Nanometer antibody for specifically recognizing Delta-like ligand 3 and application thereof

Abstract

The invention relates to a DLL3 antibody and application thereof, in particular to a specific binding DLL3 antibody or an antigen binding fragment, especially a VHH antibody, a coding nucleic acid, a recombinant vector and a host cell thereof, a preparation method, a pharmaceutical composition and application in disease treatment, such as tumor treatment. The method is of great significance to the development of DLL3-based target treatment drugs and the development of detection reagents.

Description

Nanobodies specifically recognizing Delta-like ligand 3 and their applications

[0001] The present invention claims priority to Chinese patent application No. 202311022005.X filed on August 14, 2023, entitled “Nanoantibodies specifically recognizing Delta-like ligand 3 and their applications”. The entire contents of this application, 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 nanoantibodies that specifically recognize 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 forms exist, with the primary splice sharing 91% sequence identity between the human and monkey extracellular domains and 84% between the human and mouse extracellular domains. 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. Standard first-line treatment for patients with extensive-stage small cell lung cancer includes radiotherapy, with combinations of 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 tissues such as the nervous system, pancreas, and testes, protein expression levels are 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 or squamous cell lung carcinoma. DLL3 is also expressed in other neuroendocrine-related tumors, such as melanoma, multiforme glioma, and small cell bladder cancer. DLL3 expression H-scores exceeding 100 were 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 reported that 83% of samples from patients with small cell lung cancer tested positive for DLL3, 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 membrane surface antigen expression and toxin selection, these ADCs have not generated positive overall survival data in Phase III clinical trials. The present invention aims to generate nanobody sequences targeting multiple DLL3 epitopes to generate a wider range of DLL3-targeted drug forms.

[0006] Summary of the Invention

[0007] The present invention provides a new antibody or antigen-binding fragment that specifically binds to DLL3, in particular a VHH antibody that specifically binds to DLL3, which regulates Notch signaling by inhibiting the function of DLL3, thereby providing a new tumor treatment method.

[0008] In a first aspect of the present invention, an antibody or antigen-binding fragment that specifically binds to human Delta-Like Ligand 3 (DLL3) is provided, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein 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 NOs. 7-12, 50-52, 57-58, and 60-63.

[0009] In some specific embodiments, the antibody or antigen binding protein comprises the following HCDR1, HCDR2, and HCDR3:

[0010] (1) According to the Kabat numbering system, HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 13-30, 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. 13-30, or have a sequence identity of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%;

[0011] (2) According to the IMGT numbering system, HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 31-48, 55-56, 59, 64, 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. 31-48, 55-56, 59, 64, or have 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0012] In some specific embodiments, the heavy chain variable region sequence of the antibody or antigen-binding fragment comprises a sequence as shown in SEQ ID NO.7-12, 50-52, 57-58, 60-63, or has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence as shown in SEQ ID NO.7-12, 50-52, 57-58, 60-63.

[0013] In some specific embodiments, the antibody or antigen-binding fragment is chimeric, humanized, or fully human.

[0014] In some specific embodiments, the antibody or antigen-binding fragment can bind to Delta-Like Ligand 3 (DLL3) from human, monkey or mouse.

[0015] 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 or IgD; preferably, the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or a constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4 with a mutation; further, the antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug, a cytotoxic agent or an immunomodulator, 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; more preferably, the cytotoxic agent is selected from methotrexate, anthracycline antibiotics (doxorubicin), pyrrolobenzodiazepines, (pyrrolobenzodiazepine, PBD), gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is a taxane.

[0016] In some specific embodiments, the antigen-binding fragment is selected from one or more of nanoantibodies or affibodies.

[0017] In a second aspect, the present invention provides a multispecific antigen-binding molecule comprising the aforementioned antibody or antigen-binding fragment and an antigen-binding molecule that binds to an antigen other than DLL3, or comprising the aforementioned antibody or antigen-binding fragment and an antigen-binding molecule that binds to a DLL3 epitope different from the DLL3 epitope bound by the aforementioned antibody or antigen-binding fragment.

[0018] Preferably, the other antigen-binding molecule is an antibody or antigen-binding fragment;

[0019] Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific;

[0020] Preferably, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

[0021] The third aspect of the present invention provides an isolated nucleic acid fragment, wherein the nucleic acid fragment encodes the antibody or antigen-binding fragment, or the multispecific antigen-binding molecule.

[0022] The fourth aspect of the present invention provides a recombinant vector, wherein the vector comprises the aforementioned isolated nucleic acid fragment.

[0023] In a fifth aspect of the present invention, a host cell is provided, wherein the host cell comprises the aforementioned 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).

[0024] In a sixth aspect, the present invention provides a method for preparing a product comprising the antibody or antigen-binding fragment or the multispecific antigen-binding molecule, the method comprising culturing the aforementioned host cells, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cells.

[0025] In a seventh aspect, the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the antibody or antigen-binding fragment, multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned vector or the product obtained by the aforementioned preparation method; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.

[0026] In some specific embodiments, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0027] 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, multispecific antigen-binding molecule, nucleic acid fragment, recombinant vector, product obtained by the aforementioned method, or pharmaceutical composition; preferably, the tumor or cancer is selected from a solid tumor, a hematologic tumor, or a cancer that infiltrates and expresses DLL3;

[0028] Preferably, the tumor or cancer is selected from 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.

[0029] A ninth aspect of the present invention provides use of the antibody or antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, vector, product obtained by the aforementioned method, or pharmaceutical composition for preparing a medicament for treating tumors or cancer; preferably, the tumor or cancer is selected from a solid tumor, a hematologic tumor, or a cancer that infiltrates and expresses DLL3;

[0030] Preferably, the tumor or cancer is selected from 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.

[0031] In a tenth aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, recombinant vector, product prepared by the aforementioned method, or the aforementioned pharmaceutical composition.

