigen-binding fragment thereof that specifically binds to DLL3, regulates Notch signal transduction by inhibiting the function of DLL3, and thus provides a new tumor treatment method.

[0008] In a first aspect of the present invention, an antibody or antigen-binding fragment thereof that specifically binds to human Delta-Like Ligand 3 (DLL3) is provided, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein:

[0009] (1) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 are the LCDR1, LCDR2 and LCDR3 of the VL domain as shown in any one of SEQ ID NOs. 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 338-339, 343-346, 349-355, 359-361, 364-365, 374-375; and

[0010] (2) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are the HCDR1, HCDR2 and HCDR3 of the VH domain as shown in any one of SEQ ID NO.18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 340-342, 347, 356, 362-363, 366-370, 376-377.

[0011] In some specific embodiments, the LCDR1, LCDR2, and LCDR3 are identified according to the Kabat numbering system, the IMGT numbering system, and / or the Chothia numbering system.

[0012] In some specific embodiments, the HCDR1, HCDR2 and HCDR3 are determined according to the Kabat numbering system, the IMGT numbering system and / or the Chothia numbering system.

[0013] In some specific embodiments, the antibody or antigen-binding fragment thereof comprises the following LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3:

[0014] (1) According to the Kabat numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 50-145, 357-358, 371-373, 378, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 50-145;

[0015] (2) LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 146-241 according to the IMGT numbering system, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 146-241;

[0016] (3) According to the Chothia numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 242-337, or a combination of sequences having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 242-337.

[0017] In some specific embodiments, the antibody or antigen-binding fragment thereof, wherein:

[0018] (1) The light chain variable region sequence comprises the sequence shown in SEQ ID NO. 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 338-339, 343-346, 349-355, 359-361, 364-365, 374-375, or the sequence shown in SEQ ID NO. NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 338-339, 343-346, 349-355, 359-361, 364-365, 374-375 have 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; and,

[0019] (2) The heavy chain variable region sequence comprises a sequence as shown in SEQ ID NO. 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 340-342, 347, 356, 362-363, 366-370, 376-377, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to a sequence as shown in SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 340-342, 347, 356, 362-363, 366-370, 376-377.

[0020] In some specific embodiments, the antibody or antigen-binding fragment thereof is chimeric, humanized or fully human.

[0021] In some specific embodiments, the antibody or antigen-binding fragment thereof can bind to human, monkey or mouse Delta-Like Ligand 3 (DLL3).

[0022] In some specific embodiments, the antibody or antigen-binding fragment thereof may further comprise any constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; preferably, it comprises the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4 with a mutation;

[0023] Furthermore, the antibody or antigen-binding fragment thereof is also coupled with a therapeutic agent or a tracer;

[0024] Preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent, a cytotoxic agent or an immunomodulatory agent, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer;

[0025] More preferably, the cytotoxic agent is selected from the group consisting of alkaloids, methotrexate, doxorubicin, pyrrolobenzodiazepines, (pyrrolobenzodiazepine, PBD) or gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is a taxane.

[0026] In some specific embodiments, the antigen-binding fragment is selected from one or more of F(ab')2, Fab', Fab, Fv, scFv, nanobody or affibody.

[0027] A second aspect of the present invention provides a multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises the antibody or antigen-binding fragment thereof against DLL3, and an antigen-binding molecule that binds to an antigen other than DLL3, and / or an antigen-binding molecule that binds to a DLL3 epitope different from the DLL3 epitope bound by the antibody or antigen-binding fragment thereof.

[0028] Preferably, the other antigen-binding molecule is an antibody or an antigen-binding fragment thereof;

[0029] Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific;

[0030] Preferably, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

[0031] In a third aspect, the present invention provides an isolated nucleic acid fragment, wherein the nucleic acid fragment encodes any one of the above-mentioned antibodies or antigen-binding fragments thereof or the above-mentioned multispecific antigen-binding molecule.

[0032] The fourth aspect of the present invention provides a recombinant vector, wherein the recombinant vector comprises the above-mentioned isolated nucleic acid fragment.

[0033] In a fifth aspect of the present invention, a host cell is provided, wherein the host cell comprises the above-mentioned recombinant vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

[0034] In a sixth aspect, the present invention provides a method for preparing a product comprising the aforementioned DLL3 antibody, antigen-binding fragment thereof, or multispecific antigen-binding molecule, the method comprising culturing the host cells, and isolating the antibody, antigen-binding fragment, or multispecific antigen-binding molecule expressed by the cells.

[0035] In a seventh aspect, the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the DLL3 antibody or antigen-binding fragment thereof, the multispecific antigen-binding molecule, the nucleic acid fragment, the vector, or a product prepared according to the aforementioned method; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0036] In some specific embodiments, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0037] In an eighth aspect, the present invention provides a method for treating a tumor or cancer, comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof, the multispecific antigen-binding molecule, the nucleic acid fragment, the vector, the product prepared according to the aforementioned method, or the pharmaceutical composition; preferably, the tumor or cancer is selected from a solid tumor, a hematological tumor, or a cancer that infiltrates and expresses DLL3;

[0038] Preferably, the tumor or cancer is selected from at least one of small cell lung cancer, glioma, pancreatic cancer, melanoma, breast cancer, pituitary tumor, endometrioma, acute myeloid leukemia, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer and esophageal cancer.

[0039] In a ninth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, the multispecific antigen-binding molecule, the nucleic acid fragment, the vector, the product obtained according to the aforementioned method, or the pharmaceutical composition in the preparation of a medicament for treating tumors or cancer; preferably, the tumor or cancer is selected from a solid tumor, a hematological tumor, or a cancer that infiltrates and expresses DLL3;

[0040] Preferably, the tumor or cancer is selected from at least one of small cell lung cancer, glioma, pancreatic cancer, melanoma, breast cancer, pituitary tumor, endometrioma, acute myeloid leukemia, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer and esophageal cancer.

[0041] In a tenth aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof, the multispecific antigen-binding molecule, the nucleic acid fragment, the vector, the product prepared according to the aforementioned method, and the pharmaceutical composition.

[0042] In an eleventh aspect of the present invention, a method for detecting DLL3 expression in a biological sample is provided, the method comprising contacting the biological sample with an antibody or antigen-binding fragment thereof under conditions enabling formation of a complex between the antibody or antigen-binding fragment thereof and DLL3; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of DLL3 in the sample.

[0043] The twelfth aspect of the present invention provides use of the antibody or antigen-binding fragment thereof in preparing a DLL3 detection reagent.

[0044] Definitions and Explanations of Terms

[0045] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0046] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0047] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that other elements may be present in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0048] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0049] Delta-like ligand 3 (DLL3) is a single-pass transmembrane protein attached to the cell surface and belongs to the Notch ligand family. The human DLL3 gene is located on chromosome 19q13, and its open reading frame is approximately 1800 base pairs long. The human DLL3 protein is composed of 619 amino acids, and its complete structure includes a DSL domain, an intracellular domain, and six epidermal growth factor-like domains. The DSL gene sequence at the N-terminus of the extracellular domain is highly conserved within the ligand family and is essential for binding to the Notch receptor. The DLL3 intracellular domain is shorter, and its function remains unclear. Studies have found that DLL3 is highly expressed in SCLC and other neuroendocrine tumors, but is rarely expressed in normal tissues. Activation of DLL3 can exert either pro-oncogenic or tumor-suppressive effects. DLL3 is widely expressed in human cancers, including small cell lung cancer, glioma, pancreatic cancer, melanoma, breast cancer, pituitary tumors, endometriomas, acute myeloid leukemia, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer and esophageal cancer.

