DLL3 antigen binding proteins and uses thereof

Abstract

The invention relates to a DLL3 antigen binding protein and application thereof, in particular to a separated antigen binding protein capable of binding DLL3, the antigen binding protein comprises antibody heavy chain variable regions VH and VL, and the VH and the VL comprise at least one CDR. The invention also provides a chimeric antigen receptor comprising the DLL3 antigen binding protein, a modified immune cell, an immunoconjugate, a pharmaceutical composition, a pharmaceutical combination, a nucleic acid encoding the antigen binding protein, a carrier comprising the nucleic acid molecule, and a cell comprising the carrier. The invention also provides application of the antigen binding protein in prevention and / or treatment of diseases.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and specifically relates to an antigen-binding protein capable of binding to DLL3 and its uses. Background Art

[0002] Delta-like ligand 3 (DLL3) is a single-pass transmembrane protein belonging to the Notch ligand family and is expressed in various human cancers. In normal tissues, DLL3 is expressed at low levels and is mostly confined to the inner cell membrane, most notably the Golgi apparatus, while it is highly expressed on the cell surface of tumors of neuroendocrine origin such as small cell lung cancer (SCLC), melanoma, and glioblastoma multiforme, and will be transported to the cell membrane, making it an ideal tumor treatment target with relatively good specificity and homogeneity. DLL3 is closely related to the enhancement of the proliferation, migration, and invasion abilities of tumor cells by inhibiting the Notch signaling pathway in tumor cells.

[0003] Therefore, as a highly promising target in tumor treatment, there is still a need to develop more therapeutic drugs that can effectively bind to it, such as antibodies, ADCs, etc., and to study more strategies targeting DLL3 to meet the treatment needs of a wide range of diseases and bring more treatment options for tumor patients. Summary of the Invention

[0004] This application provides an antigen-binding protein targeting DLL3. In this application, the antigen-binding protein has one or more of the following properties: (1) capable of specifically binding to DLL3 and having good binding activity; (2) capable of having good internalization ability. This application also provides an antibody-drug conjugate (ADC) comprising the antigen-binding protein. In this application, the ADC comprising has one or more of the following properties: (1) capable of specifically binding to DLL3 and having good binding activity; (2) capable of having good internalization ability.

[0005] On the one hand, the present application provides a separated antigen-binding protein capable of binding to DLL3, wherein the antigen-binding protein comprises an antibody heavy-chain variable region VH, the VH comprises HCDR3, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:5. In certain embodiments, the VH comprises HCDR2, and the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:4. In certain embodiments, the VH comprises HCDR1, and the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:3. In certain embodiments, the antigen-binding protein comprises an antibody heavy-chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:5. In certain embodiments, the amino acid sequence of the VH is as shown in SEQ ID NO:2.

[0006] In certain embodiments, the antigen-binding protein comprises an antibody light-chain variable region VL, the VL comprises LCDR3, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:13. In certain embodiments, the VL comprises LCDR2, and the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:12. In certain embodiments, the VL comprises LCDR1, and the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:11. In certain embodiments, the antigen-binding protein comprises an antibody light-chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:13. In certain embodiments, the amino acid sequence of the VL is as shown in SEQ ID NO:10.

[0007] In certain embodiments, the antigen-binding protein comprises a VH and a VL, the VH comprises HCDR1, HCDR2, and HCDR3, the VL comprises LCDR1, LCDR2, and LCDR3, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:5, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0008] In certain embodiments, the antigen-binding protein comprises a VH and a VL, the amino acid sequence of the VH is as shown in SEQ ID NO:2, and the amino acid sequence of the VL is as shown in SEQ ID NO:10.

[0009] In certain embodiments, the antigen-binding protein further comprises an immunoglobulin constant region. In certain embodiments, the immunoglobulin constant region is a heavy chain constant region and / or a light chain constant region of a human antibody. In certain embodiments, the heavy chain constant region is a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the light chain constant region of the human antibody is a human kappa (Kappa) or lambda (Lambda) light chain constant region.

[0010] In certain embodiments, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody. In certain embodiments, the antigen-binding protein is an scFv, the scFv comprises a VH and a VL, the VH comprises HCDR1, HCDR2, and HCDR3, the VL comprises LCDR1, LCDR2, and LCDR3, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:5, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13. In certain embodiments, the antigen-binding protein is an scFv, the scFv comprises a VH and a VL, the amino acid sequence of the VH is as shown in SEQ ID NO:2, and the amino acid sequence of the VL is as shown in SEQ ID NO:10.

[0011] In certain embodiments, the antigen-binding protein is a chimeric antibody, a humanized antibody or a fully human antibody. In certain embodiments, the antigen-binding protein is a monospecific antibody, a bispecific antibody or a multispecific antibody. In certain embodiments, the antigen-binding protein is a monovalent antibody, a bivalent antibody or a multivalent antibody.

[0012] On the other hand, the present application provides an immunoconjugate comprising the antigen-binding protein.

[0013] In certain embodiments, the immunoconjugate comprises the antigen-binding protein, a linker and a payload. In certain embodiments, the payload is a cytotoxic drug, a polymer, a protein, a radioisotope, a nucleic acid compound and / or a glucocorticoid. In certain embodiments, the payload is a cytotoxic drug. In certain embodiments, the payload is a chemotherapeutic drug, a tubulin inhibitor, a DNA damaging agent and / or a topoisomerase Ι inhibitor. In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC). In certain embodiments, the linker is an uncleavable linker, an enzyme-cleavable linker, an acid-cleavable linker, a GSH-cleavable reducing linker, an Fe(II)-cleavable linker, a light-responsive cleavable linker and / or a bioorthogonal cleavable linker.

[0014] On the other hand, the present application provides a chimeric antigen receptor comprising the antigen-binding protein.

[0015] On the other hand, the present application provides a modified immune cell comprising the chimeric antigen receptor.

[0016] On the other hand, the present application provides an isolated nucleic acid molecule encoding the antigen-binding protein.

[0017] On the other hand, the present application provides a vector comprising the nucleic acid molecule.

[0018] On the other hand, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0019] On the other hand, the present application provides a pharmaceutical composition comprising the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0020] On the other hand, the present application provides a kit, which comprises the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition, and the kit is used for detecting the presence and / or content of DLL3 in a sample or a subject.

[0021] On the other hand, the present application provides the use of the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease and / or disorder.

[0022] On the other hand, the present application provides a method for preventing and / or treating a disease and / or disorder, which comprises administering the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition to a subject in need thereof.

[0023] On the other hand, the present application provides the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition for preventing and / or treating a disease and / or disorder.

[0024] In certain embodiments, the disease and / or disorder is a tumor.

[0025] In certain embodiments, the tumor is a DLL3-positive tumor.

[0026] In certain embodiments, the tumor is small cell lung cancer, neuroendocrine carcinoma, glioblastoma and / or melanoma.

[0027] Those skilled in the art can easily insight into other aspects and advantages of the present application from the following detailed description. Only the exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of the present application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in the present application. Accordingly, the descriptions in the drawings and the specification of the present application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The specific features of the invention involved in the present application are shown as in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the exemplary embodiments and the drawings described in detail below. A brief description of the drawings is as follows:

[0029] Figure 1 The figure shows the flow cytometry assay results of the DLL3 antibody described in the present application.

[0030] Figure 2 The figure shows the staining results of the endocytosis experiment of the DLL3 antibody described in the present application.

[0031] Figure 3A The figure shows the flow cytometry assay results of the DLL3 antibody described in the present application and a control antibody; Figure 3B The figure shows the ELISA results of the DLL3 antibody described in the present application.