[0032] 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 under reaction conditions capable of forming a complex between the antibody or antigen-binding fragment and DLL3; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of DLL3 in the sample.

[0033] The twelfth aspect of the present invention provides use of the antibody or antigen-binding fragment in preparing a DLL3 detection reagent.

[0034] Beneficial Effects: The present invention screened and obtained high-affinity specific antibodies against DLL3, particularly VHH antibodies, and further derived humanized antibodies based on these antibodies. These antibodies bind to DLL3 with high specificity, have a small molecular weight, and are highly druggable. They are also particularly suitable for use in combination with other antibodies to form bispecific or polyspecific antibodies. In vivo studies have shown that these antibodies have low in vivo toxicity and a good safety profile. They can be used to treat various types of cancer, particularly small cell lung cancer, filling a gap in clinical treatment.

[0035] Definitions and Explanations of Terms

[0036] 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.

[0037] 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.

[0038] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements 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.

[0039] 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."

[0040] 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.

[0041] 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 -9 The 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.

[0042] 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.

[0043] 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) (the contents of which are 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.

[0044] 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.

[0045] 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.

[0046] 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).

[0047] 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.

[0048] The term "multi-specificity" herein refers to the ability of an antibody or 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 therefore can 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 therefore can bind to at least two different epitopes. As described herein, "complementary 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 having 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.

[0049] 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.

[0050]

[0014] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody.

[0051] "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 that has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0052] The terms "nanoantibody" and "single-domain antibody" are used interchangeably in this article. They refer to the natural heavy chain antibodies lacking light chains that exist in camels. Cloning their variable regions can obtain nanoantibodies consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0053] The term "chimeric antibody" herein refers to an antibody having variable sequences of an immunoglobulin from one source organism (e.g., rat, mouse, rabbit, or alpaca) and constant regions of an immunoglobulin from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art.

[0054] 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.

[0055] 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 that are 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 onto human framework sequences.

[0056] 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, each 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) (the contents of which are incorporated herein by reference). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0057] 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 hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT), while being considered to be outside the hypervariable region under a different set of criteria (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 CDRs can be abbreviated as HCDRs, and the light chain variable region can be abbreviated as LCDRs. 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), which form a loop connecting the sheet structure and, in some cases, form a portion of the sheet structure. The CDRs in each chain are held together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and contribute to the formation of the antibody antigen-binding site with the CDRs from other antibody chains.

[0058] "CDRs" herein may 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 tools such as, 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). The CDRs herein encompass overlaps and subsets of amino acid residues defined using different methods. (The foregoing is incorporated herein by reference.)

[0059] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat.

[0060] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the location of structural loop regions.

[0061] The term "IMGT numbering system" herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003 (incorporated herein by reference).

[0062] 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.

[0063] 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.

[0064] 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 the IgG CH2 and IgG CH3 domains, and optionally, may further comprise a complete or partial hinge region, but does not comprise 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 in the Fc region at the C-terminus of the CH2 domain (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 in one chain and a corresponding "hole" introduced in the other chain; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference). As described herein, such variant CH3 domains can be used to promote heterodimerization of two different antibody heavy chains.

[0065] 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 called the EU index.

[0066] 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 through mutations, such as space-filling effects, electrostatic guidance, hydrogen bonding, and hydrophobic interactions. These interactions contribute to the formation of stable heterodimeric proteins. Preferred mutational designs are "knob-into-hole" mutational designs.

[0067] 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 use of electrostatic steering by Amgen technicians to form Fc-containing heterodimers (US20100286374A1); the heterodimers (SEEDbodies) formed by IgG / Ig chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp. 1-8, 2010); the bispecific molecules formed by Genmab's DuoBody (Science, 2007. 317 (5844)) platform technology; and the use of structural calculations and Fc amino acid mutations by Xencor technicians to combine different modes of action to form heterodimeric protein forms (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 perform a knob mutation and the other to perform a hole mutation. (The aforementioned contents are incorporated herein by reference).

[0068] 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:

[0069] 1) Alanine (A), serine (S), threonine (T);

[0070] 2) Aspartic acid (D), glutamic acid (E);

[0071] 3) Asparagine (N), glutamine (Q);

[0072] 4) Arginine (R), Lysine (K), Histidine (H);

[0073] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0074] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0075] 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.

[0076] Mathematical algorithms can be used to compare sequences between two sequences and calculate percent identity. For example, the Needlema and Wunsch algorithms (available at www.gcg.com) that have been integrated into the GAP program of the GCG software package are used, using a Blossum 62 matrix or a PAM250 matrix and gap weights 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 (available at www.gcg.com) in the GCG software package is used, using the NWSgapdna.CMP matrix and gap weights 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 a parameter set that should be used unless otherwise specified) is a Blossum 62 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).

[0077] 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 (the aforementioned contents are incorporated herein by reference).

[0078] The term "chimeric antigen receptor (CAR)" herein refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells and specifically binds to an antigen, which comprises at least (1) an extracellular antigen binding domain, such as an antibody heavy chain variable region and / or light chain variable region, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. CAR is able to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding domain.

[0079] 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. The example of non-naturally occurring nucleotides includes the nucleotide bases of the modification of the residue of the sugar or phosphate backbone bonding or chemical modification with derivative.Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for directly expressing antibodies of the present disclosure in vitro and / or in vivo, such as in a host or patient.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 the 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) (its contents are incorporated herein by reference).

[0080] As used herein, "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 ordinarily 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.

[0081] 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."

[0082] 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.

[0083] 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.