[0050] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, a high affinity antibody typically refers to an antibody with a specific affinity of about 10 -6 M or lower, 10 -7 M or lower, about 10 -8 M or lower, about 10 -9The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0051] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0052] The term "antibody" herein is used in the broadest sense and refers to a polypeptide or polypeptide combination comprising sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region, so as to be capable of specific binding to an antigen. "Antibody" herein encompasses various forms and structures, as long as they exhibit desired antigen binding activity. "Antibody" herein includes alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the antibody three-dimensional structure) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer, IP, Beste, G. & Skerra, A. (2003). Proteins, 53, 121-129.; Roque, ACA, Lowe, CR & Taipa, MA Antibodies and genetically engineered related molecules: production and purification. Biotechnol. Prog. 20, 639-654 (2004) (which is incorporated herein by reference). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0053] The term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. "Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Differences in the amino acid composition and order of arrangement of the constant region of immunoglobulins' heavy chains result in varying antigenicity. Consequently, "immunoglobulins" can be classified into five classes, or isotypes, herein: IgM, IgD, IgG, IgA, and IgE, corresponding to their corresponding heavy chains: μ, δ, γ, α, and ε. Within the same class, Ig can be further divided into subclasses based on differences in the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

[0054] The term "antibody" herein also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0055] The term "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

[0056] The term "heavy chain antibody" herein refers to an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0057] The term "multi-specificity" herein refers to the ability of an antibody or its antigen-binding fragment to bind to, for example, different antigens or at least two different epitopes on the same antigen. Therefore, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes that an antibody can bind to. For example, conventional monospecific IgG antibodies have two identical antigen-binding sites (paratopes) and can therefore only bind to the same epitope (rather than binding to different epitopes). In contrast, multispecific antibodies have at least two different types of paratopes / binding sites and can therefore bind to at least two different epitopes. As described herein, "complementarity determining region" refers to the antigen-binding site of an antibody. In addition, a single "specificity" can refer to one, two, three, or more than three identical complementary determining regions (the actual number of complementary determining regions / binding sites in a single antibody molecule is referred to as "valence") in a single antibody. For example, a single natural IgG antibody is monospecific and bivalent because it has two identical paratopes. Accordingly, a multispecific antibody comprises at least two (different) complementary determining regions / binding sites. Therefore, the term "multispecific antibody" refers to an antibody having more than one paratope and the ability to bind to two or more different epitopes. The term "multispecific antibody" particularly includes bispecific antibodies as defined above, but generally also includes proteins, e.g. antibodies that specifically bind three or more different epitopes, scaffolds, i.e. antibodies with three or more paratopes / binding sites.

[0058] The term "valent" herein refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0059] "Full-length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0060] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody, wherein the portion or partial variant has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabody, affibody, and single-domain antibody.

[0061] The term "chimeric antibody" herein refers to an antibody having variable sequences of an immunoglobulin from one source organism (such as rat, mouse, rabbit or alpaca) and constant regions of an immunoglobulin from a different organism (such as human). Methods for producing chimeric antibodies are known in the art. See, for example, US Pat. No. 5,807,715A; Morrison, 1985, Science 229 (4719): 1202-1207. Transfectomas Provide Novel Chimeric Antibodies; Gillies et al., J Immunol Methods. 1989 Dec 20; 125 (1-2): 191-202; herein incorporated by reference.

[0062] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity or the ability to enhance immune response, etc.

[0063] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0064] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007); which is incorporated herein by reference. A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0065] The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) found in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered as hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of standards (such as IMGT or KABAT) and outside the hypervariable region under different sets of standards (such as KABAT or IMGT). One or more of these positions can also be found in an extended hypervariable region. The present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region can be abbreviated as LCDR. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting, and in some cases forming part of, the sheet structure. The CDRs in each chain are held together tightly by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen-binding site of antibodies with the CDRs from other antibody chains (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference).

[0066] For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways. (The above contents are incorporated herein by reference).

[0067] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; which is incorporated herein by reference).

[0068] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883; which are incorporated herein by reference).

[0069] The term "IMGT numbering system" herein generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003; which is incorporated herein by reference.

[0070] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, and has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" can be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. A "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to that of a native antibody constant region, while the latter only includes "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from an Fc or CH3 domain.

[0071] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0072] The term "Fc region" herein is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. For example, the human IgG heavy chain Fc region may extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage, removing one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells through expression of a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may be present or absent. Typically, an IgG Fc region comprises an IgG CH2 and IgG CH3 domains, optionally further comprising a complete or partial hinge region, but omitting a CH1 domain. The "CH2 domain" of a human IgG Fc region typically extends from approximately amino acid residue position 231 to approximately amino acid residue position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues at the C-terminus of the CH2 domain in the Fc region (i.e., from approximately amino acid residue position 341 to approximately amino acid residue position 447 of IgG). The CH3 domain herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "knob" introduced into one chain and a corresponding "hole" introduced into the other chain; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). As described herein, such variant CH3 domains can be used to promote heterodimerization of two different antibody heavy chains.

[0073] Unless otherwise specified herein, 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, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991, which is expressly incorporated herein by reference.

[0074] The term "Fc variant" herein refers to changes in Fc structure or function caused by one or more amino acid substitutions, insertions, or deletions at appropriate sites on the Fc protein. "Inter-Fc variant interactions" refer to interactions between Fc variants engineered to form space-filling effects, electrostatic interactions, hydrogen bonding, hydrophobic interactions, and other interactions. These interactions contribute to the formation of stable heterodimeric proteins. Preferred mutational designs are "knob-into-hole" mutational designs.

[0075] The mutation design technology of Fc variants has been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include the "knob-into-hole" format proposed by Cater et al. (Protein Engineering vol. 9 no. 7 pp. 617-621, 1996); the Fc-containing heterodimer format formed by electrostatic steering by Amgen technicians (US 20100286374 A1); the heterodimer format (SEEDbodies) formed by IgG / Ig chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp. 1-8, 2010); the bispecific molecule formed by Genmab's DuoBody (Science, 2007. 317 (5844)) platform technology; and the heterodimer protein format formed by Xencor technicians using a combination of structural calculations and Fc amino acid mutations to integrate different modes of action (mAbs 3: 6, 546-557; November / December 2010). 2011); Suzhou Alphamab's charge network-based Fc modification method (CN201110459100.7) to produce a heterodimeric protein form; and other genetic engineering methods based on Fc amino acid changes or functional modification to achieve the formation of heterodimeric functional proteins. The knob / hole structure of the Fc variant fragments described herein refers to mutations in the two Fc fragments, which, after mutation, can combine in a "knob-into-hole" formation. Preferably, the "knob-into-hole" model of Cater et al. is used to perform site-specific mutations in the Fc region, so that the resulting first and second Fc variants can combine in a "knob-into-hole" formation to form a heterodimer. Selecting a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass is within the skill of those skilled in the art. Preferred are Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4, with the Fc region of human antibody IgG1 being more preferred. Randomly select one of the first Fc variant or the second Fc variant to make a knob mutation and the other to make a hole mutation. (The above contents are incorporated herein by reference).

[0076] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0077] 1) Alanine (A), serine (S), threonine (T);

[0078] 2) Aspartic acid (D), glutamic acid (E);

[0079] 3) Asparagine (N), glutamine (Q);

[0080] 4) Arginine (R), Lysine (K), Histidine (H);

[0081] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0082] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0083] The term "identity" herein can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences varies as the number of identical positions shared by the sequences changes, taking into account the number of spaces that need to be introduced and the length of each space for optimal comparison of the two sequences.

[0084] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and one that should be used unless otherwise specified) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4. (The aforementioned contents are incorporated herein by reference).