[0032] Figure 4A The figure shows the internalization results of the DLL3 antibody described in the present application and a control antibody on MB231 target cells; Figure 4B The figure shows the internalization results of the DLL3 antibody described in the present application and a control antibody on 293T-DLL3 target cells; Figure 4C The figure shows the internalization results of the DLL3 antibody described in the present application and a control antibody on SHP77 target cells. Detailed implementation manners

[0033] The following specific embodiments illustrate the implementation manners of the invention of the present application. Those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

[0034] Term definitions

[0035] In the present application, the terms "DLL3 (Delta-Like Ligand 3)" and "Delta-like ligand 3" are generally interchangeable and usually refer to a transmembrane protein belonging to the Notch ligand family. In the present application, the DLL3 may be an inhibitory Notch ligand. In the present application, the human DLL3 protein may consist of 619 amino acids and is characterized by containing an N-terminal conserved DSL (Delta, Serrate, Lag2) domain consisting of 40 amino acids, six EGF-like repeat sequences, and a transmembrane domain. In the present application, the DLL3 may be expressed on small cell lung cancer, neuroendocrine cancer, glioblastoma, and / or melanoma. In the present application, the DLL3 may be the complete DLL3 and its functionally active fragments, homologs, analogs, variants, or derivatives. For example, the DLL3 may be the full-length DLL3 or a truncated DLL3 that retains functional activity. In the present application, the DLL3 may be from any species source. For example, the DLL3 may be human DLL3. In the present application, the DLL3 may be wild-type or artificially modified. For example, the DLL3 may be a modified DLL3.

[0036] The protein and / or amino acid sequences involved in the present application should also be understood to include at least the following scope: variants or homologs having the same or similar functions as the said protein. In the present application, the said variant may be a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the said protein (for example, the DLL3 antigen-binding protein described in the present application). For example, the said functional variant may include a protein or polypeptide having an amino acid change by substitution, deletion and / or insertion of at least 1, for example, 1-30, 1-20 or 1-10, and again for example, 1, 2, 3, 4 or 5 amino acids. The said functional variant may substantially maintain the biological characteristics of the said protein or the said polypeptide before the change (for example, substitution, deletion or addition). For example, the said functional variant may maintain at least 60%, 70%, 80%, 90% or 100% of the biological activity (for example, the ability to bind to the DLL3 antigen) of the said protein or the said polypeptide before the change. For example, the said substitution may be a conservative substitution. In the present application, a part of the amino acid sequence of the said antigen-binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class. For example, both the variable region and the constant part of the antibody may be from the variable region and the constant region of an antibody of an animal species (such as a human). In the present application, the said homolog may be a protein or polypeptide having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the said protein and / or the said polypeptide (for example, the DLL3 antigen-binding protein described in the present application).

[0037] In the present application, the term "antigen-binding protein" generally refers to a protein having the ability to bind an antigen. For example, the antigen-binding protein may be a protein having the ability to bind the DLL3 antigen. In the present application, the antigen-binding protein may comprise a portion that binds the antigen and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that facilitates binding of the antigen-binding portion to the antigen. In the present application, the antigen-binding protein may be wild-type or artificially modified. In the present application, the antigen-binding protein may comprise, for example, a protein scaffold derived from an antibody or an alternative protein scaffold or an artificial scaffold having transplanted CDRs or CDR derivatives. In the present application, the CDRs can be determined by a variety of coding systems. For example, the CDRs can be determined by CCG, Kabat, Chothia, IMGT, AbM, taking into account Kabat / Chothia etc. comprehensively. In the present application, the CDRs cover CDR sequences obtained by partitioning according to any CDR partitioning method. In the present application, the CDRs may cover variants thereof. For example, the amino acid sequence of the CDRs may be subjected to substitution, deletion and / or addition of one or more amino acids, such as 1-30, 1-20 or 1-10, or for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions. For example, the CDRs may cover homologs. For example, the homolog may be an amino acid sequence having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the CDRs. In the present application, the antigen-binding protein may be synthetic. In the present application, the antigen-binding protein may be of animal or non-animal origin. In the present application, the antigen-binding protein may be an antibody or an antigen-binding fragment thereof, as well as variants, homologs, derivatives or analogs thereof. In the present application, the antigen-binding protein may include, but is not limited to, antibodies, antigen-binding fragments (Fab, Fab’, F(ab)2, Fv fragments, F(ab’)2, scFv, di-scFv, VHH and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they exhibit the required antigen-binding activity. In the present application, the antigen-binding protein may be a VHH. In the present application, the antibody may be a chimeric antibody, a humanized antibody or a fully human antibody. In the present application, the antibody may be a recombinant, hybrid, mutant or transplanted antibody.

[0038] In the present application, the terms "VHH", "nanobody", and "single-domain antibody" are generally used interchangeably and generally refer to an antibody structure consisting of a variable heavy chain region. In the present application, the VHH may consist of VH. In the present application, the VHH may comprise complementarity-determining regions (CDRs) and framework regions (FRs). In the present application, the VHH may comprise CDR1-3 and H-FR1-4. For example, the CDRs and FRs may be arranged in the following order from the amino terminus to the carboxyl terminus: FR-1, CDR1, FR-2, CDR2, FR-3, CDR3, and FR-4. In the present application, the VHH may be synthetic. In the present application, the VHH may be artificially modified.

[0039] In the present application, the terms "immunoglobulin constant region" and "constant region" are generally used interchangeably, and the constant region generally refers to the region of an antibody that is not variable. In the present application, the constant region may not directly participate in antigen binding but exhibits various effector functions. In the present application, the constant region may be the entire non-variable region of an antibody or a part of the non-variable region of an antibody. For example, the immunoglobulin constant region is the heavy chain constant region of human IgG and / or the light chain constant region of a human antibody. For example, the heavy chain constant region of human IgG is the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. For example, the light chain constant region of a human antibody is the human κ (Kappa) or λ (Lambda) light chain constant region. In the present application, the constant region may be from different species. For example, the constant region may be derived from humans, goats, rabbits, rats, or guinea pigs.

[0040] In the present application, the term "immune cell" generally refers to a cell that participates in an immune response. For example, the immune cell may be an immune cell that exerts effector functions. For example, the exertion of effector functions may include clearing foreign antigens or promoting immune effector responses, etc. In the present application, the immune effector cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, iNKT cells, dendritic cells, granulocytes, lymphocytes, white blood cells, peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells, and / or pluripotent stem cells. For example, the immune effector cell may be a T cell.

[0041] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating other active ingredients with the antigen-binding protein. In the present application, the active ingredient may be covalently linked to the antigen-binding protein through a linker molecule. In the present application, the immunoconjugate may be a conjugate of an antigen-binding protein and a payload. For example, the payload may be a toxin, a polymer, a protein, a drug, a radioisotope, a nucleic acid compound, or a glucocorticoid. In the present application, the immunoconjugate may be an antibody-drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), an antibody-oligonucleotide conjugate (AOC), or a radioligand conjugate (RDC). In the present application, the conjugate may specifically bind to an antigen on a target cell through the antigen-binding protein and deliver the payload to the target cell. For example, the target cell may be a tumor cell.

[0042] In the present application, the term "pharmaceutically acceptable carrier" generally refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. For example, the pharmaceutically acceptable carrier includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cells or mammals exposed to them at the doses and concentrations employed. For example, a physiologically acceptable carrier may be water, salts, proteins, polysaccharides, lipids, or inactive virus particles.

[0043] In the present application, the term "prevention and / or treatment" generally refers to the prevention and / or treatment of diseases. For example, the prevention and / or treatment may be to prevent the onset of the disease, slow down or reverse the disease process, prevent or slow down the onset of one or more symptoms associated with the disease, reduce or alleviate one or more symptoms associated with the disease, reduce the severity and duration of the disease and any symptoms associated therewith, or prevent a further increase in the severity of the disease and any symptoms associated therewith. In the present application, the disease may be a tumor disease. For example, the disease may be a tumor disease with positive DLL3 expression. For example, prevent or alleviate the onset of one or more symptoms associated with the tumor, and reduce the severity and duration of the tumor and the symptoms associated therewith.

