Binding molecules to DLL3 and their use

Abstract

The present invention relates to a binding molecule or an antigen-binding fragment thereof for DLL3, derivatives and pharmaceutical compositions comprising said binding molecule or an antigen-binding fragment thereof, and to the use of said binding molecule or an antigen-binding fragment thereof in the treatment and detection / diagnosis of cancer.

Description

Technical Field

[0001] The present disclosure belongs to the field of immunology. More specifically, the present disclosure relates to binding molecules to DLL3 and antigen-binding fragments thereof, derivatives comprising the binding molecules or antigen-binding fragments thereof, pharmaceutical compositions, and their use in the treatment of cancer.

Background Art

[0002] Lung cancer is one of the malignant tumors with the fastest increasing morbidity and mortality rates and poses the greatest threat to human health and life. Pathologically, it is divided into two categories: small cell lung cancer and non-small cell lung cancer.

[0003] Non-small cell lung cancer (NSCLC) is one of the most important types of lung cancer, and more than 80% of lung cancer patients belong to this type. Currently, targeted therapies, PD-1 immunotherapies, and gene sequencing for lung cancer are booming, and the remarkable progress obtained along with this has mainly focused on non-small cell lung cancer. Therefore, the number of available effective treatments that can be selected in this subgroup of lung cancer has been significantly increased, and the 5-year survival rate of non-small cell lung cancer patients has been greatly improved.

[0004] Small cell lung cancer (SCLC) is a type of lung cancer with high invasiveness, lethality, and extensive metastatic potential. It accounts for approximately 15% of lung cancers and is pathologically, molecularly, biologically, and clinically significantly different from other lung cancers. Each year, more than 234,000 patients are diagnosed with SCLC, and it is estimated that approximately 250,000 deaths occur worldwide each year. The characteristics of SCLC are rapid tumor growth, extensive vascular distribution, genomic instability, and early occurrence of disseminated metastases. In the past 30 years, due to limitations in the detection, treatment, or improvement of survival rates of SCLC, it has been classified as a refractory cancer. Local treatments such as surgery, radiotherapy, or a combination of both have a very low possibility of complete cure. Platinum-based combination chemotherapy remains the basis of treatment, and the first-choice standard regimen is the combination of a platinum-based agent (carboplatin or cisplatin) and another cytotoxic drug (e.g., etoposide). The chemotherapy for small cell lung cancer has a very high remission rate, but especially in advanced patients, it is always accompanied by recurrence. In the case of limited-stage patients, the median survival period is 14 - 20 months, in the case of advanced-stage patients, it is only 9 - 11 months, and in the case of recurrent patients, the survival period is even shorter and there are few treatment options available. The only treatment recommended by the FDA is topotecan, however, due to its limited hematotoxicity, the treatment response rate remains unsatisfactory at 5 - 24%, and the median survival period is less than 25 weeks. Currently, since no specific third-choice treatment has been recommended yet, new effective therapeutic drugs are in demand.

[0005] Delta-like 3, also known as DLL3, is a protein encoded by the DLL3 gene and is one of the ligands of the Notch family. DLL3 only has 36% homology with DLL1. Different from other deltatype DSL (delta / serate / lag-2) proteins DLL1 and DLL4, DLL3 is most highly expressed in normal tissues in the fetal brain and plays an important role in the growth and development of the axial mesoderm. It has been revealed by research that it is expressed on the surface of tumor cells in about 85% of patients with small cell lung cancer and large cell neuroendocrine cancer, and is also highly expressed in glioblastoma multiforme, melanoma, pancreatic cancer, rectal cancer, etc. However, it is not expressed in healthy tissues and non-neuroendocrine tumors. Since this protein is involved in the regulation of the Notch signaling pathway, the signal emitted by the Notch pathway ultimately promotes the unrestricted growth of cancer. In normal tissues, the mRNA expression of DLL3 is limited to the brain, esophagus, and pancreas.

[0006] In further studies, when analyzing tumor tissues and normal tissue specimens, by detecting the expression of DLL3 in the whole-transcriptome sequencing data of primary SCLC biopsy specimens, SCLC cell lines, and normal lung biopsy specimens, it is shown that the mRNA of DLL3 in SCLC is approximately 35-fold higher than that in normal lungs. These SCLC tumor samples are compared with the transcriptome data of normal tissues and other tumor types from the oncogenome, further demonstrating that the expression of DLL3 is increased in primary SCLC tumor samples and low-grade glioma (LGG), glioblastoma (GBM), melanoma (SKCM). Also, clinically, in the Illumina BeadChip data of the Lung Cancer Genome Project, it has been shown that DLL3 in primary SCLC tumor specimens is increased compared with NSCLC.

