Dll3 antigen binding proteins and uses thereof

Abstract

The present application relates to DLL3 antigen binding proteins and uses thereof, in particular to an isolated antigen binding protein capable of binding DLL3, said antigen binding protein comprising an antibody heavy chain variable region VH and VL, said VH and VL comprising at least one CDR. The present application also provides a chimeric antigen receptor comprising said DLL3 antigen binding protein, a modified immune cell, an immunoconjugate, a pharmaceutical composition, a pharmaceutical combination, a nucleic acid encoding said antigen binding protein, a vector comprising said nucleic acid molecule, a cell comprising said vector. The present application also provides the use of said antigen binding protein in preventing and / or treating a disease.

Description

Technical Field

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

[0002] Delta-like ligand 3 (DLL3) is a single-pass transmembrane protein belonging to the Notch ligand family, expressed in various human cancers. In normal tissues, DLL3 expression is low and mostly confined to the intracellular membrane, most notably the Golgi apparatus. However, it is highly expressed on the surface of neuroendocrine-derived tumor cells such as small cell lung cancer (SCLC), melanoma, and glioblastoma multiforme, and is transported to the cell membrane, making it a relatively ideal target for tumor therapy in terms of specificity and homogeneity. In tumor cells, DLL3 is closely related to enhanced tumor cell proliferation, migration, and invasion by inhibiting the Notch signaling pathway.

[0003] Therefore, as a target with great development potential in cancer treatment, DLL3 still needs to be further explored by developing more therapeutic drugs that can effectively bind to it, such as antibodies and ADCs, as well as researching more strategies targeting DLL3, in order to meet the treatment needs of a wide range of diseases and bring more treatment options to cancer 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) it can specifically bind to DLL3 and has good binding activity; (2) it has good internalization ability. This application also provides an antibody-drug conjugate (ADC) comprising the antigen-binding protein. In this application, the ADC comprises one or more of the following properties: (1) it can specifically bind to DLL3 and has good binding activity; (2) it has good internalization ability.

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

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

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

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

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

[0010] In some embodiments, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some 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 some embodiments, the antigen-binding protein is scFv, the scFv comprising VH and VL, the VH comprising HCDR1, HCDR2, and HCDR3, the VL comprising LCDR1, LCDR2, and LCDR3, the amino acid sequence of HCDR1 being as shown in SEQ ID NO:3, the amino acid sequence of HCDR2 being as shown in SEQ ID NO:4, the amino acid sequence of HCDR3 being as shown in SEQ ID NO:5, the amino acid sequence of LCDR1 being as shown in SEQ ID NO:11, the amino acid sequence of LCDR2 being as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 being as shown in SEQ ID NO:13. In some embodiments, the antigen-binding protein is scFv, which comprises VH and VL, wherein the amino acid sequence of VH is shown in SEQ ID NO:2 and the amino acid sequence of VL is shown in SEQ ID NO:10.

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

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

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

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

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

[0016] On the other hand, this application provides isolated nucleic acid molecules that encode the antigen-binding protein.

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

[0018] On the other hand, this application provides cells that contain the nucleic acid molecules and / or the vector.

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

[0020] On the other hand, this application provides a kit comprising the antigen-binding protein, the chimeric antigen receptor, the modified immune cells, the immune conjugate, the nucleic acid molecule, the carrier, the cells, and / or the pharmaceutical composition, the kit being used to detect the presence and / or content of DLL3 in a sample or subject.

[0021] On the other hand, this application provides the use of the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0022] On the other hand, this application provides a method for preventing and / or treating diseases and / or conditions, comprising administering to a subject in need the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition.

[0023] On the other hand, this application provides the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition for the prevention and / or treatment of diseases and / or conditions.

[0024] In some implementations, the disease and / or symptom is a tumor.

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

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

[0027] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

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

[0029] Figure 1 The image shown is a flow cytometry result of the DLL3 antibody described in this application.

[0030] Figure 2 The image shown is a staining result of the endocytosis experiment of the DLL3 antibody described in this application.

[0031] Figure 3A The image shown is a flow cytometry result graph of the DLL3 antibody and the control antibody described in this application. Figure 3B The image shown is an ELISA result of the DLL3 antibody described in this application.

[0032] Figure 4A The diagram shows the internalization results of the DLL3 antibody and the control antibody described in this application on MB231 target cells. Figure 4B The diagram shows the internalization results of the DLL3 antibody and the control antibody described in this application on 293T-DLL3 target cells. Figure 4C The diagram shows the internalization results of the DLL3 antibody and control antibody described in this application on SHP77 target cells. Detailed Implementation

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

[0034] Terminology Definition

[0035] In this application, the terms "DLL3 (Delta-Like Ligand 3)" and "Delta-like ligand 3" are generally used interchangeably and typically refer to a transmembrane protein belonging to the Notch ligand family. In this application, the DLL3 may be an inhibitory Notch ligand. In this application, the human DLL3 protein may consist of 619 amino acids, characterized by including a 40-amino acid N-terminal conserved DSL (Delta, Serrate, Lag2) domain, six EGF-like repeat sequences, and a transmembrane domain. In this application, the DLL3 may be expressed in small cell lung cancer, neuroendocrine carcinoma, glioblastoma, and / or melanoma. In this 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 its functional activity. In this application, the DLL3 may be of any species origin. For example, the DLL3 may be human DLL3. In this application, the DLL3 can be wild-type or artificially modified. For example, the DLL3 can be a modified DLL3.