[0044] In the present application, the term "tumor" generally refers to any new pathological tissue hyperplasia, with tumor antigens that can be recognized by the immune system. In the present application, the tumor may include benign or malignant tumors (cancers). In the present application, the cancer may be metastatic cancer and non-metastatic cancer. In the present application, the tumor may include solid tumors and / or hematological tumors. In the present application, the solid tumor generally refers to a tangible tumor that can be detected by clinical examination means. For example, the solid tumor may include neoplasms or solid lesions formed by abnormal cell growth. In the present application, the hematological tumor generally refers to a class of hematopoietic system diseases. In the present application, the hematological tumor may include various leukemias, multiple myeloma, or malignant lymphoma. In the present application, the tumor may be a DLL3-positive tumor. For example, the tumor may be small cell lung cancer, neuroendocrine carcinoma, glioblastoma, and / or melanoma.

[0045] In the present application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0046] In the present application, the term "comprising" generally means including the specifically designated features, but not excluding other elements.

[0047] In the present application, the term "about" generally means varying within a range of 0.5% - 10% above or below the specified value, for example, varying within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value. Detailed Description of the Invention

[0049] Antigen-binding protein

[0050] The CDR of an antibody, also known as the complementarity-determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or epitope. The antibody CDR can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and by considering Kabat / Chothia comprehensively, etc. These coding systems are known in the art. For specific references, see, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR encompasses the CDR sequences obtained by any CDR partitioning method; it also encompasses its variants, where the variants include the amino acid sequence of the CDR with substitution, deletion, and / or addition of one or more amino acids. For example, 1 - 30, 1 - 20, or 1 - 10 amino acids, and for another example, substitution, deletion, and / or insertion of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids; it also encompasses its homologs, where the homologs can be amino acid sequences having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the CDR. For example, the CDR of the isolated antigen-binding protein described in the sequence listing of this application can be determined using Kabat.

[0051] On the one hand, this application provides an isolated antigen-binding protein that can bind to DLL3, wherein the antigen-binding protein comprises at least one CDR in the variable region of the antibody heavy chain.

[0052] In this application, the antigen-binding protein can have one or more of the following properties: (1) It can specifically bind to DLL3 and has good binding activity; (2) It can have good internalization ability. In this application, the antigen-binding protein can bind to DLL3 with an affinity / binding activity similar to or higher than that of a control antibody. For example, the antigen-binding protein can bind to DLL3 with a KD value similar to or lower than that of a control antibody. For example, the antigen-binding protein can bind to DLL3 with an EC50 value similar to or lower than that of a control antibody. In this application, the EC50 value or KD value can be determined by conventional technical means in the art. In this application, the KD value can be determined by Octet, SPR, ELISA, competitive ELISA, or BIACORE or KINEXA. In this application, the isolated antigen-binding protein can specifically bind to DLL3. In this application, the specific binding can be determined by FACS.

[0053] In the present application, the antigen-binding protein may comprise a heavy-chain variable region of an antibody, VH, and the VH comprises heavy-chain complementarity-determining regions HCDR1, HCDR2, and / or HCDR3. For example, the VH comprises heavy-chain complementarity-determining regions HCDR1, HCDR2, and HCDR3.

[0054] In the present application, the VH may comprise HCDR3. For example, the amino acid sequence of the HCDR3 may be as shown in SEQ ID NO:5. In the present application, the VH may comprise HCDR2. For example, the amino acid sequence of the HCDR2 may be as shown in SEQ ID NO:4. In the present application, the VH may comprise HCDR1. For example, the amino acid sequence of the HCDR1 may be as shown in SEQ ID NO:3.

[0055] In the present application, the HCDR1, HCDR2, and / or HCDR3 may be wild-type sequences. In the present application, the amino acid sequences of the HCDR1, HCDR2, and / or HCDR3 may also be modified and / or altered. For example, one or more amino acid sequences in the HCDR1, HCDR2, and / or HCDR3 may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to DLL3. For example, the HCDR1, HCDR2, and / or HCDR3 may be variants thereof, and the variants include the amino acid sequences of the HCDR1, HCDR2, and / or HCDR3 with substitution, deletion, and / or addition of one or more amino acids. For example, the HCDR1, HCDR2, and / or HCDR3 may also comprise sequences having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher homology to the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4, and / or SEQ ID NO:5.

[0056] In the present application, the antigen-binding protein may comprise VH, the VH may comprise HCDR1, HCDR2, and HCDR3, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:5.

[0057] In the present application, the VH may further comprise H-FR1, H-FR2, H-FR3, and / or H-FR4. For example, the VH may comprise H-FR1, H-FR2, H-FR3, and H-FR4.

[0058] In the present application, the VH may comprise H-FR1. For example, the amino acid sequence of the H-FR1 may be as shown in SEQ ID NO:6. In the present application, the VH may comprise H-FR2. For example, the amino acid sequence of the H-FR2 may be as shown in SEQ ID NO:7. In the present application, the VH may comprise H-FR3. For example, the amino acid sequence of the H-FR3 may be as shown in SEQ ID NO:8. In the present application, the VH may comprise H-FR4. For example, the amino acid sequence of the H-FR4 may be as shown in SEQ ID NO:9.

[0059] In the present application, the H-FR1, H-FR2, H-FR3 and / or H-FR4 may be wild-type sequences. In the present application, the amino acid sequences of the H-FR1, H-FR2, H-FR3 and / or H-FR4 may also be modified and / or altered. For example, one or more amino acid sequences of the H-FR1, H-FR2, H-FR3 and / or H-FR4 may be mutated or optimized on the premise of not reducing the binding activity / affinity of the antigen-binding protein to DLL3. For example, the H-FR1, H-FR2, H-FR3 and / or H-FR4 may be variants thereof, and the variants include the amino acid sequence of the H-FR4 with one or more amino acids substituted, deleted and / or added. For example, the H-FR1, H-FR2, H-FR3 and / or H-FR4 may also comprise sequences having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher homology with the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and / or SEQ ID NO:9.

[0060] In the present application, the C-terminus of the H-FR1 may be directly or indirectly connected to the N-terminus of the HCDR1. In the present application, the H-FR2 may be located between the HCDR1 and the HCDR2. In the present application, the H-FR3 may be located between the HCDR2 and the HCDR3. In the present application, the N-terminus of the H-FR4 may be directly or indirectly connected to the C-terminus of the HCDR3. In the present application, the antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and H-FR4, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3.

[0061] In the present application, the antigen-binding protein may comprise a VH, and the VH comprises H-FR1, H-FR2, H-FR3, and H-FR4. The amino acid sequence of H-FR1 is as shown in SEQ ID NO:6, the amino acid sequence of H-FR2 is as shown in SEQ ID NO:7, the amino acid sequence of H-FR3 is as shown in SEQ ID NO:8, and the amino acid sequence of H-FR4 is as shown in SEQ ID NO:9.

[0062] In the present application, the antigen-binding protein may comprise a VH. For example, the amino acid sequence of the VH may be as shown in SEQ ID NO:2. For example, the VH may be a wild-type sequence. In the present application, the amino acid sequence of the VH may also be modified and / or altered. For example, one or more amino acid sequences in the VH may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to DLL3. For example, the VH may be a variant thereof, and the variant includes substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the VH. For example, the VH may further comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher homology to the amino acid sequence shown in SEQ ID NO:2.

[0063] In the present application, the antigen-binding protein may comprise a variable light chain (VL) of an antibody, and the VL comprises light chain complementarity-determining regions (LCDR1, LCDR2, and / or LCDR3). For example, the VL comprises LCDR1, LCDR2, and LCDR3.