[0084] 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. Except in the case of incompatibility with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] As used herein, the term "cancer" 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. As used herein, the term "tumor" or "neoplasm" refers 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" are not mutually exclusive when used herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figures 1A and 1B show the enzyme-linked immunosorbent assay (ELISA) method for detecting the binding activity of control antibodies to human or monkey DLL3-his protein;

[0090] FIG2 shows the expression level of human DLL3 protein in CHO-K1 recombinant cell line detected by flow cytometry (FACS);

[0091] FIG3 shows the expression level of monkey DLL3 protein detected by flow cytometry (FACS) in CHO-K1 recombinant cell line;

[0092] FIG4 shows the expression level of mouse DLL3 protein detected by flow cytometry (FACS) in CHO-K1 recombinant cell line;

[0093] Figures 5A-5C show the detection of the binding activity of recombinant Nanobodies to human DLL3 protein;

[0094] Figures 6A-6C show the detection of binding activity of recombinant Nanobodies to human DLL3 recombinant cell lines;

[0095] Figures 7A-7C show the detection of the binding activity of recombinant Nanobodies to SHP-77 cells;

[0096] Figures 8A-8C show the detection of the binding activity of recombinant Nanobodies to monkey DLL3 recombinant cell lines;

[0097] Figures 9A-9C show the detection of the binding activity of recombinant Nanobodies to mouse DLL3 recombinant cell lines;

[0098] Figures 10A-10E show the detection of binding activity of humanized Nanobodies to human DLL3 protein;

[0099] Figures 11A-11D show the detection of binding activity of humanized Nanobodies to SHP-77 cells;

[0100] Figure 12 shows the detection of binding activity of humanized Nanobodies to NCI-H82 cells;

[0101] Figures 13A-13D show the detection of binding activity of humanized Nanobodies to NCI-H2171 cells;

[0102] Figures 14A-14E show the detection of binding activity of humanized Nanobodies to human DLL3 recombinant cell lines;

[0103] Figures 15A-15E show the detection of binding activity of humanized Nanobodies to monkey DLL3 recombinant cell lines;

[0104] Figures 16A-16E show the detection of the binding activity of humanized Nanobodies to mouse DLL3 recombinant cell lines. DETAILED DESCRIPTION

[0105] The present invention relates to isolated antibodies or antigen-binding portions that specifically bind to Delta-like ligand 3 (DLL3) with high affinity.

[0106] Example 1: Preparation of control antibodies, preparation of full-length and truncated antigens, identification of endogenous cells, and preparation of cell lines overexpressing full-length and truncated DLL3

[0107] 1.1 Preparation of positive control antibody 52D04

[0108] 52D04 is a VHH antibody that recognizes human DLL3. It has strong binding activity to the human DLL3 protein and can also specifically bind to recombinant or endogenous cell lines expressing DLL3. The heavy chain variable region sequence of 52D04 was obtained based on patent WO2020069028A1.

[0109] The VHH of 52D04 and human IgG1 Fc were linked in order from N-terminus to C-terminus to form VHH-human IgG1 Fc (VHH-hFc). The sequences of 52D04 VHH and 52D04 VHH-hFc are shown in Table 1.

[0110] Table 1 Amino acid sequences of control antibodies

[0111] The nucleotide sequence encoding the 52D04 VHH-hFc was cloned into the pTT5 vector (purchased from U-Bio), and plasmids were prepared according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). 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 number: F081918-001) and 6g / L glucose (Sigma, catalog number: 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, it 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.

[0112] 1.2 Preparation of full-length human DLL3 protein antigen and cynomolgus monkey DLL3 protein antigen

[0113] The nucleotide coding sequences of the human DLL3 protein extracellular domain (ECD) (NCBI Accession: Q9NYJ7, shown as SEQ ID NO. 3), the cynomolgus macaque DLL3 protein extracellular domain (UniProt ID: A0A2K5WSR4, shown as SEQ ID NO. 4), and the mouse DLL3 protein extracellular domain (UniProt ID: O88516, shown as SEQ ID NO. 5) were coupled to a His-tag and cloned into the pTT5 vector. The expression method was the same as that described for the control antibody preparation described in Section 1.1 above. Cellular components were removed by centrifugation to obtain the culture supernatant. Proteins from the cell culture supernatant were purified using a Ni affinity chromatography column (purchased from GE Healthcare). The column was 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, wash the Protein A column with equilibration buffer (3-5 times the volume of the Ni affinity column bed). Elution was performed using a gradient of 0-500 mM imidazole, and the elution was monitored using a nucleic acid protein detector (A280 UV absorbance peak). The eluted protein was collected and 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 No.: DL3-H52H4) and cynomolgus monkey DLL3-His protein (purchased from Acro, Catalog No.: DL3-C52H3) were purchased commercially.

[0114] Human DLL3 extracellular segment SEQ ID NO.3

[0115] Cynomolgus monkey DLL3 extracellular segment SEQ ID NO.4

[0116] Mouse DLL3 extracellular segment amino acid SEQ ID NO.5

[0117] 1.3 Binding reaction detection of control antibodies with human DLL3-his protein and monkey DLL3-his protein

[0118] The antigen-antibody binding activity was detected using the ELISA method. The specific method is: 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 at 4°C overnight. The next day, the plate was washed twice with PBST, and blocking solution [PBS + 2% (w / w) BSA] was added and blocked at room temperature for 2 hours. The blocking solution was discarded, and 50 μl of 100 nM gradient diluted control antibody or negative control antibody 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). As shown in Figures 1A-1B , the control antibody exhibited good binding activity to both human and monkey DLL3 proteins. hIgG1 was used as a negative control antibody (derived from J Biol Chem. 1990 Jan 5;265(1):133-8, incorporated herein by reference).