[0036] The protein and / or amino acid sequences involved in this application should also be understood to include at least the following range: variants or homologs having the same or similar functions as the protein. In this application, the variant can be a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein (e.g., the DLL3 antigen-binding protein described in this application). For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide before the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., the ability to bind to the DLL3 antigen) of the protein or polypeptide before the alteration. For example, the substitution may be a conserved substitution. In this application, a portion of the amino acid sequence of the antigen-binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific category. For example, both the variable region and the constant region of the antibody may be derived from the variable region and the constant region of an antibody from an animal species (such as a human). In this application, the homolog may be a protein or polypeptide 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 to the amino acid sequence of the protein and / or the polypeptide (e.g., the DLL3 antigen-binding protein of this application).

[0037] In this application, the term "antigen-binding protein" generally refers to a protein capable of binding antigens. For example, the antigen-binding protein may be a protein capable of binding DLL3 antigens. In this application, the antigen-binding protein may include an antigen-binding portion and optionally, allow the antigen-binding portion to employ a scaffold or framework portion that promotes the conformation of the antigen-binding portion to bind antigens. In this application, the antigen-binding protein may be wild-type or artificially modified. In this application, the antigen-binding protein may include, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold with a transplanted CDR or CDR derivative. In this application, the CDR can be determined by various coding systems. For example, the CDR can be determined by CCG, Kabat, Chothia, IMGT, AbM, or a combination of Kabat / Chothia, etc. In this application, the CDR covers CDR sequences partitioned according to any CDR partitioning method. In this application, the CDR may cover its variants. For example, the amino acid sequence of the CDR can be substituted, deleted, and / or added with 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 acids. For example, the CDR can encompass homologs. For example, the homolog can 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 CDR. In this application, the antigen-binding protein can be artificially synthesized. In this application, the antigen-binding protein can be of animal or non-animal origin. In this application, the antigen-binding protein can be an antibody or its antigen-binding fragment, as well as its variants, homologs, derivatives, or analogs. In this application, antigen-binding proteins may include, but are 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, as long as they exhibit the desired antigen-binding activity. In this application, the antigen-binding protein may be a VHH. In this application, the antibody may be a chimeric antibody, a humanized antibody, or a fully human antibody. In this application, the antibody may be a recombinant, hybrid, mutated, or transplanted antibody.

[0038] In this application, the terms "VHH," "nanobody," and "single-domain antibody" are generally used interchangeably and typically refer to an antibody structure composed of a heavy chain variable region. In this application, the VHH may consist of VHs. In this application, the VHH may contain a complementarity-determining region (CDR) and a framework region (FR). In this application, the VHH may contain 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 this application, the VHH may be artificially synthesized. In this application, the VHH may be artificially modified.

[0039] In this application, the terms "immunoglobulin constant region" and "constant region" are generally used interchangeably, and the constant region typically refers to a region of the antibody's non-variable region. In this application, the constant region may not directly participate in antigen binding but exhibits multiple effector functions. In this application, the constant region may be the entire antibody non-variable region or a portion of it. 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 this application, the constant region may be of different species origin. For example, the constant region may be derived from humans, goats, rabbits, rats, or guinea pigs.

[0040] In this application, the term "immune cell" generally refers to a cell involved in an immune response. For example, the immune cell may be an immune cell that performs effector functions. For example, performing effector functions may include clearing foreign antigens or promoting immune effector responses. In this 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, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells, and / or pluripotent stem cells. For example, the immune effector cell may be a T cell.

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

[0042] In this application, the term "pharmaceuticalally acceptable carrier" generally refers to a non-toxic material that does not interfere with the effectiveness of the bioactivity of the active ingredient. For example, a pharmaceutically acceptable carrier includes pharmaceutically acceptable excipients or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. For example, a physiologically acceptable carrier may be water, salt, protein, polysaccharide, lipid, or inactive viral particles.

[0043] In this application, the term "prevention and / or treatment" generally refers to the prevention and / or treatment of a disease. For example, the prevention and / or treatment may include preventing the onset of the disease, slowing or reversing the disease progression, 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 with it, or preventing further increases in the severity of the disease and any symptoms associated with it. In this application, the disease may be a tumor disease. For example, the disease may be a tumor disease with DLL3 positive expression. For example, preventing or alleviating the onset of one or more tumor-related symptoms, reducing the severity and duration of the tumor and its associated symptoms.