[0064] In the present application, the VL may comprise LCDR3. For example, the amino acid sequence of LCDR3 may be as shown in SEQ ID NO:13. In the present application, the VL may comprise LCDR2. For example, the amino acid sequence of LCDR2 may be as shown in SEQ ID NO:12. In the present application, the VL may comprise LCDR1. For example, the amino acid sequence of LCDR1 may be as shown in SEQ ID NO:11.

[0065] In the present application, the LCDR1, LCDR2, and / or LCDR3 may be wild-type sequences. In the present application, the amino acid sequences of the LCDR1, LCDR2, and / or LCDR3 may also be modified and / or altered. For example, one or more amino acid sequences in the LCDR1, LCDR2, and / or LCDR3 may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to DLL3. For example, the LCDR1, LCDR2, and / or LCDR3 may be variants thereof, and the variants include the amino acid sequences of the LCDR1, LCDR2, and / or LCDR3 with one or more amino acids substituted, deleted, and / or added. For example, the LCDR1, LCDR2, and / or LCDR3 may also contain sequences having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher homology with the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, and / or SEQ ID NO:13.

[0066] In the present application, the antigen-binding protein may comprise a VL, and the VL may comprise LCDR1, LCDR2, and LCDR3. The amino acid sequence of the LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:13.

[0067] In the present application, the VL may further comprise L-FR1, L-FR2, L-FR3, and / or L-FR4. For example, the VL may comprise L-FR1, L-FR2, L-FR3, and L-FR4.

[0068] In the present application, the VL may comprise L-FR1. For example, the amino acid sequence of the L-FR1 may be as shown in SEQ ID NO:14. In the present application, the VL may comprise L-FR2. For example, the amino acid sequence of the L-FR2 may be as shown in SEQ ID NO:15. In the present application, the VL may comprise L-FR3. For example, the amino acid sequence of the L-FR3 may be as shown in SEQ ID NO:16. In the present application, the VL may comprise L-FR4. For example, the amino acid sequence of the L-FR4 may be as shown in SEQ ID NO:17.

[0069] In the present application, the L-FR1, L-FR2, L-FR3, and / or L-FR4 may be wild-type sequences. In the present application, the amino acid sequences of the L-FR1, L-FR2, L-FR3, and / or L-FR4 may also be modified and / or altered. For example, one or more amino acid sequences of the L-FR1, L-FR2, L-FR3, and / or L-FR4 may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to DLL3. For example, the L-FR1, L-FR2, L-FR3, and / or L-FR4 may be variants thereof, and the variants include the amino acid sequence of the L-FR4 with one or more amino acids substituted, deleted, and / or added. For example, the L-FR1, L-FR2, L-FR3, and / or L-FR4 may also contain sequences having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher homology with the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:17.

[0070] In the present application, the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1. In the present application, the L-FR2 may be located between the LCDR1 and the LCDR2. In the present application, the L-FR3 may be located between the LCDR2 and the LCDR3. In the present application, the N-terminus of the L-FR4 may be directly or indirectly connected to the C-terminus of the LCDR3. In the present application, the antigen-binding protein may comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR4, wherein the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, the L-FR2 is located between the LCDR1 and the LCDR2, the L-FR3 is located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3.

[0071] In the present application, the antigen-binding protein may comprise VL, and the VL comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein the amino acid sequence of the L-FR1 is as shown in SEQ ID NO:14, the amino acid sequence of the L-FR2 is as shown in SEQ ID NO:15, the amino acid sequence of the L-FR3 is as shown in SEQ ID NO:16, and the amino acid sequence of the L-FR4 is as shown in SEQ ID NO:17.

[0072] In the present application, the antigen-binding protein may comprise a VL. For example, the amino acid sequence of the VL may be as shown in SEQ ID NO: 10. For example, the VL may be a wild-type sequence. In the present application, the amino acid sequence of the VL may also be modified and / or altered. For example, one or more amino acid sequences in the VL may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to DLL3. For example, the VL may be a variant thereof, and the variant includes the amino acid sequence of the VL with one or more amino acids substituted, deleted, and / or added. For example, the VL may further comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher homology with the amino acid sequence shown in SEQ ID NO: 10.

[0073] In the present application, the direct or indirect connection may be connected by intermolecular forces or may be connected by a linker. In the present application, the amino acid sequence of the FR may be from any species source. For example, the FR may be from a mouse, rabbit, goat, alpaca or human source. For example, the FR may be a human-derived FR.

[0074] In the present application, the antigen-binding protein may comprise a VH and a VL. The VH comprises HCDR1, HCDR2 and HCDR3, and the VL comprises LCDR1, LCDR2 and LCDR3. The amino acid sequence of HCDR1 is as shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 4, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 5, the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 11, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 13.

[0075] In the present application, the antigen-binding protein may comprise a VH and a VL. The amino acid sequence of the VH is as shown in SEQ ID NO: 2, and the amino acid sequence of the VL is as shown in SEQ ID NO: 10.

[0076] In the present application, the antigen-binding protein may further comprise an immunoglobulin constant region. In the present application, the immunoglobulin constant region may be a heavy chain constant region and / or a light chain constant region. In the present application, the heavy chain constant region may be the heavy chain constant region of IgG. In the present application, the heavy chain constant region of IgG may be the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4. For example, the heavy chain constant region of IgG may be the IgG1 heavy chain constant region. In the present application, the light chain constant region may be the κ (Kappa) or λ (Lambda) light chain constant region.

[0077] In the present application, the immunoglobulin constant region may be a human heavy chain constant region and / or a human light chain constant region. In the present application, the human heavy chain constant region may be the heavy chain constant region of human IgG. In the present application, the heavy chain constant region of human IgG may be the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4. For example, the heavy chain constant region of human IgG may be the human IgG1 heavy chain constant region. In the present application, the human light chain constant region may be the human κ (Kappa) or human λ (Lambda) light chain constant region.

[0078] In the present application, the immunoglobulin constant region may be an immunoglobulin constant region from any species source. For example, the immunoglobulin constant region may be an immunoglobulin constant region from mouse, rabbit, goat or human. For example, the immunoglobulin constant region may be a human immunoglobulin constant region. In the present application, the immunoglobulin constant region may be the heavy chain constant region of human IgG and / or the light chain constant region of a human antibody. In the present application, the heavy chain constant region of human IgG may be the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4. For example, the heavy chain constant region of human IgG may be the human IgG1 heavy chain constant region. In the present application, the light chain constant region of the human antibody may be the human κ (Kappa) or λ (Lambda) light chain constant region.

[0079] In the present application, the antigen-binding protein may be an antibody or an antigen-binding fragment thereof. In the present application, the antigen-binding fragment may be the Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH or dAb fragment of the antibody.

[0080] In the present application, the antigen-binding protein may be a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0081] In the present application, the antigen-binding protein may be a monovalent antibody, a bivalent antibody or a multivalent antibody.

[0082] For example, the antigen-binding protein can be mutated / optimized for the FR and constant regions without reducing the binding activity / affinity of the antigen-binding protein to DLL3. For example, the antigen-binding protein can have lower immunogenicity without reducing the binding activity / affinity of the antigen-binding protein to DLL3.

[0083] In the present application, the antigen-binding protein can be a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0084] In the present application, the antigen-binding protein can be an antibody or an antigen-binding fragment thereof. In the present application, the antigen-binding fragment is a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH or dAb fragment of the antibody.

[0085] In the present application, the antigen-binding protein can be an scFv. In the present application, the scFv can comprise VH and VL, the VH comprises HCDR1, HCDR2 and HCDR3, the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:5, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13. In the present application, the scFv can comprise VH and VL, the amino acid sequence of VH is as shown in SEQ ID NO:2, and the amino acid sequence of VL is as shown in SEQ ID NO:10.

[0086] In the present application, the scFv can further comprise a linker. In the present application, the scFv can comprise VH and VL, and VH and VL can be connected by a linker. In the present application, the linker can be (GGGS)4 or (GGGGS)4. In the present application, the amino acid sequence of the scFv can be as shown in SEQ ID NO:1.