[0119] 1.4 Preparation of CHO-K1 recombinant cell lines expressing human DLL3 protein

[0120] Nucleotide sequences encoding the amino acids for the extracellular region of human DLL3 (sequence shown in SEQ ID NO. 3) and the amino acid sequences for the transmembrane and intracellular regions of EpCAM (sequence shown in SEQ ID NO. 6) were cloned into the pLVX-IRES-Puro vector (purchased from Ubao Bio, catalog number: VT1464) for lentiviral packaging and subsequently infected CHO-K1 cells. 72 hours after viral infection of CHO-K1 cells, the cells were detected by flow cytometry using the known DLL3 antibody 2G1 (an antibody that recognizes human DLL3, with heavy and light chain variable region sequences obtained according to patent WO2020180591A1 and produced in-house using conventional methods). Once transfected cells were confirmed to be expressing human DLL3 protein, 10 μg / ml Puromycin (Gibco, Cat. 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 wells 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 2 and Figure 2. The IgG isotype control was human IgG1 (hIgG1). Table 2 demonstrates the generation of a series of CHO-K1 monoclonal cell lines positive for human DLL3 expression. The abscissa of Figure 2 shows cell fluorescence intensity, and the ordinate shows cell number. The results showed that #5, #7, and #11 were cell lines with high expression of human DLL3.

[0121] EpCAM transmembrane region and intracellular region:

[0122] Table 2 FACS detection results of CHO-K1 recombinant cell line expressing human DLL3 protein

[0123] 1.5 Preparation of CHO-K1 recombinant cell line expressing monkey DLL3 protein

[0124] Nucleotide sequences encoding the extracellular amino acid region of monkey DLL3 (sequence shown in SEQ ID NO. 4) and the amino acid sequences of the transmembrane and intracellular regions of EpCAM (sequence shown in SEQ ID NO. 6) were cloned into the pLVX-IRES-Puro vector. Using the recombinant cell construction method described in Section 1.4, stable cell lines expressing monkey DLL3 were generated. The stably transfected cell lines were tested with the 2G1 antibody, and the results are shown in Table 3 and Figure 3 . The IgG subtype control was human IgG1 (i.e., hIgG1). Table 3 illustrates the generation of a series of CHO-K1 monoclonal cell lines that positively express monkey DLL3. 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 are highly expressing monkey DLL3.

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

[0126] 1.6 Preparation of CHO-K1 recombinant cell line expressing mouse DLL3 protein

[0127] The nucleotide sequences encoding the extracellular amino acid region of mouse DLL3 (sequence shown in SEQ ID NO. 5) and the amino acid sequences of the transmembrane and intracellular regions of EpCAM (sequence shown in SEQ ID NO. 6) were cloned into the pLVX-IRES-Puro vector. Using the recombinant cell construction method described in Section 1.4, stably transfected cell lines expressing mouse DLL3 were generated. The stably transfected cell lines were tested with the 2G1 antibody, and the results are shown in Table 4 and Figure 4 . The IgG subtype control was human IgG1 (i.e., hIgG1). Table 4 shows that a series of CHO-K1 monoclonal cell lines expressing mouse DLL3 were generated. Figure 4 shows cell fluorescence intensity on the horizontal axis and cell number on the vertical axis. The results indicate that cell lines #4 and #5 are highly expressing mouse DLL3.

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

[0129] Example 2: Preparation of Nanobodies Against DLL3

[0130] 2.1 Camel immunization and serum titer detection

[0131] One camel was selected for immunization, and 10 ml of blood was collected before immunization to serve as a negative control serum. The principle of doubling the first dose was adopted, and 0.5 mg of human DLL3 antigen protein was fully mixed with Freund's complete adjuvant (purchased from Sigma, F5881) and injected subcutaneously and intramuscularly at multiple points. Two weeks later, a second immunization was performed, and 0.25 mg of protein was mixed with Freund's incomplete adjuvant and injected subcutaneously and intramuscularly at multiple points. One week later, serum was collected to measure the titer. For the third immunization, 0.25 mg of protein was mixed with Freund's incomplete adjuvant and injected subcutaneously and intramuscularly at multiple points. One week later, serum was collected to measure the titer. For the fourth immunization, 0.25 mg of protein was mixed with Freund's incomplete adjuvant and injected subcutaneously and intramuscularly at multiple points. One week later, serum was collected to measure the titer. For the fifth immunization, 0.5 mg of protein was mixed with Freund's incomplete adjuvant and injected subcutaneously and intramuscularly at multiple points. One week later, serum was collected to measure the titer. For the sixth immunization, 0.25 mg of protein was mixed with Freund's incomplete adjuvant and injected subcutaneously and intramuscularly at multiple sites. Serum titers were measured one week later. The titers of antibodies against human DLL3 protein in serum were measured using enzyme-linked immunosorbent assay (ELISA).

[0132] 2.2 Construction of Nanobody Library

[0133] A total of 50 mL of peripheral blood was collected from camels after the fourth and sixth immunizations, and PBMCs were isolated using lymphocyte separation medium. RNA was extracted and transcribed, and a nanobody (VHH) fragment was amplified using cDNA as a template using a one-step PCR method. The amplified target band was approximately 500 bp in size. A nanobody library was constructed using conventional methods, and the number of transformants in the nanobody library was determined on a total of 8×10 plates. 8 clones, and the library size was calculated to be 1.6×10 9 96 clones were randomly selected from the library transformant titer plate for identification. The target protein band was approximately 500 bp in size. The results showed that all 93 clones were positive, indicating an insertion rate of 96.8%.

[0134] 2.3 Selection of Nanobodies Targeting DLL3 Protein

[0135] Human DLL3 protein was used to select nanobodies conventionally used in the art. After multiple rounds of panning, positive phages were continuously enriched during the panning process to screen out nanobodies with good specificity and high affinity.

[0136] 2.4 Screening of positive clones by enzyme-linked immunosorbent assay (ELISA)

[0137] The positive clones that bind to human DLL3 protein were screened by enzyme-linked immunosorbent assay (ELISA).

[0138] 2.5 Flow cytometry (FACS) screening of cell-bound phage clones

[0139] Flow cytometry (FACS) was used to detect phage clones that bound to cell lines expressing DLL3.