[0044] In this application, the term "tumor" generally refers to any new pathological tissue proliferation containing tumor antigens that can be recognized by the immune system. In this application, the tumor may include benign or malignant tumors (carcinomas). In this application, the carcinoma may be metastatic or non-metastatic. In this application, the tumor may include solid tumors and / or hematologic malignancies. In this application, a solid tumor generally refers to a tangible tumor that can be detected by clinical examination. For example, a solid tumor may include growths or solid lesions formed by abnormal cell growth. In this application, a hematologic malignancy generally refers to a class of hematopoietic system diseases. In this application, a hematologic malignancy may include various types of leukemia, multiple myeloma, or malignant lymphoma. In this 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 this 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 this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0047] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation 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 a specified value. Invention Details

[0049] antigen-binding proteins

[0050] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at 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 antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants of the CDR, including amino acid sequences with substitutions, deletions, and / or additions of one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; this also covers its homologs, which can be amino acid sequences 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 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 capable of binding 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 may possess 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 the control antibody. For example, the antigen-binding protein can bind to DLL3 with a KD value similar to or higher than that of the control antibody. For example, the antigen-binding protein can bind to DLL3 with an EC50 value similar to or lower than that of the control antibody. In this application, the EC50 value or KD value can be determined by conventional techniques in the art. In this application, the KD value can be determined by Octet, SPR, ELISA, competitive ELISA, 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 this application, the antigen-binding protein may include an antibody heavy chain variable region (VH), wherein the VH includes heavy chain complementarity-determining regions (HCDR1, HCDR2, and / or HCDR3). For example, the VH includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3.

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

[0055] In this application, HCDR1, HCDR2, and / or HCDR3 may be wild-type sequences. The amino acid sequences of HCDR1, HCDR2, and / or HCDR3 may also be modified and / or altered. For example, one or more amino acid sequences of 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, HCDR1, HCDR2, and / or HCDR3 may be variants thereof, which include substitutions, deletions, and / or additions of one or more amino acids to the amino acid sequences of HCDR1, HCDR2, and / or HCDR3. For example, HCDR1, HCDR2 and / or HCDR3 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:3, SEQ ID NO:4 and / or SEQ ID NO:5.

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

[0057] In this application, the VH may also include H-FR1, H-FR2, H-FR3 and / or H-FR4. For example, the VH may include H-FR1, H-FR2, H-FR3 and H-FR4.

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

[0059] In this application, H-FR1, H-FR2, H-FR3, and / or H-FR4 may be wild-type sequences. In this application, the amino acid sequences of 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 H-FR1, H-FR2, H-FR3, and / or H-FR4 may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein with DLL3. For example, H-FR1, H-FR2, H-FR3, and / or H-FR4 may be variants thereof, including those in which the amino acid sequence of H-FR4 has been substituted, deleted, and / or added to one or more amino acids. For example, H-FR1, H-FR2, H-FR3 and / or H-FR4 may also contain sequences that have 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 this application, the C-terminus of H-FR1 can be directly or indirectly connected to the N-terminus of HCDR1. In this application, H-FR2 can be located between HCDR1 and HCDR2. In this application, H-FR3 can be located between HCDR2 and HCDR3. In this application, the N-terminus of H-FR4 can be directly or indirectly connected to the C-terminus of HCDR3. In this application, the antigen-binding protein can comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4, wherein the C-terminus of H-FR1 is directly or indirectly connected to the N-terminus of HCDR1, H-FR2 is located between HCDR1 and HCDR2, H-FR3 is located between HCDR2 and HCDR3, and the N-terminus of H-FR4 is directly or indirectly connected to the C-terminus of HCDR3.

[0061] In this application, the antigen-binding protein may include VH, wherein the VH includes H-FR1, H-FR2, H-FR3 and H-FR4, the amino acid sequence of H-FR1 is shown in SEQ ID NO:6, the amino acid sequence of H-FR2 is shown in SEQ ID NO:7, the amino acid sequence of H-FR3 is shown in SEQ ID NO:8, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:9.

[0062] In this 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 this 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 with DLL3. For example, the VH may be a variant thereof, the variant comprising substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the VH. For example, the VH may also 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 this application, the antigen-binding protein may include an antibody light chain variable region (VL), wherein the VL includes light chain complementarity-determining regions (LCDR1, LCDR2, and / or LCDR3). For example, the VL includes light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3.

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

[0065] In this application, LCDR1, LCDR2, and / or LCDR3 may be wild-type sequences. The amino acid sequences of LCDR1, LCDR2, and / or LCDR3 may also be modified and / or altered. For example, one or more amino acid sequences of 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, LCDR1, LCDR2, and / or LCDR3 may be variants thereof, which include substitutions, deletions, and / or additions of one or more amino acids to the amino acid sequences of LCDR1, LCDR2, and / or LCDR3. For example, LCDR1, LCDR2 and / or LCDR3 may also contain sequences that have 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 this application, the antigen-binding protein may include VL, which may include LCDR1, LCDR2 and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12 and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13.