[0087] Immunoconjugate

[0088] On the other hand, the present application provides an immunoconjugate, which may comprise the antigen-binding protein described above. In the present application, the antigen-binding protein may be as described above. In the present application, the immunoconjugate may specifically bind to DLL3. For example, the immunoconjugate may specifically bind to DLL3-positive target cells. For example, the immunoconjugate may target DLL3. In the present application, the immunoconjugate may comprise the antigen-binding protein, a linker, and a payload.

[0089] In the present application, the various parts of the immunoconjugate are directly or indirectly linked. For example, the various parts of the immunoconjugate are indirectly linked. For example, the various parts of the immunoconjugate are linked through a linker.

[0090] In the present application, the payload may be a cytotoxic drug, a polymer, a protein, a radioisotope, a nucleic acid compound, or a glucocorticoid.

[0091] In the present application, the immunoconjugate may be an antibody-drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a radioligand-drug conjugate (RDC). For example, the immunoconjugate may be an antibody-drug conjugate.

[0092] In the present application, the PROTAC-antibody conjugate may comprise the antigen-binding protein, a linker, and a PROTAC. In the present application, the PROTAC may comprise a target protein ligand, a linker, and an E3 ligase ligand. In the present application, the E3 ligase ligand may be CRBN, VHL, IAP, MDM2, DCF15, RNF114, DCAF16, KEAP1, or FEM1B.

[0093] In the present application, the radioligand-drug conjugate may comprise the antigen-binding protein, a linker, and a radioisotope. In the present application, the radioisotope may be iodine-131, radium-223, thallium-201, or arsenic-211.

[0094] On the other hand, the present application also provides an antibody-drug conjugate comprising the antigen-binding protein, which has one or more of the following properties: (1) capable of specifically binding to DLL3 and having good binding activity; (2) capable of having good internalization ability.

[0095] In the present application, the immunoconjugate may comprise one or more payloads. In the present application, the immunoconjugate may comprise one or more payloads. For example, the immunoconjugate may comprise one, two, or more (e.g., 3, 4, 5, 6, 7, or 8) cytotoxic drugs. In the present application, the antibody-drug conjugate (ADC) may be a single-payload, dual-payload ADC, or multi-payload ADC.

[0096] In the present application, the immunoconjugate may comprise the antigen-binding protein, the linker, and the cytotoxic drug. In the present application, the cytotoxic drug may be a chemotherapeutic drug, a tubulin inhibitor, a DNA-damaging agent, or a topoisomerase Ι inhibitor. In the present application, the chemotherapeutic drug may be vinblastine, doxorubicin, or its derivatives, etc. In the present application, the tubulin inhibitor may be auristatin, eribulin, maytansine, tubulysin, cryptophycin, mitotic EG5 inhibitor, or its derivatives, etc. For example, the cytotoxic drug may be MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), DM1, DM4, or Tubulysins. In the present application, the DNA-damaging agent may be calicheamicin, esperamicin, anthramycin derivatives, pyrrolobenzodiazepines, indolobenzodiazepines, or its derivatives, etc. For example, the DNA-damaging agent may be PBD or IBD. In the present application, the cytotoxic drug may be an apoptosis inducer (such as a Bcl-xL inhibitor), talipexole and its analogs (such as thailanstatin A), amanitin (such as α-amanitin or β-amanitin), nicotinamide phosphoribosyltransferase, or camamycin (such as camamycin A and camamycin B). In the present application, the topoisomerase Ι inhibitor may be camptothecin, hydroxycamptothecin, irinotecan, topotecan, or its derivatives, etc.

[0097] In the present application, the antigen-binding protein and the cytotoxic drug are linked by a linker. In the present application, the linker may be an uncleavable linker or a cleavable linker. In the present application, the cleavable linker may break within the target cell and release the payload. In the present application, the cleavable linker may be an enzyme-cleavable linker, an acid-cleavable linker, or a GSH-cleavable reducing linker. In the present application, the linker may be an Fe(II)-cleavable linker, a light-responsive cleavable linker, or a bioorthogonal cleavable linker. In the present application, the linker may be a hydrazone, a disulfide bond, a carbonate, an acetal, a ketal, MC-VC-PABC, PEG8-VA-PABC, MC-VC-PAB, MCC, MC, GGFG (SEQ ID NO:29), or MC-GGFG, etc.

[0098] In the present application, the antibody-drug conjugate may further comprise a spacer unit. In the present application, the spacer unit may be incorporated between the cleavable linker and the payload, or be part of the cleavable linker itself. In the present application, after the cleavable linker breaks under suitable conditions, the spacer unit can spontaneously rearrange its structure, thereby releasing the payload linked thereto. In the present application, the spacer unit may be p-aminobenzyl alcohol (PAB), β-glucuronide, substituted or unsubstituted ethylenediamine, etc.

[0099] Chimeric antigen receptor

[0100] On the other hand, the present application provides a chimeric antigen receptor (CAR), which may comprise the antigen-binding protein.

[0101] In the present application, the chimeric antigen receptor may comprise an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In the present application, the chimeric antigen receptor may further comprise a hinge region. In the present application, the chimeric antigen receptor may further comprise an intracellular co-stimulatory domain. In the present application, the chimeric antigen receptor may further comprise a signal peptide.

[0102] On the other hand, the present application provides a modified immune cell, wherein the immune cell may comprise and / or express the chimeric antigen receptor. In the present application, the immune cell may comprise and / or express one or more chimeric antigen receptors. In the present application, the immune cell may comprise and / or express one or more types of chimeric antigen receptors.

[0103] In the present application, the immune cell may be a T cell, NK cell, NKT cell, dendritic cell, macrophage, tumor infiltrating lymphocyte (TIL), invariant natural killer T cell (iNKT), cytokine-induced killer cell (CIK), γδ T cell, and / or double negative T cell (DNT cell). For example, the immune cell may be an immune effector cell. For example, the immune cell may be a T cell. For example, the immune cell may be a mixture, and the mixture may contain different types of immune cells. For example, the mixture may contain one or more immune cells.

[0104] Nucleic acid molecule, vector and cell

[0105] On the other hand, the present application further provides an isolated nucleic acid molecule, which may encode the antigen-binding protein and / or the chimeric antigen receptor. In the present application, the antigen-binding protein may be as described above.

[0106] In the present application, the nucleic acid molecule may further comprise a sequence encoding a signal peptide.

[0107] In the present application, the nucleic acid molecule can be produced or synthesized by the following methods: (i) amplified in vitro, for example, by polymerase chain reaction (PCR) amplification; (ii) produced by cloning and recombination; (iii) purified, for example, by digestion with enzymes and fractionation by gel electrophoresis; or (iv) synthesized, for example, by chemical synthesis. In the present application, the nucleic acid molecule can be DNA and / or RNA. In the present application, the nucleic acid molecule can be an artificially synthesized nucleic acid analogue. In the present application, the nucleic acid molecule can be a modified nucleic acid molecule.

[0108] On the other hand, the present application provides a vector, which can contain the nucleic acid molecule.

[0109] In the present application, the vector can contain one or more of the nucleic acid molecules. In the present application, the vector can contain one or more of the nucleic acid molecules. In the present application, the vector can be an expression vector or a cloning vector. In the present application, the vector can be a viral vector or a non-viral vector. In the present application, the vector can be a viral vector, a plasmid vector, a phage vector or other vectors commonly used in, for example, genetic engineering. For example, the viral vector can be an adenovirus, an adeno-associated virus, a retrovirus (including a lentivirus). In the present application, the vector can be a fusion vector or a non-fusion vector.

[0110] In the present application, the vector can also contain other genes. For example, the other gene can be a marker gene.

[0111] In the present application, the vector can contain a variety of elements that control expression. For example, the vector can include a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and / or a reporter gene. For example, the vector can also contain an origin of replication. For example, the vector can include components that assist in entering the cell. In order for the nucleic acid molecule to replicate in the vector, the 5' end and 3' end of the nucleic acid molecule can also contain long terminal repeats.