[0085] Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present disclosure can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0086] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules as described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as vectors for in vitro and / or in vivo, For example, in a host or patient, directly expressing the antibodies of the present disclosure. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP2101823B1), which are incorporated herein by reference.

[0087] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0088] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0089] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0090] The term "pharmaceutical composition" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0091] The term "pharmaceutically acceptable carrier" herein includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, which are known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition. Mack Printing Company, 1990, pp. 1289-1329). Except in cases where it is incompatible with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

[0092] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0093] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Exemplarily, a "subject" includes a mammal, such as a human, primate (e.g., monkey), or non-primate mammal, being treated for a disease or condition.

[0094] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0095] The term "cancer" herein refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. The terms "tumor" or "neoplasm" herein refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" as used herein are not mutually exclusive. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figures 1A and 1B show the binding activity of control antibodies to human or monkey DLL3-his proteins detected by enzyme-linked immunosorbent assay (ELISA), wherein Figure 1A shows the binding activity of the control antibody to human DLL3-his protein, and Figure 1B shows the binding activity of the control antibody to monkey DLL3-his protein;

[0097] Figure 2: Flow cytometry (FACS) analysis of the expression of human DLL3 protein in the CHO-K1 recombinant cell line.

[0098] Figure 3: Flow cytometry fluorescence sorting (FACS) assay to detect the expression of monkey DLL3 protein in CHO-K1 recombinant cell lines;

[0099] Figure 4: Flow cytometry (FACS) analysis of the expression of mouse DLL3 protein in the CHO-K1 recombinant cell line.

[0100] Figure 5A to Figure 5E: Enzyme-linked immunosorbent assay (ELISA) to detect the binding activity of chimeric antibodies to human DLL3 protein;

[0101] Figure 6A to Figure 6E: Flow cytometry fluorescence sorting experiment (FACS) detection of the binding activity of chimeric antibodies to SHP-77 cells;

[0102] Figure 7A to Figure 7E: Flow cytometric fluorescence sorting experiment (FACS) detection of the binding activity of chimeric antibodies to monkey DLL3 overexpressing cell line CHO K1-cyno DLL3 cells;

[0103] Figure 8A to Figure 8G: Enzyme-linked immunosorbent assay (ELISA) to detect the binding activity of humanized antibodies to human DLL3 protein;

[0104] Figures 9A to 9F: Flow cytometry (FACS) analysis of the binding activity of humanized antibodies to SHP-77 cells;

[0105] Figures 10A to 10F: Flow cytometry fluorescence sorting (FACS) assay to detect the binding activity of humanized antibodies to NCI-H82 cells;

[0106] Figures 11A to 11F: Flow cytometric fluorescence sorting (FACS) assay to detect the binding activity of humanized antibodies to human DLL3 recombinant cell lines;

[0107] Figures 12A to 12F: Flow cytometric fluorescence sorting (FACS) assay to detect the binding activity of humanized antibodies to monkey DLL3 recombinant cell lines;

[0108] Figure 13A to Figure 13F: Flow cytometric fluorescence sorting experiment (FACS) detection of the binding activity of humanized antibodies to mouse DLL3 recombinant cell lines. DETAILED DESCRIPTION

[0109] The present disclosure is further described below with reference to specific examples, and the advantages and features of the present disclosure will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0110] The embodiments of the present disclosure are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications and replacements shall fall within the scope of protection of the present disclosure.

[0111] Example 1: Control Antibody, Full-Length Antigen, Endogenous Cell Identification, and Preparation of Cell Lines Overexpressing DLL3

[0112] 1.1 Preparation of control antibodies

[0113] SC16.56, AMG757-4, 2G1, and BI3 are antibodies that recognize human DLL3 and have strong binding activity to human DLL3 protein. They can also specifically bind to recombinant or endogenous cell lines expressing DLL3.

[0114] The heavy chain variable region and light chain variable region sequences of SC16.56 were obtained according to patent US20200138969A1, the heavy chain variable region and light chain variable region sequences of AMG757-4 were obtained according to patent WO2017021349A1, the heavy chain variable region and light chain variable region sequences of 2G1 were obtained according to patent WO2020180591A1, and the heavy chain variable region and light chain variable region sequences of BI3 were obtained according to patent WO2019234220A1.

[0115] SC16.56 and BI3 express complete IgG. The VH and VL of AMG757-4 and 2G1, along with a human IgG1 Fc, are linked from N-terminus to C-terminus to form a scFv-human IgG1 Fc (scFv-hFc) format. The sequences of SC16.56 VH, SC16.56 VL, SC16.56 HC, SC16.56 LC, AMG757-4 scFv, AMG757-4 scFv-hFc, 2G1 scFv, 2G1 scFv-hFc, BI3 VH, BI3 VL, BI3 HC, and BI3 LC are shown in Table 1.

[0116] The nucleotide sequences were cloned into the pTT5 vector (purchased from U-Bio), and plasmids were prepared according to established standard molecular biology methods. For details, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press), which is incorporated herein by reference. The expression vector and transfection reagent PEI (purchased from Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed, and allowed to stand for 15 minutes. The cells were then added to Expi293F cells (Thermofisher, Catalog No. A14527) and cultured in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No.: F081918-001) and 6g / L glucose (Sigma, Catalog No.: G7528) were added. On the sixth day of transfection, the cell supernatant was collected. The culture supernatant was loaded onto a Protein A chromatography column (Protein A filler AT Protein A Diamond, purchased from Bogelon), washed with PBS phosphate buffer (pH 7.4) and then with 20mM PB, 1M NaCl, pH 7.2. Finally, the column was eluted with pH 3.4 citrate buffer. The Fc-tagged antibody eluted from the Protein A chromatography column was collected, neutralized with 1 / 10 column volume of 1M Tris, pH 8.0, and dialyzed against PBS at 4°C overnight. Protein aggregates were removed using molecular sieves (BXK16 / 26, purchased from Bogelon). The antibody concentration was determined using Nanodrop, the antibody purity was determined using HPLC-SEC, and the antibody endotoxin content was detected using an endotoxin detection kit. Finally, the control antibody was sterile filtered through 0.22 μm and then packaged and stored at -80°C.

[0117] Table 1 Amino acid sequences of control antibodies

[0118] 1.2 Preparation of full-length human DLL3 protein antigen and cynomolgus monkey DLL3 protein antigen

[0119] The amino acid sequence of the extracellular domain (ECD) of human DLL3 protein (UniProt ID: Q9NYJ7, the specific sequence is shown in SEQ ID NO. 13) and the amino acid sequence of the extracellular domain of cynomolgus monkey DLL3 protein (UniProt ID: A0A2K5WSR4, the specific sequence is shown in SEQ ID NO. 14) were coupled to a His tag and cloned into the pTT5 vector. The expression method was the same as the preparation process of the control antibody in Section 1.1 above. Cellular components were removed by centrifugation to obtain the culture supernatant. The protein in the cell culture supernatant was purified using a Ni affinity chromatography column (purchased from GE Healthcare). The column was first equilibrated with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.4). The clarified culture supernatant was then loaded onto the Ni affinity chromatography column at a flow rate of 5 mL / min. After loading, the Protein A column was washed with equilibration buffer at a volume of 3-5 times the column bed volume. Contaminants were eluted using PBS + 10 mM imidazole and PBS + 20 mM imidazole, respectively, and the elution was monitored using a nucleic acid protein detector (A280 UV absorption peak). The protein was then eluted and collected using PBS + 250 mM imidazole. The protein was dialyzed into PBS phosphate buffer using a dialysis card (purchased from Thermo Scientific) at 4°C. Sterile filtration was performed using a 0.22 μm filter (purchased from Millipore) and stored aseptically to obtain the purified protein. Simultaneously, human DLL3-His protein (purchased from Acro, catalog number: DL3-H52H4) and cynomolgus monkey DLL3-His protein (purchased from Acro, catalog number: DL3-C52H3) were purchased commercially.