[0140] After multiple rounds of optimization and screening, six positive clones were selected that could simultaneously recognize the full-length or truncated DLL3 protein and cells, and the amino acid sequences of the heavy chain variable regions of these positive clones were determined. Table 5 shows the amino acid sequences of the Nanobody molecules, and Tables 6 and 7 show the CDR sequences of the Nanobody molecules.

[0141] Table 5 Variable region amino acid sequences of anti-DLL3 nanobodies

[0142] According to the Kabat numbering system, the results of the CDR sequence analysis of the above-mentioned Nanobody variable region are shown in Table 6:

[0143] Table 6 Kabat analysis results of the variable region CDR sequences of anti-DLL3 nanobodies

[0144] According to the IMGT numbering system, the results of the CDR sequence analysis of the above-mentioned Nanobody variable region are shown in Table 7:

[0145] Table 7 IMGT analysis results of the variable region CDR sequences of anti-DLL3 nanobodies

[0146] Example 3: Identification of DLL3 Nanobodies

[0147] 3.1 Expression and purification of recombinant nanobodies

[0148] The obtained nanobody sequences were cloned into the eukaryotic expression vector pTT5 with an Fc tag (Fc sequence, EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, SEQ ID NO.49), and transiently transfected into Expi293F cells (purchased from Gibco, A14527) by PEI. After 6 days of culture, the cell culture supernatant expressing the antibody was collected by high-speed centrifugation. The antibody was purified according to the purification method described in Section 1.1 of Example 1 to obtain the corresponding recombinant nanobody.

[0149] 3.2 ELISA detection of the binding of nanoantibodies to human DLL3 protein

[0150] The Nanobodies obtained above were subjected to ELISA detection and data analysis according to the method in Section 1.3 of Example 1. The OD450nm value was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The binding activity results of the Nanobodies to human DLL3 protein are shown in Figures 5A-5C. The results show that the Nanobodies have good binding activity to human DLL3 protein. The IgG control is hIgG1, 52D04 is the positive control, and the data in the table are OD 450nm value.

[0151] 3.3 Enzyme-linked immunosorbent assay (ELISA) detection of the binding of nanobodies to human DLL1 and DLL4 proteins

[0152] The Nanobodies obtained above were subjected to ELISA detection and data analysis according to the method in Section 1.3 of Example 1. The OD450nm value was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The maximum binding values ​​of the Nanobodies to human DLL1 (purchased from Acro, Catalog No.: DL1-H52H8) or DLL4 (purchased from Acro, Catalog No.: DL4-H5227) proteins at a concentration of 100 nM are shown in Table 8. The results show that DLL3-VHH2 and DLL3-VHH5 bind to hDLL1 or DLL4, while the remaining antibodies have little binding to human DLL1 or DLL4. The negative control is hIgG1, and 52D04 is the positive control. The data in the table are OD 450nm value.

[0153] Table 8 ELISA detection of the binding reaction between nanobodies and human DLL1 and DLL4 proteins

[0154] 3.4 Flow cytometry (FACS) assay to detect the binding activity of nanoantibodies to cells overexpressing human DLL3

[0155] The desired human DLL3 overexpressing cell line, CHO K1-hDLL3 cells, were expanded in a T-75 cell culture flask to the logarithmic growth phase. The culture medium was aspirated, the cells were washed twice with PBS buffer, and the cells were trypsinized. The digestion was then terminated with complete culture medium and the cells were pipetted to a single cell suspension. After cell counting, the cells were centrifuged and the cell pellet was resuspended in FACS buffer (PBS + 2% fetal bovine serum) to a concentration of 2 × 10 6For 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 to detect and analyze the results. 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-6C, indicating that all Nanobodies can bind to CHO K1-hDLL3 cells.

[0156] Table 9 FACS detection of nanobody binding reaction with CHO K1-hDLL3 cells

[0157] 3.5 Flow cytometry (FACS) assay to detect the binding activity of nanoantibodies to cells endogenously expressing DLL3

[0158] SHP77 is a human small cell lung cancer cell line, and the detection method refers to the method in Section 3.4 of Example 3. The FACS results of the binding of Nanobodies to SHP77 cells are shown in Table 10 and Figures 7A-7C, indicating that all Nanobodies can bind to SHP77 cells.

[0159] Table 10 FACS detection of the binding activity of nanobodies to SHP77 cells

[0160] Example 4: Detection of cross-binding activity of DLL3 nanobodies

[0161] 4.1 FACS detection of the binding activity of nanobodies to monkey DLL3

[0162] The detection method was similar to that described in Section 3.4 of Example 3. The FACS results for the binding of Nanobodies to the monkey DLL3-overexpressing cell line CHO K1-cyno DLL3 are shown in Table 11 and Figures 8A-8C , demonstrating that all Nanobodies bind to CHO K1-cyno DLL3 cells. The IgG control was hIgG1, and 52D04 was the positive control. The data in the table are mean fluorescence intensity (MFI) values.

[0163] Table 11 FACS detection of the binding reaction between nanobodies and CHO K1-cynoDLL3 cells

[0164] 4.2 FACS detection of the binding activity of nanobodies to mouse DLL3

[0165] The detection method was similar to that described in Section 3.4 of Example 3. The FACS results of Nanobodies binding to the mouse DLL3-overexpressing cell line CHO K1-mDLL3 are shown in Table 12 and Figures 9A-9C . The results demonstrate that, with the exception of DLL3-VHH1 and DLL3-VHH3, which weakly bound to mouse DLL3, all other Nanobodies bound to CHO K1-mDLL3 cells, demonstrating mouse cross-reactivity. The IgG control was hIgG1, and 52D04 was the positive control. Data in the table represent mean fluorescence intensity (MFI) values.