[0112] In the present application, the nucleic acid molecule can contain a nucleic acid sequence encoding a promoter. In the present application, the sequence encoding the promoter is usually operably linked to the coding sequence of the protein to be expressed. In the present application, the promoter can be any nucleic acid sequence that shows transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. In the present application, the promoter can be selected from one or more of the following groups: EF1α, CMV, MSCV, SSFV and UbC.

[0113] On the other hand, the present application provides a cell, which contains the nucleic acid molecule and / or the vector.

[0114] In the present application, the cells may include the progeny of a single cell. Due to natural, accidental or intentional mutations, the progeny may not necessarily be identical to the original parent cell (either morphologically or genomically in the total DNA complement). In the present application, the cells may be prokaryotic cells (e.g., bacterial cells), CHO cells, NS / 0 cells, HEK293T cells or HEK293A cells, or other eukaryotic cells such as fungal or yeast cells, etc.

[0115] In the present application, the cells may contain one or more of the nucleic acid molecules and / or one or more vectors. In the present application, the vectors may contain one or more of the nucleic acid molecules and / or one or more vectors.

[0116] In the present application, the vectors may be introduced into the cells by methods known in the art. For example, the method may be electroporation, Lipofectine transfection or Lipofectamin transfection.

[0117] On the other hand, the present application provides a method for preparing the antigen-binding protein, the chimeric antigen receptor and / or the immunoconjugate. In the present application, the method comprises culturing the cells under conditions that allow expression of the antigen-binding protein and / or the chimeric antigen receptor.

[0118] Pharmaceutical composition

[0119] On the other hand, the present application also provides a pharmaceutical composition, which may contain the antigen-binding protein, the chimeric antigen receptor, the modified immune cells, the immunoconjugate, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0120] In the present application, the pharmaceutical composition may contain a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the doses and concentrations used. The pharmaceutical compositions of the present application may include liquid, frozen and lyophilized compositions.

[0121] In the present application, the pharmaceutically acceptable carrier may contain any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with drug administration, and are generally safe, non-toxic and neither biologically nor otherwise undesirable.

[0122] In the present application, the pharmaceutical composition may be administered parenterally, transdermally, intracavitary, intraarterially, intrathecally, and / or intranasally or directly injected into a tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

[0123] Drug combination

[0124] On the other hand, the present application provides a pharmaceutical combination comprising the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, and / or the cell, and the pharmaceutical combination may further comprise one or more additional active ingredients. For example, the pharmaceutical combination may further comprise substances related to immune responses. For example, the pharmaceutical combination may further comprise drugs related to immune responses.

[0125] On the other hand, the present application also provides a scheme for the combined use of the antigen-binding protein with one or more other active ingredients. For example, the antigen-binding protein is used in combination with one or more other therapeutic agents. In the present application, the pharmaceutical combination may be administered separately, simultaneously, or sequentially. In the present application, the pharmaceutical combination may be administered in the same or different doses or administration methods. For example, the active ingredients in the pharmaceutical combination are administered to a patient as separate entities in the same / different doses and administration methods. In the present application, the components in the pharmaceutical combination may be administered to a patient simultaneously in the form of a single entity or dose. For example, the components in the pharmaceutical combination are administered to a patient simultaneously, jointly, or sequentially as separate entities. In the present application, the specific administration method may be determined according to the category of the active ingredient, and the specific administration dose may be adjusted according to the degree of the subject's illness, the subject's physical condition, etc.

[0126] In the present application, the different active ingredients in the pharmaceutical combination may be placed together or separately. For example, the active ingredients may be placed in the same container. For example, the active ingredients may be placed in different containers.

[0127] Use

[0128] On the other hand, the present application also provides the use of the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition, and / or the pharmaceutical combination in the preparation of a drug, and the drug may be used for preventing, diagnosing, and / or treating diseases and / or disorders.

[0129] On the other hand, the present application also provides a method for preventing, diagnosing, and / or treating a disease and / or disorder, which may include administering to a subject in need thereof the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition, and / or the pharmaceutical combination.

[0130] On the other hand, the present application also provides the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition, and / or the pharmaceutical combination, which can be used for preventing, diagnosing, and / or treating a disease and / or disorder.

[0131] In the present application, the disease and / or disorder may be a DLL3-related disease and / or disorder. In the present application, the disease and / or disorder may be a tumor. In the present application, the tumor may be a solid tumor and / or a hematologic tumor. In the present application, the tumor may be a DLL3-positive tumor. In the present application, the hematologic tumor may include various types of leukemia, multiple myeloma, and / or malignant lymphoma. In the present application, the tumor may be small cell lung cancer, neuroendocrine carcinoma, glioblastoma multiforme, and / or melanoma.

[0132] In the present application, the prevention, diagnosis, and / or treatment may be preventing the onset of the disease, slowing or reversing the disease process, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing a further increase in the severity of the disease and any symptoms associated therewith.

[0133] On the other hand, the present application provides the use of the antigen-binding protein according to the present application for preparing a diagnostic agent, wherein the diagnostic agent is used for diagnosing a disease and / or disorder related to the expression of DLL3 protein.

[0134] On the other hand, the present application provides the antigen-binding protein according to the present application, which can be used for diagnosing a disease and / or disorder related to the expression of DLL3 protein.

[0135] In the present application, the diagnostic agent may be used alone or may be used in combination with an instrument, apparatus, device, or system. During the prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation, and prediction of genetic diseases of a disease, the diagnostic agent can be used to perform in vitro detection on a human sample (e.g., various body fluids, cells, tissue samples, etc.).

[0136] On the other hand, the present application also provides a kit, which may include the antigen-binding protein, and the kit is used for detecting the presence and / or content of DLL3 in a sample or a subject. For example, the kit can be used for preventing, diagnosing, and / or treating diseases and / or disorders.

[0137] For example, the present application relates to an immunoassay kit using the antigen-binding protein of the present application in combination with immunoassay methods such as ELISA, immunohistochemistry, Western blotting, flow cytometry, etc.

[0138] On the other hand, the use of the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition, and / or the drug combination of the present application in the preparation of a kit for diagnosis.

[0139] In the present application, the container component of the kit generally will include at least one vial, test tube, flask, bottle, syringe, or other container component into which an antibody or preferably an appropriate aliquot of the antibody can be placed. The kit of the present application will typically also include a component for containing the antibody, antigen, and any other reagent containers, which are in a sealed form for commercial sale. Such containers can include injection-molded or blow-molded plastic containers in which the above vials are retained.

[0140] On the other hand, the present application also provides a method for detecting the presence and / or content of DLL3, which may include using the antigen-binding protein.

[0141] On the other hand, the present application provides a method for diagnosing a disease and / or disorder related to the expression of DLL3 protein in a subject, the method comprising: contacting a sample derived from the subject with the antigen-binding protein, and determining the presence and / or content of a substance in the sample that can specifically bind to the antigen-binding protein.

[0142] On the other hand, the present application provides a method for detecting DLL3 in a sample or a subject, the method comprising administering the antigen-binding protein. In the present application, the administration can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

[0143] Without being limited by any theory, the following examples are merely for explaining the antigen-binding protein, preparation method, and uses, etc. of the present application, and are not used to limit the scope of the invention of the present application.

[0144] Examples

[0145] Example 1 Preparation and Binding Force Verification of a Humanized Antibody

[0146] 1.1 Screening of antibodies targeting DLL3

[0147] Commission Xunyao Biotech to screen for antibody sequences targeting DLL3. Specifically, utilize the antibody library EMPRESS TM to screen for specific antibodies, and verify the positive ones by ELISA and analyze the high-affinity clones by sequencing alignment. The obtained DLL3 Fab fragment contains VH and VL. The VH contains HCDR1, HCDR2, and HCDR3, and the VL contains LCDR1, LCDR2, and LCDR3. The amino acid sequence of the VH is shown in SEQ ID NO:2, the amino acid sequence of the HCDR1 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:4, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:5, the amino acid sequence of the VL is shown in SEQ ID NO:10, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:13. Prepare scFv A1 according to the obtained Fab, and the amino acid sequence of the obtained scFv is shown in SEQ ID NO:1.