[0067] In this application, the VL may also include L-FR1, L-FR2, L-FR3 and / or L-FR4. For example, the VL may include L-FR1, L-FR2, L-FR3 and L-FR4.

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

[0069] In this application, L-FR1, L-FR2, L-FR3, and / or L-FR4 can be wild-type sequences. The amino acid sequences of L-FR1, L-FR2, L-FR3, and / or L-FR4 can also be modified and / or altered. For example, one or more amino acid sequences of L-FR1, L-FR2, L-FR3, and / or L-FR4 can be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein with DLL3. For example, L-FR1, L-FR2, L-FR3, and / or L-FR4 can be variants thereof, including those in which one or more amino acids of the L-FR4 amino acid sequence have been substituted, deleted, and / or added. For example, L-FR1, L-FR2, L-FR3 and / or L-FR4 may also contain sequences that have 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 this application, the C-terminus of L-FR1 can be directly or indirectly connected to the N-terminus of LCDR1. In this application, L-FR2 can be located between LCDR1 and LCDR2. In this application, L-FR3 can be located between LCDR2 and LCDR3. In this application, the N-terminus of L-FR4 can be directly or indirectly connected to the C-terminus of LCDR3. In this application, the antigen-binding protein can comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR4, where the C-terminus of L-FR1 is directly or indirectly connected to the N-terminus of LCDR1, L-FR2 is located between LCDR1 and LCDR2, L-FR3 is located between LCDR2 and LCDR3, and the N-terminus of L-FR4 is directly or indirectly connected to the C-terminus of LCDR3.

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

[0072] In this 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 this 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 with DLL3. For example, the VL may be a variant thereof, the variant comprising substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the VL. For example, the VL may also 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:10.

[0073] In this application, the direct or indirect linkage can be achieved through intermolecular forces or through linkers. In this application, the amino acid sequence of the FR can be any species-derived FR. For example, the FR can be derived from mouse, rabbit, goat, alpaca, or human. For example, the FR can be a human FR.

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

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

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

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

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

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

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

[0081] In this 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 in terms of the FR and constant regions without reducing its binding activity / affinity with DLL3. For example, the antigen-binding protein can exhibit low immunogenicity without reducing its binding activity / affinity with DLL3.

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

[0084] In this application, the antigen-binding protein may be an antibody or an antigen-binding fragment thereof. In this 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 this application, the antigen-binding protein may be an scFv. In this application, the scFv may comprise VH and VL, where VH comprises HCDR1, HCDR2, and HCDR3, and VL comprises LCDR1, LCDR2, and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is shown in SEQ ID NO:4, the amino acid sequence of HCDR3 is shown in SEQ ID NO:5, the amino acid sequence of LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13. In this application, the scFv may comprise VH and VL, where the amino acid sequence of VH is shown in SEQ ID NO:2, and the amino acid sequence of VL is shown in SEQ ID NO:10.

[0086] In this application, the scFv may further include a linker. In this application, the scFv may include VH and VL, which are linked by a linker. In this application, the linker may be (GGGS)4 or (GGGGS)4. In this application, the amino acid sequence of the scFv may be as shown in SEQ ID NO:1.

[0087] Immunoconjugates

[0088] On the other hand, this application provides an immunoconjugate that may contain the antigen-binding protein described above. In this application, the antigen-binding protein may be as described previously. In this 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 this application, the immunoconjugate may contain the antigen-binding protein, a linker, and a payload.

[0089] In this application, the portions of the immunoconjugate are directly or indirectly connected. For example, the portions of the immunoconjugate are indirectly connected. For example, the portions of the immunoconjugate are connected by connectors.

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

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

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

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

[0094] On the other hand, this application also provides an antibody-drug conjugate containing the antigen-binding protein, which has 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.

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

[0096] In this application, the immune conjugate may comprise the antigen-binding protein, a linker, and a cytotoxic drug. In this application, the cytotoxic drug may be a chemotherapeutic agent, a tubulin inhibitor, a DNA damaging agent, or a topoisomerase I inhibitor. In this application, the chemotherapeutic agent may be vinblastine, doxorubicin, or derivatives thereof. In this application, the tubulin inhibitor may be oliquitin, eribulin, maytansine, tubulolysin, cryptomycin, an antimitotic EG5 inhibitor, or derivatives thereof. For example, the cytotoxic drug may be MMAE (monomethyloliquitin E), MMAF (monomethyloliquitin F), DM1, DM4, or tubulysins. In this application, the DNA damaging agent may be chachiin, pamoatetin, atrazomycin derivatives, pyrrole benzodiazepines, indolechlorobenzodiazepines, or derivatives thereof. For example, the DNA damaging agent may be PBD or IBD. In this application, the cytotoxic drug may be an apoptosis inducer (e.g., a Bcl-xL inhibitor), thailanstatin or its analogues (e.g., thailanstatin A), amatoxins (e.g., α-amatoxin or β-amatoxin), nicotinamide phosphoribosyltransferase, or carbamycin (e.g., carbamycin A and carbamycin B). In this application, the topoisomerase I inhibitor may be camptothecin, hydroxycamptothecin, irinotecan, topotecan, or its derivatives.