[0166] Table 12 FACS detection of nanobody binding reaction with CHO K1-mDLL3 cells

[0167] Example 5: Affinity detection of DLL3 nanobodies

[0168] The experiment used a Biacore 8K (GE) instrument and multi-cycle kinetics to determine the affinity of the tested anti-DLL3 nanobody to the DLL3 antigen protein.

[0169] 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.

[0170] The experimental data were fitted with the (1:1) Langmuir model using GE Biacore 8K Evaluation version 2.0 software to obtain the association rate (Ka), dissociation rate (Kd), and affinity value (KD) of the humanized antibody to the human DLL3 protein, as shown in Table 13.

[0171] Table 13 Binding affinity of nanobodies to human DLL3 protein

[0172] Example 6: Humanized design of anti-human DLL3 nanobody

[0173] By comparing the IMGT (http: / / imgt.cines.fr) human antibody variable region germline gene database and MOE (Molecular Operating Environment) software, heavy chain variable region germline genes with high homology to camel nanobodies were selected as templates. The CDRs of the camel nanobodies were transplanted into the corresponding human templates (where CDRs are identified and annotated using the IMGT numbering system), forming a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Back mutations and / or mutations at sites prone to modification were performed as needed.

[0174] 6.1 Humanization of DLL3-VHH4

[0175] 1. DLL3-VHH4 germline sequence selection

[0176] The humanized templates of antibody DLL3-VHH4 are IGHV3-23*04 and IGHJ3*01. The CDRs of antibody DLL3-VHH4 are transplanted into the corresponding humanized templates to obtain humanized antibodies.

[0177] 2. Design of humanized DLL3-VHH4 antibody

[0178] As needed, key amino acids in the FR region sequence of the DLL3-VHH4 humanized antibody were back-mutated to maintain the original affinity. At the same time, given the presence of a high-risk modification site DG, amino acid mutations were performed on DG using computational simulation based on the antibody structure to eliminate modification risks. Specific mutation designs are shown in Table 14.

[0179] Table 14 Humanized antibody design of DLL3-VHH4

[0180] Note: S35G indicates a mutation from S to G at position 35, and the same applies to the remaining residues. Amino acid residues are numbered in natural order. Underlined residues are mutations at sites prone to modification; the remaining residues are back mutations.

[0181] The amino acid sequence of the humanized heavy chain variable region is shown in Table 15:

[0182] Table 15 Variable region amino acid sequence and germline gene sequence of DLL3-VHH4 humanized antibody

[0183] According to the IMGT numbering system, the results of the CDR sequence analysis of the above humanized antibody heavy chain variable region are shown in Table 16.

[0184] Table 16 IMGT analysis results of the variable region CDR sequences of the DLL3-VHH4 humanized antibody

[0185] 6.2 Humanization of DLL3-VHH5

[0186] 1. DLL3-VHH5 germline sequence selection

[0187] The humanization templates of antibody DLL3-VHH5 are IGHV3-23*01 and IGHJ2*01. The CDRs of antibody DLL3-VHH5 are transplanted into the corresponding humanization templates to obtain humanized antibodies.

[0188] 2. Humanized antibody design of DLL3-VHH5

[0189] As needed, key amino acids in the FR region sequence of the DLL3-VHH5 humanized antibody were backmutated to maintain the original affinity. At the same time, given the presence of a site NG that is prone to deamidation in the antibody, amino acid mutations of NG were performed using computational simulation based on the antibody structure to eliminate modification risks. Specific mutation designs are shown in Table 17 (backmutations are in natural numbering order).

[0190] Table 17 Humanized antibody design of DLL3-VHH5

[0191] Note: V37Y indicates a V to Y mutation at position 37, and the same applies to the remaining residues. Amino acid residues are numbered in natural order. Underlined residues are mutations at sites prone to modification; the remaining residues are back mutations.

[0192] The amino acid sequence of the humanized heavy chain variable region is shown in Table 18:

[0193] Table 18 DLL3-VHH5 humanized antibody backmutated variable region amino acid sequence

[0194] According to the IMGT numbering system, the results of the CDR sequence analysis of the heavy chain variable region of the above humanized antibody are shown in Table 19:

[0195] Table 19 IMGT analysis results of the variable region CDR sequences of the DLL3-VHH5 humanized antibody

[0196] 6.3 Humanization of DLL3-VHH2

[0197] 1. DLL3-VHH2 germline sequence selection

[0198] The humanized heavy chain templates of the VHH nanoantibody DLL3-VHH2 are IGHV3-23*04 and IGHJ2*01. The CDRs of the VHH nanoantibody DLL3-VHH2 are transplanted into their human templates respectively to obtain the corresponding humanized versions.

[0199] 2. Design of DLL3-VHH2 humanized antibody

[0200] As needed, the key amino acids in the FR region sequence of the humanized antibody of DLL3-VHH2 were back-mutated to the amino acids corresponding to the VHH nanobody to ensure the original affinity. The specific mutations are shown in Table 20.

[0201] Table 20 Humanized antibody design of DLL3-VHH2

[0202] Note: V37Y indicates a V to Y mutation at position 37, and the same applies to the remaining residues. Amino acid residues are numbered in natural order. Underlined residues are mutations at sites prone to modification; the remaining residues are back mutations.

[0203] The amino acid sequence of the humanized heavy chain variable region is shown in Table 21:

[0204] Table 21 DLL3-VHH2 humanized antibody backmutated variable region amino acid sequence

[0205] According to the IMGT numbering system, the results of the CDR sequence analysis of the heavy chain variable region of the above humanized antibody are shown in Table 22:

[0206] Table 22 IMGT analysis results of the variable region CDR sequences of the DLL3-VHH2-hFc humanized antibody

[0207] 6.4 DLL3-VHH6

[0208] 1. DLL3-VHH6 germline sequence selection

[0209] The humanized templates for antibody DLL3-VHH6 are IGHV3-9*01 / IGHV3-66*01 and IGHJ3*01. The CDRs of antibody DLL3-VHH6 are transplanted into the corresponding humanized templates to obtain humanized antibodies.