[0148] 1.2 Cell culture

[0149] The human small cell lung cancer cell line SHP-77, breast cancer cell lines MB231 and MB453, and human embryonic kidney cells HEK-293T were all purchased from ATCC in the United States. The suspension-cultured Expi293F cells were purchased from ThermoFisher Scientific (Cat no. A14527). To facilitate the detection of tumor cell killing, the target cells were stably transfected with GFP by lentiviral infection. The SHP-77, MB231, and MB453 cell lines were maintained in complete medium (IMEM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine). Some of the cells were suspended in the medium, and the culture medium was changed every three days. The HEK-293T-DLL3 cell line with overexpressed DLL3 was obtained by lentiviral infection of HEK-293T cells containing the DLL3 transgene and was maintained in adherent culture in complete medium (DMEM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine). Subculture was performed when the cell density reached 95%. The Expi293F cells used for antibody expression were inoculated in serum-free medium (Expi293 expression medium, Cat no. A1435101) and cultured with shaking in a constant temperature incubator at 37 °C, 8% CO2, and 125 rpm. Subculture was performed once the cell density reached 3×10 6 / mL.

[0150] 1.3 Flow cytometry

[0151] Flow cytometry was performed using a Beckman Coulter flow cytometer in semi-automatic or plate mode, and the data were analyzed using FlowJo software. The MB453, SHP-77, or HEK-293T-DLL3 cells were washed once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0), resuspended to 1-5×10 6 cells / mL, and placed on ice before staining. Recombinant antibodies with His or Fc tags were added for staining, and the cells were incubated in the dark at 4 °C for 30 minutes. Then, a large amount of FACS buffer was added to wash the cells to remove unbound antibodies. Subsequently, staining was performed with a fluorescently labeled secondary antibody, and the cells were incubated in the dark at 4 °C for 30 minutes. After staining, a large amount of FACS buffer was added to wash away unbound antibodies, and the cells were centrifuged at 300 x g for 3 minutes to remove the supernatant. After washing and resuspending with FACS buffer, the cells were loaded onto the flow cytometer for analysis. The flow cytometry data were analyzed and organized using FlowJo software.

[0152] Experimental results:

[0153] The results of flow cytometry are as follows Figure 1 As shown, compared with the negative control (PBS), both A1 of the present application and the positive control (Invitrogen, MA5-43156) showed stronger binding. The experimental results indicate that the DLL3 antibody obtained in the present application has a strong ability to bind to target cells and has good affinity.

[0154] Example 2 Immunofluorescence Staining to Verify Antibody Endocytosis Function

[0155] 2.1 Preparation and staining of immunofluorescence staining slides

[0156] Preparation for cell culture: Immerse round glass coverslips (18 mm) in ethanol for sterilization. Transfer the coverslips to a 12-well plate and add 1 ml of 50 μg / ml poly-D-lysine treatment to each well. Incubate at room temperature for 3 hours, remove the poly-D-lysine solution, rinse the coverslips with sterile water, and then air dry naturally. Culture the positive cell line SHP77 (DLL3+) on the poly-D-lysine-coated coverslips as needed.

[0157] Immunofluorescence staining: Rinse the cells once with PBS (1 ml per well), and then fix the cells with a solution containing 3% paraformaldehyde in PBS for 10 minutes. Remove the fixing solution and rinse the cells once with PBS-GSA (1 ml per well) to terminate the reaction. Incubate the cells with fresh PBS-GSA (1 ml per well) for 5 minutes. Treat the cells with PBS-GSA containing 0.5% Triton X-100 (freshly prepared) for 3 minutes to make them permeable. Remove the permeabilizing solution and quickly rinse the cells with PBS-GSA (1 ml per well). Incubate the cells with fresh PBS-GSA (1 ml per well) for 5 minutes. Dilute the DLL3 antibody A1 in PBS-GSA containing 1% BSA, and only prepare the amount required for the experiment (50 μl per coverslip). Incubate the antibody dilution with the cells. Co-culture at 37°C for 3 hours. Quickly rinse with PBS-GSA (1 ml per well). Use BD flow cytometry permeabilization reagent (BD Perm / Wash TMFix and permeabilize with buffer). A1 is tagged with His and can be captured by anti-HIS antibody-FITC, while CD107a-PE stains lysosome-related proteins on the cell membrane, which will be endocytosed into the cell. DAPI, as a background dye, can label the DNA in the nucleus as the cell background. Dilute anti-His FITC, anti-CD107-PE, and DAPI in PBS-GSA containing 1% and incubate with the cells in the dark for 30 minutes. Place the coverslip with the cell side facing up back into the tissue culture plate. Rinse quickly with PBS-GSA (1 ml per well). Incubate the cells with fresh PBS-GSA (1 ml per well) in the dark for 10 to 15 minutes. To mount each coverslip, place a drop of mounting medium (Vectashield) containing DAPI on the slide. Gently blot the back of the coverslip dry with a low-lint wipe (Kimwipe), and then place each coverslip with the cell side facing down on the mounting medium.

[0158] 2.2 Fluorescence microscopy and statistical analysis

[0159] Use a 20x objective of the Olympus IX73 microscope to find a clear field of view for confocal imaging. Randomly select four fields of view for each experimental condition. Calculate the number of cells that have internalized the antibody based on the fluorescence signals presented by the cells. Analyze the captured field-of-view images using ImageView (Version 4.11), and calculate the ratio of the number of antibody-internalized cells (His+CD107a+, yellow) to the total number of cells (DAPI, blue) in any four fields of view. Statistical analysis was performed by t-test (Student t-test), *p < 0.05.

[0160] Experimental results:

[0161] The internalization results are as Figure 2 shown. By statistically analyzing the number of internalized cells, the experimental results show that the DLL3 antibody described in this application has a high proportion of cells that internalize into SHP77 (DLL3+), showing good internalization effects and being suitable for the development of antibody-drug conjugates (ADCs).

[0162] Example 3 Affinity Comparison between Antibody A1 and Amgen Bispecific Antibody AMG757

[0163] 3.1 Expression and purification of recombinant antibodies

[0164] Clone antibody A1 containing the human IgG Fc tag and AMG757 into the eukaryotic expression vector pcDNA3.1 (Thermo Fisher Scientific) respectively, and verify by sequencing that the target sequences have been correctly inserted. Seed Expi293F cells at 1×10 6Inoculate at a density of / mL into a 125 mL Erlenmeyer flask (BioFIL, Cat no. TAB112125) and culture overnight. Then, transfect the expression plasmid into Expi293F cells at a ratio of DNA:PEI = 1:3 by mass, and culture with shaking in a constant temperature incubator at 37 °C, 8% CO2, and 125 rpm for 6 days. Centrifuge the cell suspension at 4200 rpm to obtain the supernatant, and then purify the antibody containing the Fc tag from the culture supernatant using Protein G affinity chromatography agar (Thermo Scientific, Cat no A50585). After concentration and washing, the obtained antibody is measured for concentration using a NanoDrop One spectrophotometer (Thermo Scientific) for subsequent experiments.

[0165] 3.2 Flow cytometry

[0166] The process of flow cytometry is as shown in Example 1.