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

[0098] In this application, the antibody-drug conjugate may further include a spacer unit. In this application, the spacer unit may be embedded between the cleavable linker and the payload, or may be part of the cleavable linker itself. In this application, the spacer unit can spontaneously rearrange its structure after the cleavable linker breaks under suitable conditions, thereby releasing the payload attached to it. In this application, the spacer unit may be an aminobenzyl alcohol (PAB), a β-glucuronide, or a substituted or unsubstituted ethylenediamine, etc.

[0099] Chimeric antigen receptor

[0100] On the other hand, this application provides a chimeric antigen receptor (CAR) that may include the antigen-binding protein.

[0101] In this application, the chimeric antigen receptor may include an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. In this application, the chimeric antigen receptor may also include a hinge region. In this application, the chimeric antigen receptor may also include an intracellular co-stimulatory domain. In this application, the chimeric antigen receptor may also include a signal peptide.

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

[0103] In this application, the immune cells may be T cells, NK cells, NKT cells, dendritic cells, macrophages, TIL cells, iNKT cells, CIK cells, γδT cells, and / or DNT cells. For example, the immune cells may be immune effector cells. For example, the immune cells may be T cells. For example, the immune cells may be a mixture, and the mixture may contain different types of immune cells; for example, the mixture may contain one or more types of immune cells.

[0104] Nucleic acid molecules, vectors and cells

[0105] On the other hand, this application also provides isolated nucleic acid molecules that can encode the antigen-binding protein and / or the chimeric antigen receptor. In this application, the antigen-binding protein can be as described above.

[0106] In this application, the nucleic acid molecule may also contain a sequence encoding a signal peptide.

[0107] In this application, the nucleic acid molecule may be generated or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR); (ii) clonal recombination; (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, such as by chemical synthesis. In this application, the nucleic acid molecule may be DNA and / or RNA. In this application, the nucleic acid molecule may be an artificially synthesized nucleic acid analog. In this application, the nucleic acid molecule may be a modified nucleic acid molecule.

[0108] On the other hand, this application provides a carrier that can contain the nucleic acid molecule.

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

[0110] In this application, the vector may also contain other genes. For example, the other genes may be marker genes.

[0111] In this application, the vector may contain various elements that control expression. For example, the vector may include a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and / or a reporter gene. For example, the vector may also contain a replication initiation site. For example, the vector may include components that facilitate entry into the cell. To enable the nucleic acid molecule to replicate in the vector, the 5' and 3' ends of the nucleic acid molecule may also contain long terminal repeat sequences.

[0112] In this application, the nucleic acid molecule may contain a nucleic acid sequence encoding a promoter. In this application, the sequence encoding the promoter is typically operatively linked to the coding sequence of the protein to be expressed. In this application, the promoter may be any nucleic acid sequence exhibiting transcriptional activity in a selected host cell, including mutated, truncated, and heterozygous promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to that of the host cell. In this application, the promoter may be selected from one or more of the following: EF1α, CMV, MSCV, SSFV, and UbC.

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

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

[0115] In this application, the cell may contain one or more of the aforementioned nucleic acid molecules and / or one or more vectors. In this application, the vector may contain one or more of the aforementioned nucleic acid molecules and / or one or more vectors.

[0116] In this application, the vector can be introduced into the cells using methods known in the art. For example, the method may be electroporation, Lipofectine transfection, or Lipofectamin transfection.

[0117] On the other hand, this application provides a method for preparing the antigen-binding protein, the chimeric antigen receptor, and / or the immunoconjugate. In this application, the method includes culturing the cells under conditions that cause the antigen-binding protein and / or the chimeric antigen receptor to be expressed.

[0118] Pharmaceutical Composition

[0119] On the other hand, this application also provides pharmaceutical compositions that may comprise the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0120] In this application, the pharmaceutical composition may comprise suitable formulations 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 dosage and concentration used. The pharmaceutical compositions of this application may include liquid, freeze-dried, and lyophilized compositions.

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

[0122] In this application, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally.

[0123] Drug combination

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

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

[0126] In this application, the different active ingredients in the drug combination can be placed together or separately. For example, the active ingredients can be placed in the same container. Or, the active ingredients can be placed in different containers.

[0127] use

[0128] On the other hand, this application also provides the use of the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and / or the pharmaceutical combination in the preparation of a medicament that can be used for the prevention, diagnosis and / or treatment of diseases and / or conditions.

[0129] On the other hand, this application also provides a method for preventing, diagnosing, and / or treating diseases and / or conditions, which may include administering the antigen-binding protein, the chimeric antigen receptor, the modified immune cells, the immune conjugate, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition, and / or the pharmaceutical combination to a subject in need.

[0130] On the other hand, this application also provides the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and / or the pharmaceutical combination, which can be used for the prevention, diagnosis and / or treatment of diseases and / or conditions.