[0210] 2. Design of DLL3-VHH6 humanized antibody

[0211] As needed, key amino acids in the FR region sequence of the DLL3-VHH6 humanized antibody were back-mutated to maintain the original affinity. At the same time, given the presence of a site DG prone to deamidation in the antibody, amino acid mutations were performed on DG using computational simulation based on the antibody structure to eliminate modification risks. Specific mutation designs are shown in Table 23 (back mutations are in natural numbering order).

[0212] Table 23 Humanized antibody design of DLL3-VHH6

[0213] Note: H35D indicates that the H at position 35 is changed to a D, and so on. Amino acid residues are numbered in natural order. Underlined residues are mutations at sites prone to modification; the rest are back mutations.

[0214] The amino acid sequence of the humanized heavy chain variable region is shown in Table 24:

[0215] Table 24 DLL3-VHH6 humanized antibody backmutated variable region amino acid sequence

[0216] According to the IMGT numbering system, the results of the CDR sequence analysis of the heavy chain variable region of the above humanized antibody are shown in Table 25:

[0217] Table 25 IMGT analysis results of the variable region CDR sequences of the DLL3-VHH6 humanized antibody

[0218] Example 7: Identification of humanized antibodies against DLL3

[0219] The obtained humanized Nanobody sequences were cloned into the eukaryotic expression vector pTT5 with an Fc tag (the Fc sequence is shown in SEQ ID NO. 49), and transiently transfected into Expi293F cells (purchased from Gibco, A14527) using PEI. After 6 days of culture, the cell culture supernatant expressing the antibody was collected by high-speed centrifugation. The antibodies were then purified according to the purification method described in Section 1.1 of Example 1 to obtain the corresponding recombinant humanized Nanobodies.

[0220] 7.1 ELISA assay to detect the binding of humanized antibodies to human DLL3 protein

[0221] The humanized antibodies obtained above were 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 humanized antibodies to human DLL3 protein is shown in Table 26 and Figures 10A-10E. The results show that the humanized antibodies have good binding activity to human DLL3 protein. The IgG control was hIgG1, and 522D04 was the positive control. The data in the table are OD 450nm value.

[0222] Table 26 ELISA detection of binding reaction between humanized antibodies and human DLL3 protein

[0223] 7.2 ELISA assay for binding of humanized antibodies to human DLL1 and DLL4 proteins

[0224] The humanized antibodies obtained above were subjected to ELISA testing and data analysis 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 27. The results show that the humanized antibodies have essentially no binding to human DLL1 or DLL4. The negative control is hIgG1, and 52D04 is the positive control. The data in the table are OD 450nm value.

[0225] Table 27 ELISA detection of binding reaction between humanized antibodies and human DLL1 and DLL4 proteins

[0226] 7.3 Flow cytometry (FACS) assay to detect the binding activity of humanized antibodies to cells endogenously expressing DLL3

[0227] The humanized antibodies obtained above were subjected to FACS analysis and data analysis according to the method in Section 3.4 of Example 3. Data analysis was performed using FlowJo software to obtain the mean fluorescence intensity (MFI) of the cells. Tables 28-30 and Figures 11A-11D, 12, and 13A-13D demonstrate that the humanized antibodies maintained substantial activity against the high-DLL3-expressing SHP77 cell line and the low- to medium-DLL3-expressing NCI-H82 and NCI-H2171 cell lines.

[0228] Table 28 FACS detection of binding activity of humanized antibodies to SHP77 cells

[0229] Table 29 FACS detection of binding activity of humanized antibodies to NCI-H82 cells

[0230] Table 30 FACS detection of binding activity of humanized antibodies to NCI-H2171 cells

[0231] 7.4 Flow cytometry (FACS) assay for binding activity of humanized antibodies to cells overexpressing DLL3

[0232] The humanized antibodies obtained above were subjected to FACS analysis and data analysis according to the method in Section 3.4 of Example 3. Data analysis was performed using FlowJo software to obtain the mean fluorescence intensity (MFI) of the cells. Table 31 and Figures 14A-14E demonstrate that all humanized antibodies can bind to CHO-K1 hDLL3 cells.

[0233] Table 31 FACS detection of binding activity of humanized antibodies to CHO-K1 hDLL3 cells

[0234] Example 8: Detection of cross-binding activity of DLL3 humanized antibodies

[0235] 8.1 FACS detection of humanized antibody binding activity to monkey DLL3

[0236] The humanized antibodies obtained above were subjected to FACS analysis and data analysis according to the method described in Section 3.4 of Example 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 32 and Figures 15A-15E . These results demonstrate that the humanized antibodies maintain substantial binding activity to CHO K1-cyno DLL3 cells. The IgG control is hIgG1, and the data in the table represent mean fluorescence intensity (MFI) values.

[0237] Table 32 FACS detection of humanized antibody binding reaction with CHO K1-cyno DLL3 cells

[0238] 8.2 FACS detection of binding activity of humanized antibodies to mouse DLL3

[0239] The humanized antibodies obtained above were subjected to FACS analysis and data analysis according to the method described in Section 4.2 of Example 4. The FACS results of binding of the humanized antibodies to the mouse DLL3-overexpressing cell line CHO K1-mDLL3 are shown in Table 33 and Figures 16A-16E . These results demonstrate that the humanized antibodies maintain substantial binding activity to CHO K1-mDLL3 cells and exhibit mouse cross-reactivity. The IgG control is hIgG1, and the data in the table represent mean fluorescence intensity (MFI) values.