[0120] Table 2 Human, monkey, and mouse DLL3 antigen sequences and EpCAM transmembrane and intracellular domain sequences

[0121] 1.3 Binding reaction detection of control antibodies with human DLL3-his protein and monkey DLL3-his protein

[0122] The antigen-antibody binding activity was detected using the ELISA method. The specific method is as follows: the antigen protein was diluted with PBS to a final concentration of 0.5 μg / mL, and then 50 μL was added to each well of a 96-well ELISA plate. Seal with plastic film and incubate overnight at 4°C. The next day, the plate was washed twice with PBST and blocked with blocking solution [PBS + 2% (w / w) BSA] at room temperature for 2 hours. The blocking solution was discarded and 50 μL of control antibody or negative control antibody with an initial concentration of 100 nM and 10-fold serial dilution was added to each well. After incubation at room temperature for 1 hour, the plate was washed 3 times with PBS. HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Merck, catalog number: AP113P) was added, incubated at room temperature for 1 hour, and then washed 5 times with PBS. 50 μL of TMB substrate was added to each well, incubated at room temperature for 10 minutes, and then 50 μL of stop solution (1.0 M HCl) was added to each well. OD450nm values ​​were read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The results are shown in Figures 1A-1B , showing that the control antibody has good binding activity to both human and monkey DLL3 proteins. hIgG1 served as a negative control.

[0123] 1.4 Preparation of CHO-K1 recombinant cell lines expressing human DLL3 protein

[0124] Nucleotide sequences encoding the amino acids of the extracellular region of human DLL3 (UniProt ID: Q9NYJ7, shown as SEQ ID NO. 13) and the transmembrane and intracellular regions of EpCAM (UniProt ID: P16422, shown as SEQ ID NO. 15; see Table 2 for detailed sequence information) were cloned into the pLVX-IRES-Puro vector (purchased from U-Bio, Catalog No. VT1464) for lentiviral packaging and subsequently infected CHO-K1 cells. 72 hours after infection, the cells were detected by flow cytometry using the known DLL3 antibody 2G1. Once transfected cells were confirmed to be expressing human DLL3 protein, 10 μg / ml Puromycin (Gibco, Catalog No. A1113802) was added for selection. After recovery, cells were subcloned using limiting dilution in 96-well culture plates and cultured at 37°C, 5% (v / v) CO₂. After approximately two weeks, a subset of single-clone cells were selected and expanded into 6-well plates. These expanded clones were then screened using flow cytometry using the 2G1 antibody. Cell lines with good growth, high fluorescence intensity, and monoclonal activity were selected for further expansion, retested by flow cytometry, and frozen in liquid nitrogen. These stably transfected cell lines expressing human DLL3 were obtained. The results are shown in Table 3 and Figure 2. The IgG isotype control was a human IgG1 control. Table 3 shows that a series of CHO-K1 monoclonal cell lines expressing human DLL3 have been generated. Figure 2 shows cell fluorescence intensity on the abscissa and cell number on the ordinate. The results indicate that cell lines #5, #7, and #11 all express high levels of human DLL3.

[0125] Table 3 FACS detection results of CHO-K1 recombinant cell line expressing human DLL3 protein

[0126] 1.5 Preparation of CHO-K1 recombinant cell line expressing monkey DLL3 protein

[0127] The nucleotide sequence encoding the extracellular amino acid region of monkey DLL3 (UniProt ID: A0A2K5WSR4, the specific sequence is shown in SEQ ID NO. 14) and the amino acid sequences of the transmembrane and intracellular regions of EpCAM (see Table 2) were cloned into the pLVX-IRES-Puro vector. Using the recombinant cell construction method described in Section 1.4 above, a stable cell line expressing monkey DLL3 was generated. The results are shown in Table 4 and Figure 3 , with a human IgG1 control as the IgG isotype control. Table 4 shows that a series of CHO-K1 monoclonal cell lines #1, #2, and #3 expressing monkey DLL3 were generated. Figure 3 shows cell fluorescence intensity on the abscissa and cell number on the ordinate. The results indicate that cell lines #1, #2, and #3 all highly express monkey DLL3.

[0128] Table 4 FACS detection results of CHO-K1 recombinant cell line expressing monkey DLL3 protein

[0129] 1.6 Preparation of CHO-K1 recombinant cell line expressing mouse DLL3 protein

[0130] The nucleotide sequence encoding the extracellular amino acid region of mouse DLL3 (UniProt ID: 088516, the specific sequence is shown in SEQ ID NO. 16) and the amino acid sequences of the transmembrane and intracellular regions of EpCAM (see Table 2) were cloned into the pLVX-IRES-Puro vector. Using the recombinant cell construction method described in Section 1.4, stable mouse DLL3-expressing cell lines #4 and #6 were generated. The results are shown in Table 5 and Figure 4 , with a human IgG1 control as the IgG isotype control. Table 5 demonstrates the generation of a series of mouse DLL3-positive CHO-K1 monoclonal cell lines. Figure 4 shows cell fluorescence intensity on the abscissa and cell number on the ordinate. The results demonstrate that both #4 and #6 are highly mouse DLL3-expressing cell lines.

[0131] Table 5 FACS detection results of CHO-K1 recombinant cell line expressing mouse DLL3 protein

[0132] Example 2 Preparation of hybridoma antibodies against DLL3

[0133] 2.1 Mouse immunization and serum titer detection

[0134] The animal immunization experiment was divided into nine groups. The experimental animals were 6-8 week old female Balb / c, SJL, MRL / lpr, and C57 BL / 6J mice (purchased from Shanghai Slake Co., Ltd.) housed in an SPF-grade environment. Orbital blood was collected from the mice before immunization to serve as negative serum.

[0135] For the first, second, and third immunization groups, human DLL3 antigen was emulsified with Alum (Thermo Fisher Scientific, Catalog No. 77161) and CpG (synthesized by Sangon, Catalog No. ODN1826) and injected intraperitoneally (0.1 ml). TiterMax (Sigma, Catalog No. T2684) and CpG were then emulsified and injected subcutaneously and at multiple sites in the paws, for a total of 50 μg of immunogen per mouse. Subsequently, booster immunizations were performed weekly, with antigen emulsified with Alum or TiterMax and injected subcutaneously at multiple sites, for a total of 25 μg of immunogen per mouse, for a total of six booster immunizations. Group 3 was then boosted three times with CHO cells expressing the DLL3 protein. TiterMax was emulsified with saline and injected intraperitoneally (0.1 ml), followed by intraperitoneal injection of the cell and CpG suspension, for a total of 1E+7 cells per mouse.

[0136] For the fourth and fifth immunization groups, for the initial immunization, 0.1 mL of TiterMax was emulsified with saline and injected intraperitoneally. Subsequently, a cell and CpG suspension was injected intraperitoneally, with 1E+7 cells injected per mouse. For the second immunization, 25 μg of the immunogen was emulsified with Alum and CpG and injected subcutaneously at multiple sites. Thereafter, booster immunizations were administered weekly, alternating with cell protein.

[0137] For the sixth to ninth immunization groups, for the initial immunization, 0.1 mL of TiterMax was emulsified with saline and injected intraperitoneally. Subsequently, a cell and CpG suspension was injected intraperitoneally, with 1E+7 cells injected per mouse. For the second immunization, 25 μg of the immunogen was emulsified with Alum and CpG and injected subcutaneously at multiple sites. Thereafter, booster immunizations were administered weekly, alternating with cell protein.