[0167] 3.3 Enzyme-linked immunosorbent assay (ELISA)

[0168] Dissolve the target antigen DLL3 in ELISA coating buffer, dispense it into a 96-well plate at a quantity of 50 ng per well, and incubate overnight in the dark at 4 °C in a refrigerator. First, wash the 96-well plate with PBST (PBS + 0.1% Tween-20), and then incubate with a blocking solution (PBS containing 1% BSA) at 37 °C for one hour. Add the antibody to be tested to the 96-well plate and dilute it in a 3-fold gradient with the blocking solution, and then incubate at 37 °C for one hour. Discard the antibody to be tested, wash three times with PBST, add a goat anti-human IgG Fc HRP secondary antibody (Goat anti-Human IgG Fc HRP secondary antibody, Invitrogen, Cat no. A18817, 1:5000), and then incubate at 37 °C for one hour. Discard the secondary antibody, wash three times with PBST, add 50 μl of TMB substrate (Thermo Scientific, Cat no. N301), react at room temperature for 30 minutes, and then add 0.18 M H2SO4 to terminate the reaction. Measure the absorbance value at OD450nm using a spectrophotometer (Thermo Scientific, Multiskan Sky). Determine the affinity of the antibody for soluble DLL3 by ELISA.

[0169] Experimental results:

[0170] The results of flow cytometry are as Figure 3AAs shown, antibody A1 and AMG757 do not bind to the negative cell line MB453 (DLL3-), specifically bind to the DLL3-positive cells 293T-DLL3 and SHP77 (DLL3+), and the binding abilities of the two antibodies to the membrane antigen on the cell surface are comparable.

[0171] The ELISA results are as Figure 3B shown. Compared with the negative control, the half-maximal effective concentration (EC50) of antibody A1 is 38.9 ng / mL, and it has a relatively high affinity for soluble DLL3.

[0172] The experimental results show that the DLL3 antibody obtained in this application has a strong ability to bind to target cells, has a high affinity for the DLL3 antigen, and can bind well to target cells and target antigens.

[0173] Example 4 pH-Sensitive Dye Labeling Assay for Antibody Internalization

[0174] 4.1 Antibody internalization assay

[0175] Harvest the target cells to be inoculated and count them, dilute them with the medium to a cell concentration of 2×10 5 / mL, and then inoculate 50 μl of the cell suspension into each well of a 96-well flat-bottom cell culture plate. Place it in a constant temperature incubator at 37°C and 5% CO2 and let the cells settle and adhere to the bottom of the wells for 4-6 hours. Mix the antibody to be tested with the pH-sensitive dye at a molar ratio of 1:3, adjust the concentration of the antibody to twice the final concentration, and then incubate it in the 37°C constant temperature incubator in the dark for 15 minutes. Add 50 μl of the dye-labeled antibody to be tested to the 96-well plate already inoculated with target cells. Place the 96-well plate in an Incucyte scanning imager, set the color channel, and scan it once every 30 minutes to capture cell images. When the labeled antibody internalizes into intracellular lysosomes, the pH-sensitive dye shows a corresponding color due to the acidic environment, so the degree of antibody internalization can be determined according to the color signal.

[0176] Experimental results:

[0177] The internalization results are as Figure 4A shown in -C. In antigen-negative target cells (MB231), the antibodies to be tested did not internalize. In the DLL3-positive cell lines (293T-DLL3, SHP77), compared with the negative control, both antibody A1 and AMG757 showed rapid internalization, and the internalization ability of A1 was comparable to or even stronger than that of AMG757 (SHP77). For SHP77 target cells, A1 has a better internalization function compared to the positive antibody AMG757.

[0178] The experimental results show that the DLL3 antibody obtained in this application has good internalization ability and can enter cells better. The DLL3 antibody of this application is more suitable for the development of ADC drugs.

Claims

1. A isolated antigen-binding protein that is capable of binding to DLL3, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, the VH comprises HCDR3, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

5.

2. The antigen-binding protein according to claim 1, wherein the VH comprises HCDR2, and the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:

4.

3. The antigen-binding protein according to any one of claims 1-2, wherein the VH comprises HCDR1, and the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:

3.

4. The antigen-binding protein according to any one of claims 1-3, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

5.

5. The antigen-binding protein according to any one of claims 1-5, wherein the amino acid sequence of the VH is as shown in SEQ ID NO:

2.

6. The antigen-binding protein according to any one of claims 1-5, wherein the antigen-binding protein comprises an antibody light chain variable region VL, the VL comprises LCDR3, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:

13.

7. The antigen-binding protein according to claim 6, wherein the VL comprises LCDR2, and the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:

12.

8. The antigen-binding protein according to any one of claims 6-7, wherein the VL comprises LCDR1, and the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:

11.

9. The antigen-binding protein according to any one of claims 6-8, wherein the antigen-binding protein comprises an antibody light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:

13.

10. The antigen-binding protein according to any one of claims 6-9, wherein the amino acid sequence of the VL is as shown in SEQ ID NO:

10.

11. An antigen-binding protein according to any one of claims 1-10, wherein the antigen-binding protein comprises a VH and a VL, the VH comprises HCDR1, HCDR2 and HCDR3, the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:5, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

13.

12. An antigen-binding protein according to any one of claims 1-11, wherein the antigen-binding protein comprises a VH and a VL, the amino acid sequence of the VH is as shown in SEQ ID NO:2, and the amino acid sequence of the VL is as shown in SEQ ID NO:

10.

13. An antigen-binding protein according to any one of claims 1-12, wherein the antigen-binding protein further comprises an immunoglobulin constant region.

14. The antigen-binding protein according to claim 13, wherein the immunoglobulin constant region is a heavy chain constant region and / or a light chain constant region.

15. The antigen-binding protein according to claim 14, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

16. The antigen-binding protein according to any one of claims 14-15, wherein the light chain constant region is human κ (Kappa) or λ (Lambda) light chain constant region.

17. An antigen-binding protein according to any one of claims 1-16, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

18. The antigen-binding protein according to claim 17, wherein the antigen-binding fragment is the Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH or dAb fragment of the antibody.

19. An antigen-binding protein according to any one of claims 1-18, wherein the antigen-binding protein is an scFv, and the amino acid sequence of the scFv is as shown in SEQ ID NO:

1.

20. An immunoconjugate comprising the antigen-binding protein according to any one of claims 1-19.

21. The immunoconjugate according to claim 20, which comprises the antigen-binding protein, a linker and a payload.

22. The immunoconjugate according to claim 21, wherein the payload is a cytotoxic drug, a polymer, a protein, a radioisotope, a nucleic acid compound and / or a glucocorticoid.

23. The immunoconjugate according to any one of claims 20-22, wherein the immunoconjugate is an antibody-drug conjugate (ADC).

24. A chimeric antigen receptor comprising the antigen-binding protein according to any one of claims 1-19.

25. An isolated nucleic acid molecule encoding the antigen-binding protein according to any one of claims 1-19.

26. A vector comprising the nucleic acid molecule according to claim 25.

27. A cell comprising the nucleic acid molecule according to claim 25 and / or the vector according to claim 26.

28. A pharmaceutical composition comprising the antigen-binding protein according to any one of claims 1-19, the immunoconjugate according to any one of claims 20-23, the chimeric antigen receptor according to claim 24, the nucleic acid molecule according to claim 25, the vector according to claim 26, and / or the cell according to claim 27, and optionally a pharmaceutically acceptable carrier.

29. Use of the antigen-binding protein according to any one of claims 1-19, the immunoconjugate according to any one of claims 20-23, the chimeric antigen receptor according to claim 24, the nucleic acid molecule according to claim 25, the vector according to claim 26, the cell according to claim 27, and / or the pharmaceutical composition according to claim 28 in the preparation of a medicament for the prevention and / or treatment of a disease and / or disorder.

30. The use according to claim 29, wherein the disease and / or disorder is a tumor.

31. The use according to claim 30, wherein the disease and / or disorder is a DLL3-positive tumor.

32. The use according to any one of claims 30-31, wherein the tumor is small cell lung cancer, neuroendocrine carcinoma, glioblastoma, and / or melanoma.

Images