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

[0132] In this application, the prevention, diagnosis and / or treatment may be to prevent the onset of the disease, slow down or reverse the disease progression, 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 further increase in the severity of the disease and any symptoms associated therewith.

[0133] On the other hand, this application provides the use of the antigen-binding protein in the preparation of diagnostic agents, wherein the diagnostic agents are used to diagnose diseases and / or conditions associated with the expression of the DLL3 protein.

[0134] On the other hand, this application provides that the antigen-binding protein can be used to diagnose diseases and / or conditions related to the expression of the DLL3 protein.

[0135] In this application, the diagnostic agent can be used alone or in combination with instruments, appliances, devices, or systems. In the prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation, and prediction of hereditary diseases, the diagnostic agent can be used to perform in vitro testing on human samples (e.g., various body fluids, cells, tissue samples, etc.).

[0136] On the other hand, this application also provides a kit that may include the antigen-binding protein, the kit being used to detect the presence and / or level of DLL3 in a sample or subject. For example, the kit may be used for the prevention, diagnosis, and / or treatment of diseases and / or conditions.

[0137] For example, this application relates to immunoassay kits used in immunoassay methods such as antigen-binding protein binding ELISA, immunohistochemistry, Western blotting, and flow cytometry.

[0138] On the other hand, the use of the antigen-binding protein, the chimeric antigen receptor, the modified immune cell, the immune conjugate, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and / or the pharmaceutical combination described in this application in the preparation of a kit for diagnostic purposes.

[0139] In this application, the container components of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container components in which antibodies or preferably appropriately aliquots of antibodies can be placed. The kit of this application will also typically include components for containing antibodies, antigens, and any other reagents in a sealed form for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the aforementioned vials are held.

[0140] On the other hand, this 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, this application provides a method for diagnosing diseases and / or conditions 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 capable of specifically binding the antigen-binding protein.

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

[0143] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the antigen-binding protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0144] Example

[0145] Example 1: Preparation and Binding Ability Verification of Humanized Antibodies

[0146] 1.1 Screening for antibodies targeting DLL3

[0147] We commissioned Xunyao Biotechnology to screen antibody sequences targeting DLL3. Specifically, we utilized the antibody library EMPRESS. TM Specific antibodies were screened, and positive clones were verified by ELISA and high-affinity clones were obtained through sequencing analysis. The obtained DLL3 Fab fragment contained VH and VL. VH contained HCDR1, HCDR2, and HCDR3, and VL contained LCDR1, LCDR2, and LCDR3. The amino acid sequences of VH, HCDR1, HCDR2, and LCDR3 are shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, VL, LCDR1, LCDR2, and LCDR3, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively. scFv A1 was prepared based on the obtained Fab fragment, and the amino acid sequence of the obtained scFv is shown in SEQ ID NO:1.

[0148] 1.2 Cell Culture

[0149] 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 (American Academy of Sciences). Suspension-cultured Expi293F cells were purchased from Thermo Fisher Scientific (Cat no. A14527). To facilitate the detection of tumor cell killing, all target cells were stably infected with lentivirus to express GFP. SHP-77, MB231, and MB453 cell lines were maintained in complete culture medium (IMEM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine), with some cells suspended in the medium. The culture medium was changed every three days. HEK-293T-DLL3 cell lines overexpressing DLL3 were obtained by infecting HEK-293T cells with a lentivirus containing the DLL3 transfer gene. These cells were then cultured in DMEM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine, and passaged until reaching 95% confluence. Expi293F cells for antibody expression were seeded in serum-free medium (Expi293 expression medium, Cat no. A1435101) and cultured with shaking at 37°C, 8% CO2, and 125 rpm until a cell density of 3 × 10⁻⁶ cells / year was reached. 6 After passing the culture at a rate of / mL, perform one subculture.

[0150] 1.3 Flow Cytometry Assay

[0151] Flow cytometry was performed using a Beckman Coulter instrument in semi-automatic or plate mode, and data were analyzed using FlowJo software. MB453, SHP-77, or HEK-293T-DLL3 cells were washed once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2mM EDTA, pH 7.0) and resuspended to 1-5 × 10⁻⁵. 6 Cells / mL, placed on ice before staining. Add recombinant antibody with His or Fc tag for staining, incubate at 4°C for 30 minutes in the dark, then wash with a large amount of FACS buffer to remove unbound antibodies. Then stain with fluorescently labeled secondary antibody, incubate at 4°C for 30 minutes in the dark. After staining, wash with a large amount of FACS buffer to remove unbound antibodies, centrifuge at 300xg for 3 minutes, and remove the supernatant. After washing and resuspending with FACS buffer, run on a flow cytometer. Flow cytometry data are analyzed and processed using FlowJo software.

[0152] Experimental results:

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

[0154] Example 2: Immunofluorescence staining to verify antibody endocytosis function

[0155] 2.1 Preparation and staining of immunofluorescence staining slides

[0156] Cell culture preparation: Sterilize round glass coverslips (18 mm) by immersing them in ethanol. Transfer the coverslips to 12-well plates and add 1 mL of 50 μg / mL poly-D-lysine 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. Culture the positive cell line SHP77(DLL3+) on the poly-D-lysine-coated coverslips as needed.