[0138] After the second, sixth, and seventh booster immunizations, blood was collected from the mouse orbitals in each of the immunization groups. The serum batches were designated TB1, TB2, and TB3, respectively. ELISA was used to test the binding titers of antibodies to the human DLL3-His protein in the mouse sera. The results showed that the sera from the immunized mice all showed varying degrees of binding to the immunogen, demonstrating an antigen-antibody reaction.

[0139] After booster immunization, mice with high antibody titers in serum were selected for spleen cell fusion. Three days before spleen cell fusion, booster immunization was performed and 50 μg / mouse antigen solution in normal saline was injected subcutaneously, in the plantar and intraperitoneally.

[0140] 2.2 Spleen cell fusion and hybridoma screening

[0141] ACK Lysing Buffer (purchased from Gibco, catalog number: A1049201) was added to lyse the red blood cells mixed in the spleen cells to obtain a spleen cell suspension. The cells were washed three times by centrifugation at 1000 rpm with DMEM (purchased from Gibco, catalog number: 12800017) basal medium, and then mixed with mouse myeloma cells SP2 / 0 (purchased from ATCC) at a ratio of 2:1 in terms of the number of living cells. Cell fusion was performed using the BTX ECM2001+ high-efficiency electric fusion method (see METHODS IN ENZYMOLOGY, VOL. 220). The fused cells were diluted into DMEM culture medium containing 20% ​​fetal bovine serum (purchased from ExCell Bio, catalog number: FND500) and 1×HAT (purchased from Sigma, catalog number: H0262-10VL). The percentages are weight percentages. Then, 2×10 4 200 μl was added to each well of a 96-well cell culture plate and placed in a 5% CO2, 37°C incubator. Percentages are by volume. After 14 days, the supernatant from the cell fusion plate was screened by ELISA. Clones positive for human DLL3 protein binding were expanded to 24-well plates and cultured in DMEM supplemented with 10% HT (Sigma, Catalog No. H0137-10VL) fetal bovine serum at 37°C, 5% CO2. After 3 days of culture, the culture fluid from the expanded 24-well plate was centrifuged and the supernatant collected. Binding activity was assayed using ELISA with monkey DLL3 protein and using FACS with CHO-K1 hDLL3. Based on the results of the 24-well plate screening, hybridoma cells that positively bound to the hybridoma culture supernatant were selected as qualified positive clones. Hybridomas from these positive wells were subcloned in 6-well plates using Medium D (STEMCELL, Catalog No. 03810). Seven days after subcloning, single colonies were picked and cultured in DMEM supplemented with 10% FBS and 1x HT at 37°C, 5% CO2 for two days. Initial screening was performed using ELISA, and single positive clones were selected and expanded into 24-well plates for further culture. Three days later, binding activity was assayed using ELISA with human and monkey DLL3 proteins, and by FACS with CHO-K1 hDLL3 cells, SHP-77 cells, or NCI-H82 cells. Binding specificity was also confirmed by FACS with negative CHO-K1 or FlpinCHO cells.

[0142] Based on the test results of the 24-well plate samples, the best clone was selected and cultured in DMEM medium containing 10% FBS at 37° C. and 5% CO 2 , and then frozen in liquid nitrogen to obtain the hybridoma cells of the present invention.

[0143] Example 3 Determination of amino acid sequences of light and heavy chain variable regions of hybridoma positive clones

[0144] Hybridoma cells were collected during the logarithmic growth phase, thoroughly lysed with Trizol (Invitrogen, Cat No. 15596-018), and stored at -80°C. The amino acid sequences of the light and heavy chain variable regions of positive hybridoma clones were determined. The variable region sequences are shown in Table 6. The sequencing results were analyzed using MOE software. An phylogenetic tree was constructed based on the amino acid sequences of the variable region-encoded proteins. Candidate clones were screened after eliminating similar sequences. The light chain variable region sequence and heavy chain variable region sequence of the candidate clone were cloned into the expression vector pTT5 containing the signal peptide and the human antibody IgG1 Fc fragment in the order of VL-linker-VH (Fc sequence, EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, SEQ ID NO.17), an expression vector for human-mouse chimeric antibodies was obtained and antibodies were prepared according to the method in Section 1.1 of Example 1 to obtain 16 scFv-hFc chimeric antibodies, whose VH and VL sequences are shown in Table 6, and CDR sequences are shown in Table 7.

[0145] Table 6 Antibody variable region amino acid sequences

[0146] Table 7 DLL3 chimeric antibody heavy and light chain CDR sequences

[0147] Example 4 Identification of DLL3 Human-Mouse Chimeric Antibodies

[0148] 4.1 ELISA assay to detect the binding of chimeric antibodies to human DLL3 protein

[0149] The chimeric antibody obtained above was subjected to ELISA testing and data analysis according to the method described in Section 1.3 of Example 1. OD450nm values ​​were read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The binding activity of the chimeric antibody to human DLL3 protein is shown in Figures 5A-5E , demonstrating strong binding activity of the chimeric antibody to human DLL3 protein. The IgG control was hIgG1, and the data in the table are OD450nm values.

[0150] 4.2 ELISA detection of binding of chimeric antibodies to human DLL1 and DLL4 proteins

[0151] The chimeric antibodies obtained above were assayed and the data analyzed by ELISA according to the method described in Section 1.3 of Example 1. OD 450 nm values ​​were measured using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The maximum binding values ​​of the chimeric antibodies to human DLL1 (purchased from Acro, Catalog No. DL1-H52H8) or DLL4 (purchased from Acro, Catalog No. DL4-H5227) at concentrations of 0.1, 1, 10, and 100 nM are shown in Table 8. The results showed that mab010 scFv-hFc bound to hDLL1, mab013 scFv-hFc and mab015 scFv-hFc bound weakly to hDLL1, and mab015 scFv-hFc bound to hDLL4. The remaining chimeric antibodies showed little binding to human DLL1 or DLL4. The negative control was hIgG1, and the positive controls were anti-DLL1 antibody (purchased from R&D, catalog number: MAB0108-100) and anti-DLL1 antibody (purchased from R&D, catalog number: MAB1506). The data in the table are OD450nm values.

[0152] Table 8 ELISA detection of the binding reaction between chimeric antibodies and human DLL1 and DLL4 proteins

[0153] 4.3 Flow cytometry (FACS) assay to detect the binding activity of chimeric antibodies to SHP77 cells endogenously expressing DLL3

[0154] SHP77 is a human small cell lung cancer cell line that moderately expresses DLL3 protein on its cell surface. The desired SHP77 cells were cultured in a T-75 cell culture flask until the logarithmic growth phase. The medium was aspirated and the cells were washed twice with PBS buffer. The cells were trypsinized and then digested with complete medium. The cells were pipetted to a single cell suspension. After counting the cells, the cells were centrifuged and the cell pellet was resuspended in FACS buffer (PBS + 2% fetal bovine serum) to a concentration of 2 × 10 6 For each milliliter of cells, 50 μL per well was added to a 96-well FACS reaction plate, and 50 μL of the chimeric antibody sample to be tested was added to each well and incubated at 4 degrees for 1 hour. Washed three times by centrifugation with PBS buffer, 50 μL of goat anti-human IgG H+L antibody (Jackson, Cat. No.: 109605088) was added to each well and incubated on ice for 1 hour. Washed three times by centrifugation with PBS buffer, resuspended in 100 μL PBS and analyzed by FACS (FACS Canto TM , purchased from BD Biosciences) were used for detection and analysis. Data were analyzed using FlowJo software to obtain the mean fluorescence intensity (MFI) of the cells. The results are shown in Table 9 and Figures 6A-6E, demonstrating that all chimeric antibodies bind to SHP77 cells.