[0240] Table 33 FACS detection of humanized antibody binding reaction with CHO K1-mDLL3 cells

[0241] Example 9: Affinity detection of DLL3 humanized antibodies

[0242] This experiment used a Biacore 8K (GE) instrument to determine the affinity of the humanized anti-DLL3 antibody for the DLL3 antigen using multi-cycle kinetics. The 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. The reaction signal was monitored in real time on the Biacore 8K instrument (GE) to generate association and dissociation curves. After each dissociation cycle, the antigen-antibody complex was washed and regenerated with glycine-HCl regeneration solution (Cat. BR-1003-54, Cytiva), pH 1.5. Binding was monitored by injecting various concentrations of DLL3 antigen in solution for 180 seconds at a flow rate of 30 μL / min, starting with 20 nM (see detailed results for the actual concentrations tested), followed by a 1:1 dilution series. Dissociation was continued for 600 seconds, and the chip surface was finally regenerated by washing with 10 mM glycine-HCl solution (pH 1.5) for 30 seconds at a flow rate of 30 μL / min.

[0243] The experimental data were fitted with the (1:1) Langmuir model using GE Biacore 8K Evaluation version 2.0 software to obtain the association rate (Ka), dissociation rate (Kd), and affinity value (KD) of the humanized antibody to the human DLL3 protein, as shown in Table 34.

[0244] Table 34 Binding affinity of humanized antibodies to human DLL3 protein

Claims

1. An antibody or antigen-binding fragment that specifically binds to human Delta-Like Ligand 3 (DLL3), wherein: The antibody or antigen-binding fragment comprises a heavy chain variable region, wherein 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 NOs. 7-12, 50-52, 57-58, 60-63.

2. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen binding comprises the following HCDR1, HCDR2, HCDR3: (1) According to the Kabat numbering system, HCDR1-3 has an amino acid sequence as shown in SEQ ID NO.13-30, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence as shown in SEQ ID NO.13-30, or having a sequence identity of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%; (2) According to the IMGT numbering system, HCDR1-3 has an amino acid sequence as shown in SEQ ID NO.31-48, 55-56, 59, 64, or a combination of sequences having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences as shown in SEQ ID NO.31-48, 55-56, 59, 64, or having a sequence identity of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

3. The antibody or antigen-binding fragment according to any one of claims 1 to 2, wherein: The heavy chain variable region sequence comprises a sequence as shown in SEQ ID NO.7-12, 50-52, 57-58, 60-63, or has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence as shown in SEQ ID NO.7-12, 50-52, 57-58, 60-63.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein The antibody or antigen-binding fragment is chimeric, humanized or fully human.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein The antibody or antigen-binding fragment can bind to Delta-Like Ligand 3 (DLL3) derived from humans, monkeys or mice.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein It may also contain any constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or 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; further, the antibody or antigen-binding fragment thereof is also coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioactive isotopes, chemotherapeutic drugs, cytotoxic agents and immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from methotrexate, anthracycline antibiotics (doxorubicin), pyrrolobenzodiazepines (pyrrolobenzodiazepine, PBD), gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is a taxane.

7. The antigen-binding fragment according to any one of claims 1 to 6, wherein The antibody or antigen-binding fragment is selected from one or more of nanobodies or affibodies.

8. A multispecific antigen-binding molecule, wherein: The multispecific antigen-binding molecule comprises the antibody or antigen-binding fragment of any one of claims 1 to 7 and an antigen-binding molecule that binds to an antigen other than DLL3, or comprises the antibody or antigen-binding fragment of any one of claims 1 to 7 and an antigen-binding molecule that binds to a DLL3 epitope different from the DLL3 epitope bound by the antibody or antigen-binding fragment of any one of claims 1 to 7; Preferably, the other antigen-binding molecule is an antibody or an antigen-binding fragment; 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.

9. An isolated nucleic acid fragment, wherein The nucleic acid fragment encodes the antibody or antigen-binding fragment of any one of claims 1 to 7 or the multispecific antigen-binding molecule of claim 8.

10. A recombinant vector, wherein: The vector comprises the isolated nucleic acid fragment of claim 9.

11. A host cell, wherein The host cell comprises the recombinant vector as claimed in claim 10; 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).

12. A method for preparing a product comprising the antibody or antigen-binding fragment of any one of claims 1 to 7 or the multispecific antigen-binding molecule of claim 8; the method comprising the steps of culturing the host cell of claim 11, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the host cell.

13. A pharmaceutical composition, wherein: The pharmaceutical composition comprises the antibody or antigen-binding fragment according to claims 1 to 7, the multispecific antigen-binding molecule according to claim 8, the nucleic acid fragment according to claim 9, the recombinant vector according to claim 10, or a product prepared by the method according to claim 12; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.

14. The pharmaceutical composition according to claim 13, wherein The pharmaceutical composition may further comprise an additional anti-tumor agent.

15. 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 of claims 1 to 7, the multispecific antigen-binding molecule of claim 8, the nucleic acid fragment of claim 9, the recombinant vector of claim 10, the product prepared according to the method of claim 12, or the pharmaceutical composition of any one of claims 13 to 14; 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 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.

16. Use of the antibody or antigen-binding fragment of claims 1 to 7, the multispecific antigen-binding molecule of claim 8, the nucleic acid fragment of claim 9, the recombinant vector of claim 10, the product obtained by the method of claim 12, or the pharmaceutical composition of any one of claims 13 to 14 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 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.

17. A kit comprising the antibody or antigen-binding fragment of claims 1 to 7, the multispecific antigen-binding molecule of claim 8, the nucleic acid fragment of claim 9, the recombinant vector of claim 10, the product prepared according to the method of claim 12, or the pharmaceutical composition of any one of claims 13 to 14.

18. A method for detecting DLL3 expression in a biological sample, the method comprising contacting the biological sample with the antibody or antigen-binding fragment as claimed in claims 1 to 7 under reaction conditions capable of forming a complex between the antibody or antigen-binding fragment and DLL3; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of DLL3 in the sample.

19. Use of the antibody or antigen-binding fragment according to claims 1 to 7 in the preparation of a DLL3 detection reagent.

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