[0157] Immunofluorescence staining: Rinse cells once with PBS (1 mL per well), then fix cells for 10 min with PBS containing 3% paraformaldehyde. Remove the fixative and rinse cells once with PBS-GSA (1 mL per well) to terminate the reaction. Incubate cells with fresh PBS-GSA (1 mL per well) for 5 min. Treat cells with PBS-GSA containing 0.5% Triton X-100 (freshly prepared) for 3 min to permeabilize them. Remove the permeabilization solution and quickly rinse cells with PBS-GSA (1 mL per well). Incubate cells with fresh PBS-GSA (1 mL per well) for 5 min. Dilute DLL3 antibody A1 in PBS-GSA containing 1% BSA to prepare only the required experimental volume (50 μL per coverslip), and co-incubate the antibody dilution with the cells. Co-culture at 37°C for 3 h. Rinse rapidly with PBS-GSA (1 mL per well). Use BD flow cytometry permeabilization reagent (BD Perm / Wash). TMCells were fixed and perforated using a buffer solution. A1 cells, carrying a His marker, were captured by the anti-HIS antibody-FITC, while CD107a-PE stained lysosome-associated proteins on the cell membrane, which then endophagically entered the cell. DAPI was used as a background dye to label DNA in the cell nucleus. Anti-His FITC, anti-CD107-PE, and DAPI were diluted in 1% PBS-GSA and incubated with the cells in the dark for 30 minutes. The coverslips were placed back into the tissue culture plate with the cell side up. The cells were quickly rinsed with PBS-GSA (1 mL per well). The cells were then incubated in the dark with fresh PBS-GSA (1 mL per well) for 10 to 15 minutes. To mount each coverslip, a drop of mounting medium containing DAPI was placed on the slide. The back of the coverslip was gently blotted dry with a lint wipe, and then each coverslip was placed on the mounting medium with the cell side down.

[0158] 2.2 Fluorescence microscopy examination and statistical analysis

[0159] Using an Olympus IX73 microscope with a 20x objective lens, confocal imaging was performed to find a clear field of view. Four fields of view were randomly selected for each experimental condition, and the number of cells endocytosed by the antibody was calculated based on the fluorescence signal presented by the cells. ImageView (Version 4.11) was used to analyze the captured images, and the ratio of the number of antibody-endocytosed cells (His+CD107a+, yellow) to the total number of cells (DAPI, blue) in any four fields of view was calculated. Statistical analysis using a Student t-test showed *p<0.05.

[0160] Experimental results:

[0161] Internalization results as follows Figure 2 As shown, statistical analysis of the number of endocytic cells revealed that the DLL3 antibody described in this application had a high proportion of cells that entered SHP77 (DLL3+) through internalization, demonstrating a good internalization effect and making it suitable for the development of antibody-drug conjugates (ADCs).

[0162] Example 3: Comparison of affinity between antibody A1 and Amgen's bispecific antibody AMG757

[0163] 3.1 Expression and purification of recombinant antibodies

[0164] Antibodies A1 and AMG757, containing human IgG Fc tags, were cloned into the eukaryotic expression vector pcDNA3.1 (Thermo Fisher Scientific), and sequencing verified that the target sequences had been correctly inserted. Expi293F cells were then used at a rate of 1 × 10⁻⁶ cells / year. 6Cells were seeded at a density of [missing value] / mL in 125mL Erlenmeyer flasks (Erlenmeyer flask, BioFIL, Cat no. TAB112125) and cultured overnight. The expression plasmid was then transfected into Expi293F cells at a DNA:PEI ratio of 1:3 (mass ratio). Cells were incubated at 37°C, 8% CO2, and 125rpm with shaking for 6 days. The cell suspension was centrifuged at 4200rpm to collect the supernatant. The Fc-tagged antibody was then purified from the culture supernatant using Protein G affinity chromatography agar (Thermo Scientific, Cat no. A50585). After concentration and washing, the antibody concentration was determined using a NanoDrop One spectrophotometer (Thermo Scientific) for subsequent experiments.

[0165] 3.2 Flow cytometry assay

[0166] The flow cytometry assay procedure is as shown in Example 1.

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

[0168] Dissolve the target antigen DLL3 in ELISA coating buffer and aliquot 50 ng per well into a 96-well plate. Incubate overnight at 4°C, protected from light. Wash the 96-well plate with PBST (PBS + 0.1% Tween-20), then incubate with blocking buffer (PBS containing 1% BSA) at 37°C for one hour. Add the test antibody to the 96-well plate and perform a 3-fold serial dilution with blocking buffer, then incubate at 37°C for one hour. Discard the test antibody, wash three times with PBST, add goat anti-human IgG Fc HRP secondary antibody (Goat anti-Human IgG Fc HRP secondary antibody, Invitrogen, Cat no. A18817, 1:5000), and incubate at 37°C for one hour. The secondary antibody was discarded, and the sample was washed three times with PBST. 50 μl of TMB substrate (Thermo Scientific, Catno. N301) was added, and the reaction was stopped by adding 0.18 M H₂SO₄ after reacting at room temperature for 30 minutes. The absorbance at OD450 nm was measured using a spectrophotometer (Thermo Scientific, Multiskan Sky). The affinity of the antibody for soluble DLL3 was determined by ELISA.