[0155] Table 9 FACS detection of the binding activity of chimeric antibodies to SHP77 cells

[0156] Example 5 Detection of cross-binding activity of DLL3 chimeric antibodies

[0157] (A) FACS detection of the binding activity of chimeric antibodies to monkey DLL3

[0158] The detection method was similar to that described in Section 4.3 of Example 4. The FACS results of chimeric antibody binding to the monkey DLL3-overexpressing cell line CHO K1-cyno DLL3 are shown in Table 10 and Figures 7A-7E . The results demonstrate that all chimeric antibodies bind to CHO K1-cyno DLL3 cells. The IgG control is hIgG1, and the data in the table represent mean fluorescence intensity (MFI) values.

[0159] Table 10 FACS detection of chimeric antibody binding reaction with CHO K1-cyno DLL3 cells

[0160] Example 6 Humanized Design of DLL3 Antibody

[0161] By comparing the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database and MOE (Molecular Operating Environment) software, heavy and light chain variable region germline genes with high homology to murine antibodies were selected as templates. The CDRs of the murine antibodies were transplanted into the corresponding human templates, forming a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Back mutations and / or hotspot mutations were performed as needed. The antibody sequences and CDR sequences in this example are numbered according to the Kabat numbering system.

[0162] 6.1 Humanization of mab006

[0163] 6.1.1 mab006 germline sequence selection

[0164] The humanized light chain templates for antibody mab006 are IGKV1-39*01 or IGKV4-1*01, and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDRs of the murine antibody mab006 were transplanted into the corresponding humanized templates to obtain the humanized antibody of mab006.

[0165] 6.1.2 Back mutation and hotspot mutation design of mab006 humanized antibody

[0166] As needed, key amino acids in the FR region sequence of the mab006 humanized antibody were backmutated to maintain the original affinity. The specific mutation designs are shown in Table 11 (backmutations are in natural numbering order).

[0167] Table 11 Humanized antibody backmutation design of mab006

[0168] 6.1.3 mab006 Humanized Antibody Sequence Combination

[0169] The humanized antibody back mutation designs in Table 11 were combined to obtain a variety of mab006 humanized antibodies (see Table 12 for details).

[0170] Table 12 Corresponding design combinations of mab006 humanized antibodies

[0171] The amino acid sequence of the humanized heavy and light chain variable regions is shown in Table 13:

[0172] Table 13 Amino acid sequences of the variable regions of the mab006 humanized antibody backmutation

[0173] According to the Kabat numbering system, the results of the CDR sequence analysis of the heavy and light chain variable regions of the above humanized antibody are shown in Table 14:

[0174] Table 14 Kabat analysis results of the CDR sequences of the heavy and light chain variable regions of the mab006 humanized antibody

[0175] 6.2 Humanization of mab009

[0176] 6.2.1 mab009 germline sequence selection

[0177] The humanized light chain templates of the antibody mab009 are IGKV2-28*01 and IGKJ2*01, and the humanized heavy chain template is IGHV1-3*01. The CDRs of the murine antibody mab009 were transplanted into the corresponding humanized templates to obtain the humanized antibody of mab009.

[0178] 6.2.2 Back mutation and hotspot mutation design of mab009 humanized antibody

[0179] As needed, key amino acids in the FR region sequence of the mab009 humanized antibody were backmutated to maintain the original affinity. Given the presence of a high-risk modification site, NG, in the mab009 light chain, computational simulation was used to perform amino acid mutations on NG based on the antibody structure to eliminate modification risks. Specific mutation designs are shown in Table 15 (backmutations are in natural numbering order).

[0180] Table 15 Back mutation and hotspot mutation design of humanized antibody mab009

[0181] 6.2.3 mab009 humanized antibody sequence combination

[0182] The humanized antibody back mutations and hotspot mutation designs of mab009 in Table 15 were combined to ultimately obtain a variety of humanized antibodies (see Table 16 for details).

[0183] Table 16 Corresponding design combinations of mab009 humanized antibodies

[0184] The amino acid sequence of the humanized heavy and light chain variable regions is shown in Table 17:

[0185] Table 17 Amino acid sequences of the variable regions of the mab009 humanized antibody backmutation

[0186] According to the Kabat numbering system, the results of the CDR sequence analysis of the heavy and light chain variable regions of the above humanized antibody are shown in Table 18:

[0187] Table 18 Kabat analysis results of the CDR sequences of the heavy and light chain variable regions of the mab009 humanized antibody

[0188] 6.3 Humanization of mab013

[0189] 6.3.1 mab013 germline sequence selection

[0190] The humanized light chain templates for the antibody mab013 are IGKV3-11*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01. The CDRs of the murine antibody mab013 were transplanted into the corresponding humanized templates to obtain the humanized antibody mab013.

[0191] 6.3.2 Back mutation and hotspot mutation design of mab013 humanized antibody

[0192] As needed, key amino acids in the FR region of the humanized antibody were backmutated to maintain the original affinity. Given the presence of a high-risk modification site (NG) in the mab013 heavy chain, computational simulation was used to perform amino acid mutations based on the antibody structure to eliminate modification risks. Specific mutation designs are shown in Table 19 (backmutations are in natural numbering order).

[0193] Table 19 Back mutation and hotspot mutation design of humanized antibody mab013

[0194] 6.3.3 mab013 humanized antibody sequence combination

[0195] The humanized antibody back mutations and hotspot mutation designs of mab013 in Table 19 were combined to ultimately obtain a variety of humanized antibodies (see Table 20 for details).

[0196] Table 20 Corresponding design combinations of mab013 humanized antibodies

[0197] The amino acid sequence of the humanized heavy and light chain variable regions is shown in Table 21:

[0198] Table 21 Amino acid sequences of the variable regions of the mab013 humanized antibody backmutation

[0199] According to the Kabat numbering system, the results of the CDR sequence analysis of the heavy and light chain variable regions of the above humanized antibody are shown in Table 22:

[0200] Table 22 Kabat analysis results of the CDR sequences of the heavy and light chain variable regions of the mab013 humanized antibody

[0201] 6.4 Humanization of mab014

[0202] 6.4.1 mab014 germline sequence selection

[0203] The humanized light chain templates for antibody mab014 are IGKV1-27*01, IGKV4-1*01, and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDRs of the murine antibody mab014 were transplanted into the corresponding humanized templates to obtain the humanized antibody of mab014.

[0204] 6.4.2 Back mutation and hotspot mutation design of mab014 humanized antibody

[0205] As needed, key amino acids in the FR region sequence of the mab014 humanized antibody were backmutated to maintain the original affinity. The specific mutation designs are shown in Table 23 (backmutations are in natural numbering order).

[0206] Table 23 Humanized antibody backmutation design of mab014

[0207] 6.4.3 mab014 humanized antibody sequence combination

[0208] The humanized antibody back mutation designs of mab014 in Table 23 were combined to obtain a variety of mab014 humanized antibodies (see Table 24 for details).

[0209] Table 24 Corresponding design combinations of mab014 humanized antibodies

[0210] The amino acid sequence of the humanized heavy and light chain variable regions is shown in Table 25:

[0211] Table 25 Amino acid sequences of the variable regions of the mab014 humanized antibody backmutation

[0212] According to the Kabat numbering system, the results of the CDR sequence analysis of the heavy and light chain variable regions of the above humanized antibody are shown in Table 26:

[0213] Table 26 Kabat analysis results of the CDR sequences of the heavy and light chain variable regions of the mab014 humanized antibody

[0214] 6.5 Humanization of mab015

[0215] 6.5.1 mab015 germline sequence selection

[0216] The humanized light chain templates for the antibody mab015 are IGKV3-11*01, IGKV1-39*01, and IGKJ4*01, and the humanized heavy chain templates are IGHV3-15*01 and IGHJ6*01. The CDRs of the murine antibody mab015 were transplanted into the corresponding humanized templates to obtain the humanized antibody of mab015.