[0169] Experimental results:

[0170] Flow cytometry results as follows Figure 3AAs shown, antibodies A1 and AMG757 do not bind to the negative cell line MB453 (DLL3-), but specifically bind to DLL3-positive cells 293T-DLL3 and SHP77 (DLL3+), and the two antibodies have comparable binding ability to cell surface membrane antigens.

[0171] ELISA results as follows Figure 3B As shown, compared with the negative control, the half-maximal effective concentration (EC50) of antibody A1 was 38.9 ng / mL, indicating a higher affinity for soluble DLL3.

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

[0173] Example 4: pH-sensitive dye labeling assay for antibody internalization

[0174] 4.1 Antibody internalization assay

[0175] Harvest and count the target cells to be inoculated, and dilute with culture medium to 2 × 10⁻⁶. 5 Cells were cultured at a concentration of 100 μL / mL, and then seeded into 96-well flat-bottom cell culture plates with 50 μL of cell suspension per well. The plates were incubated at 37°C with 5% CO2 for 4-6 hours to allow cell settling and adsorption to the bottom of the wells. The test antibody and pH-sensitive dye were mixed at a molar ratio of 1:3, and the antibody concentration was adjusted to twice the final concentration. The mixture was then incubated at 37°C for 15 minutes in the dark. 50 μL of the dye-labeled test antibody was added to each well of the pre-seedled 96-well plate. The 96-well plate was placed in an Incucyte scanning imager, the color channels were set, and the plates were scanned every 30 minutes to capture cell images. When the labeled antibody internalized into intracellular lysosomes, the pH-sensitive dye displayed a corresponding color due to the acidic environment, thus determining the degree of antibody internalization based on the color signal.

[0176] Experimental results:

[0177] Internalization results as follows Figure 4A As shown in Figure C, the test antibodies did not internalize in antigen-negative target cells (MB231). However, in DLL3-positive cell lines (293T-DLL3, SHP77), both antibodies A1 and AMG757 showed rapid internalization compared to the negative control, with A1 exhibiting comparable to or even stronger internalization capacity than AMG757 (SHP77). For SHP77 target cells, A1 demonstrated better internalization function than the positive antibody AMG757.

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

Claims

1. An isolated antibody or antigen-binding fragment thereof capable of binding DLL3, wherein the antibody or antigen-binding fragment comprises an antibody heavy chain variable region VH and an antibody light chain variable region VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 4, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 5, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

13.

2. The antibody or antigen-binding fragment thereof capable of binding DLL3 according to claim 1, wherein the amino acid sequence of VH is as shown in SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof capable of binding DLL3 according to claim 1, wherein the amino acid sequence of DLL3 is shown in SEQ ID NO:

10.

4. The antibody or antigen-binding fragment thereof capable of binding DLL3 according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises VH and VL, the amino acid sequence of VH is shown in SEQ ID NO: 2, and the amino acid sequence of VL is shown in SEQ ID NO:

10.

5. The antibody or antigen-binding fragment thereof capable of binding DLL3 according to claim 1, wherein the antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region.

6. The antibody or antigen-binding fragment thereof capable of binding DLL3 according to claim 5, wherein the immunoglobulin constant region is a heavy chain constant region and / or a light chain constant region.

7. The antibody or antigen-binding fragment thereof capable of binding DLL3 according to claim 6, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

8. The antibody or antigen-binding fragment thereof capable of binding DLL3 according to claim 6, wherein the light chain constant region is a human κ (Kappa) or λ (Lambda) light chain constant region.

9. The antibody capable of binding DLL3 or its antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv or Fv fragment of the antibody.

10. The antibody or antigen-binding fragment thereof capable of binding DLL3 according to claim 1, wherein the antibody or antigen-binding fragment thereof is scFv, and the amino acid sequence of the scFv is shown in SEQ ID NO:

1.

11. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof capable of binding DLL3 as described in any one of claims 1-10.

12. A carrier comprising the nucleic acid molecule of claim 11.

13. A host cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof capable of binding DLL3 according to any one of claims 1-10, a nucleic acid molecule according to claim 11, a vector according to claim 12 or a host cell according to claim 13, and a pharmaceutically acceptable carrier.

15. Use of any one of the antibodies or antigen-binding fragments of DLL3 capable of binding to DLL3 according to claims 1-10, the nucleic acid molecule according to claim 11, the vector according to claim 12, or the host cell according to claim 13 in the preparation of a medicament for the prevention and / or treatment of small cell lung cancer.

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