[0217] 6.5.2 Back Mutation and Hotspot Mutation Design of the Humanized mAb015 Antibody

[0218] As needed, key amino acids in the FR region sequence of the mab015 humanized antibody were backmutated to maintain the original affinity. Given the presence of a high-risk modification site, DD, in the mab015 heavy chain, computational simulation was used to perform amino acid mutations on DD based on the antibody structure to eliminate modification risks. Specific mutation designs are shown in Table 27 (backmutations are numbered in natural order).

[0219] Table 27 Humanized antibody back mutation and hotspot mutation design of mab015

[0220] 6.5.3 mab015 Humanized Antibody Sequence Combination

[0221] The humanized antibody back mutations and hotspot mutation designs of mab015 in Table 27 were combined to obtain a variety of mab015 humanized antibodies (see Table 28 for details).

[0222] Table 28 Corresponding design combinations of mab015 humanized antibodies

[0223] The amino acid sequence of the humanized heavy and light chain variable regions is shown in Table 29:

[0224] Table 29 Amino acid sequences of the variable regions of the mab015 humanized antibody backmutation

[0225] According to the Kabat numbering system, the results of the CDR sequence analysis of the heavy and light chain variable regions of the above humanized antibody are shown in Table 30:

[0226] Table 30 Kabat analysis results of the CDR sequences of the heavy and light chain variable regions of the mab015 humanized antibody

[0227] 6.6 Humanization of mab008

[0228] 6.6.1 mab008 germline sequence selection

[0229] The humanized light chain templates of antibody mab008 are IGKV1-39*01, IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDRs of the murine antibody mab008 were transplanted into the corresponding humanized templates to obtain the humanized antibody of mab008.

[0230] 6.6.2 Back mutation and hotspot mutation design of mab008 humanized antibody

[0231] As needed, key amino acids in the FR region sequence of the mab008 humanized antibody were backmutated to maintain the original affinity. Given the presence of a high-risk modification site, NG, in the mab008 heavy chain, computational simulation was used to perform amino acid mutations on NG based on the antibody structure to eliminate modification risks. Specific mutation designs are shown in Table 31 (backmutations are in natural numbering order).

[0232] Table 31 Humanized antibody back mutation and hotspot mutation design of mab008

[0233] 6.6.3 mab008 Humanized Antibody Sequence Combination

[0234] The humanized antibody back mutations and hotspot mutation designs of mab008 in Table 31 were combined to obtain a variety of mab008 humanized antibodies (see Table 32 for details).

[0235] Table 32 Corresponding design combinations of mab008 humanized antibodies

[0236] The amino acid sequences of the humanized heavy and light chain variable regions are shown in Table 33:

[0237] Table 33 Amino acid sequences of the variable regions of the mab008 humanized antibody backmutation

[0238] According to the Kabat numbering system, the results of the CDR sequence analysis of the heavy and light chain variable regions of the above humanized antibody are shown in Table 34:

[0239] Table 34 Kabat analysis results of the CDR sequences of the heavy and light chain variable regions of the mab008 humanized antibody

[0240] 6.6 Construction, Expression, and Purification of a Humanized Full-Length Antibody Against DLL3

[0241] PCR primers were designed to construct the VH / VL gene segments of each humanized antibody, which were then homologously recombined with the vector to construct a full-length humanized antibody expression vector. The humanized antibody was expressed in a VL-linker-VH sequence linked to a human IgG1 Fc fragment. After plasmid construction, Expi293F cells were transiently transfected. After 7 days, the supernatant was collected by centrifugation and the antibody was purified according to the purification method described in Section 1.1 of Example 1.

[0242] Example 7 Identification of Humanized Antibodies to DLL3

[0243] 7.1 ELISA assay to detect the binding of humanized antibodies to human DLL3 protein

[0244] The humanized antibodies obtained above were assayed and analyzed by ELISA according to the method described in Section 1.3 of Example 1. OD450nm values ​​were read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The binding activity of the humanized antibodies to human DLL3 protein is shown in Figures 8A-8G , demonstrating strong binding activity between the humanized antibodies and human DLL3 protein. The IgG control was hIgG1, and the data in the table are OD450nm values.

[0245] 7.2 ELISA assay for binding of humanized antibodies to human DLL1 and DLL4 proteins

[0246] The humanized antibodies obtained above were assayed and the data analyzed by ELISA according to the method in Section 1.3 of Example 1. OD450nm values ​​were read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The maximum binding values ​​of the humanized antibodies to human DLL1 (purchased from Acro, Catalog No. DL1-H52H8) or DLL4 (purchased from Acro, Catalog No. DL4-H5227) proteins at concentrations of 0.1, 1, 10, and 100 nM are shown in Table 35. The results show that the humanized antibodies showed little binding to human DLL1 or DLL4.

[0247] Table 35 ELISA detection of binding reaction between humanized antibodies and human DLL1 and DLL4 proteins

[0248] 7.3 Flow cytometric fluorescence sorting (FACS) assay for binding activity of humanized antibodies to cells endogenously expressing DLL3. The humanized antibodies obtained above were subjected to FACS analysis and data analysis according to the method of Example 4.3. Data analysis was performed using FlowJo software to obtain the mean fluorescence intensity (MFI) of the cells. Tables 36-37 and Figures 9A-10F demonstrate that the humanized antibodies maintained substantially the same binding activity as the chimeric antibodies to SHP77 and NCI-H82 cells.

[0249] Table 36 FACS detection of binding activity of humanized antibodies to SHP77 cells

[0250] Table 37 FACS detection of binding activity of humanized antibodies to NCI-H82 cells

[0251] 7.4 Flow cytometric analysis (FACS) of the binding activity of humanized antibodies to cells overexpressing DLL3. The humanized antibodies obtained above were subjected to FACS analysis and data analysis according to the method in Example 4.3. Data analysis was performed using FlowJo software to obtain the mean fluorescence intensity (MFI) of the cells. Table 38 and Figures 11A-11F demonstrate that all humanized antibodies bind to CHO-K1 hDLL3 cells.

[0252] Table 38 FACS detection of binding activity of humanized antibodies to CHO-K1 hDLL3 cells

[0253] Example 8 Detection of cross-binding activity of DLL3 humanized antibodies

[0254] (A) FACS detection of the binding activity of humanized antibodies to monkey DLL3

[0255] The humanized antibodies obtained above were subjected to FACS analysis and data analysis according to the methods of Example 4.3. The FACS results of binding of the humanized antibodies to the monkey DLL3-overexpressing cell line CHO K1-cyno DLL3 are shown in Table 39 and Figures 12A-12F . These results demonstrate that the humanized antibodies maintain substantial binding activity to CHO K1-cyno DLL3 cells compared to the chimeric antibodies. The IgG control is hIgG1, and the data in the table represent mean fluorescence intensity (MFI) values.

[0256] Table 39 FACS detection of humanized antibody binding reaction with CHO K1-cyno DLL3 cells

[0257] (B) FACS detection of the binding activity of humanized antibodies to mouse DLL3

[0258] The humanized antibodies obtained above were subjected to FACS analysis and data analysis according to the methods of Example 4.3. The FACS results of binding of the humanized antibodies to the mouse DLL3-overexpressing cell line CHO K1-mDLL3 are shown in Table 40 and Figures 13A-13G. These results demonstrate that the humanized antibodies maintain substantial binding activity to CHO K1-mDLL3 cells compared to the chimeric antibodies, demonstrating mouse cross-reactivity. The humanized Hab008 molecule exhibits no mouse cross-reactivity. The IgG control is hIgG1, and the data in the table represent mean fluorescence intensity (MFI) values.

[0259] Table 40 FACS detection of humanized antibody binding reaction with CHO K1-mDLL3 cells

[0260] Example 9 Affinity detection of DLL3 humanized antibodies

[0261] The experiment used a Biacore 8K (GE) instrument and multi-cycle kinetics to determine the affinity of the tested anti-DLL3 humanized antibody to the DLL3 antigen protein.

[0262] The experimental running buffer consisted of 1× HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, Cat. BR-1006-69, Cytiva). The flow-through cell temperature was set at 25°C, and the sample chamber temperature was set at 16°C. Both were pre-treated with running buffer. A specific amount of the test antibody was affinity-captured using a Protein A biosensor chip (Cat. #29-1275-56, Cytiva). A specific concentration of DLL3 antigen was then passed over the chip surface. Binding and dissociation curves were generated using a Biacore 8K instrument (GE) for real-time signal monitoring. After each dissociation cycle, the antigen-antibody complex was washed and regenerated with glycine-HCl regeneration solution (Cat. BR-1003-54, Cytiva) at pH 1.5. The binding process was detected by injecting different concentrations of DLL3 antigen in solution for 180 seconds at a flow rate of 30 μL / min, starting from 20 nM (the actual concentration tested is shown in the detailed results), and diluted 1:1 to set up a series of concentration gradients; the dissociation time was up to 600 seconds, and finally the chip surface was regenerated by washing with 10 mM glycine-HCl solution (pH 1.5) for 30 seconds at a flow rate of 30 μL / min.

[0263] The experimental data were fitted with a (1:1) Langmuir model using GE Biacore 8K Evaluation version 2.0 software to obtain the association rate (Ka), dissociation rate (Kd), and affinity (KD) of the humanized antibody for human DLL3 protein, as shown in Table 41. The results demonstrate that the humanized antibody binds well to the human DLL3 protein.

[0264] Table 41 Binding affinity of humanized antibodies to human DLL3 protein

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human Delta-Like Ligand 3 (DLL3), wherein: The antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein: (1) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 are the LCDR1, LCDR2 and LCDR3 of the VL domain as shown in any one of SEQ ID NO.19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 338-339, 343-346, 349-355, 359-361, 364-365, 374-375; and (2) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are the HCDR1, HCDR2 and HCDR3 of the VH domain shown in any one of SEQ ID NO.18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 340-342, 347, 356, 362-363, 366-370, 376-377.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The LCDR1, LCDR2 and LCDR3 are determined according to the Kabat numbering system, the IMGT numbering system and / or the Chothia numbering system.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The HCDR1, HCDR2 and HCDR3 are determined according to the Kabat numbering system, the IMGT numbering system and / or the Chothia numbering system.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or antigen-binding fragment thereof comprises the following LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3: (1) LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 50-145, 348, 357-358, 371-373, 378 according to the Kabat numbering system, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 50-145; (2) LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 146-241 or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 146-241 according to the IMGT numbering system; (3) According to the Chothia numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 242-337, or a combination of sequences having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 242-337.

5. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein: (1) The light chain variable region sequence comprises the sequence shown in SEQ ID NO. 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 338-339, 343-346, 349-355, 359-361, 364-365, 374-375, or the sequence shown in SEQ ID NO. NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 338-339, 343-346, 349-355, 359-361, 364-365, 374-375 have 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity; and, (2) The heavy chain variable region sequence comprises a sequence as shown in SEQ ID NO.18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 340-342, 347, 356, 362-363, 366-370, 376-377, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with a sequence as shown in SEQ ID NO:18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 340-342, 347, 356, 362-363, 366-370, 376-377.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The antibody or antigen-binding fragment thereof is chimeric, humanized or fully human.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein It can bind to human, monkey or mouse Delta-Like Ligand 3 (DLL3).

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein It may also contain any constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; preferably, it contains the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4 with mutation; Furthermore, the antibody or antigen-binding fragment thereof is also coupled with a therapeutic agent or a tracer; Preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent, a cytotoxic agent or an immunomodulatory agent. The tracer is selected from the group consisting of radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from the group consisting of alkaloids, methotrexate, anthracycline antibiotics (doxorubicin), pyrrolobenzodiazepines (pyrrolobenzodiazepine, PBD) or gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is a taxane.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein The antigen binding fragment is selected from one or more of F(ab')2, Fab', Fab, Fv, scFv, nanobody or affibody.

10. A multispecific antigen-binding molecule, wherein: The multispecific antigen-binding molecule comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, an antigen-binding molecule that binds to an antigen other than DLL3, and / or an antigen-binding molecule that binds to a DLL3 epitope different from the DLL3 epitope bound by the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; Preferably, the other antigen-binding molecule is an antibody or an antigen-binding fragment thereof; Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific; Preferably, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

11. An isolated nucleic acid fragment, wherein: The nucleic acid fragment encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the multispecific antigen-binding molecule according to claim 12.

12. A recombinant vector, wherein the recombinant vector comprises the isolated nucleic acid fragment of claim 11.

13. A host cell, wherein The host cell comprises the recombinant vector of claim 12; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

14. A method for preparing a product comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the multispecific antigen-binding molecule according to claim 10, the method comprising culturing the host cell according to claim 13, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cell.

15. A pharmaceutical composition, wherein The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to claims 1 to 9, the multispecific antigen-binding molecule according to claim 10, the nucleic acid fragment according to claim 11, the vector according to claim 12 or a product prepared by the method according to claim 14; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.

16. The pharmaceutical composition according to claim 15, wherein The pharmaceutical composition may further comprise an additional anti-tumor agent.

17. A method for treating a tumor or cancer, wherein: The method comprises administering to a subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the multispecific antigen-binding molecule according to claim 10, the nucleic acid fragment according to claim 11, the vector according to claim 12, the product prepared by the method according to claim 14, or the pharmaceutical composition according to claim 15 or 16; preferably, the tumor or cancer is selected from a solid tumor, a hematological tumor, or a cancer infiltrating and expressing DLL3; Preferably, the tumor or cancer is selected from at least one of small cell lung cancer, glioma, pancreatic cancer, melanoma, breast cancer, pituitary tumor, endometrioma, acute myeloid leukemia, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer and esophageal cancer.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the multispecific antigen-binding molecule according to claim 10, the nucleic acid fragment according to claim 11, the vector according to claim 12, the product obtained by the method according to claim 14, or the pharmaceutical composition according to claim 15 or 16 in the preparation of a drug for treating tumors or cancer; preferably, the tumor or cancer is selected from solid tumors, hematological tumors, or cancers infiltrating and expressing DLL3; Preferably, the tumor or cancer is selected from at least one of small cell lung cancer, glioma, pancreatic cancer, melanoma, breast cancer, pituitary tumor, endometrioma, acute myeloid leukemia, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer and esophageal cancer.

19. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the multispecific antigen-binding molecule according to claim 10, the nucleic acid fragment according to claim 11, the vector according to claim 12, the product prepared by the method according to claim 14, or the pharmaceutical composition according to claim 15 or 16.

20. A method for detecting DLL3 expression in a biological sample, the method comprising contacting the biological sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 under conditions in which a complex can be formed between the antibody or antigen-binding fragment thereof and DLL3; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of DLL3 in the sample.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a DLL3 detection reagent.

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