[0007] Data obtained from the Cancer Cell Line Encyclopedia database further demonstrates that the expression of DLL3 mRNA is particularly elevated in SCLC cell lines. In short, as shown in these expression data obtained from multiple technical platforms and samples, DLL3 mRNA is overexpressed in primary SCLC tumors, SCLC PDX, conventional SCLC cell lines, and LCNEC PDX, and mRNA expression in normal tissues is mainly limited to the brain. Summary of the Invention Problems to be Solved by the Invention

[0008] The present disclosure aims to provide a binding molecule to DLL3 and an antigen-binding fragment thereof, wherein the binding molecule or its antigen-binding fragment can specifically bind to DLL3 and does not non-specifically bind to the proteins DLL1 and DLL4 of the same family. Means for Solving the Problems

[0009] A first aspect of the present disclosure provides a molecule that binds to DLL3 or an antigen-binding fragment thereof, and the molecule that binds to DLL3 or its antigen-binding fragment i) has a heavy-chain complementarity-determining region 1 (HCDR1) whose amino acid sequence is selected from SEQ ID NO: 6, 9, 12, 15, or 18, ii) has a heavy-chain complementarity-determining region 2 (HCDR2) whose amino acid sequence is selected from SEQ ID NO: 7, 10, 13, 16, or 19, and iii) includes a heavy-chain complementarity-determining region 3 (HCDR3) whose amino acid sequence is selected from SEQ ID NO: 8, 11, 14, 17, 20, 110, or 111.

[0010] In one embodiment, the molecule that binds to DLL3 or its antigen-binding fragment includes a heavy-chain variable region (VH), the heavy-chain variable region includes HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively a) SEQ ID NO: 6, 7, and 8, or b) SEQ ID NO: 9, 10, and 11, or c) SEQ ID NO: 12, 13, and 14, or d) SEQ ID NO: 15, 16, and 17, or e) SEQ ID NO: 18, 19, and 20, or f) SEQ ID NO: 6, 7, and 110, or g) SEQ ID NO: 6, 7, and 111.

[0011] In one embodiment, the heavy chain variable region comprises any amino acid sequence selected from SEQ ID NOs: 1-5, 21-29, or comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any of SEQ ID NOs: 1-5, 21-29.

[0012] A second aspect of the present disclosure provides a molecule that binds to another DLL3 or an antigen-binding fragment thereof, and the molecule that binds to DLL3 or an antigen-binding fragment thereof i) a heavy chain complementarity determining region 1 (HCDR1) whose amino acid sequence is selected from SEQ ID NOs: 57, 63, 69, 72, 78, 84, 93, or 99, ii) a heavy chain complementarity determining region 2 (HCDR2) whose amino acid sequence is selected from SEQ ID NOs: 58, 64, 70, 73, 79, 85, 94, or 100, iii) a heavy chain complementarity determining region 3 (HCDR3) whose amino acid sequence is selected from SEQ ID NOs: 59, 65, 71, 74, 80, 86, 95, or 101, iv) a light chain complementarity determining region 1 (LCDR1) whose amino acid sequence is selected from SEQ ID NOs: 60, 66, 75, 81, 87, 90, 96, or 102, v) a light chain complementarity determining region 2 (LCDR2) whose amino acid sequence is selected from SEQ ID NOs: 61, 67, 76, 82, 88, 91, 97, or 103, and vi) a light chain complementarity determining region 3 (LCDR3) whose amino acid sequence is selected from SEQ ID NOs: 62, 68, 77, 83, 89, 92, 98, 104, 112, 113, or 114.

[0013] In one embodiment, the molecule or antigen-binding fragment thereof that binds to DLL3 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), the heavy-chain variable region comprises HCDR1, HCDR2, and HCDR3, the light-chain variable region comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are, respectively, a) SEQ ID NO: 57, 58, 59, 60, 61, and 62, or b) SEQ ID NO: 63, 64, 65, 66, 67, and 68, or c) SEQ ID NO: 69, 70, 71, 66, 67, and 68, or d) SEQ ID NO: 72, 73, 74, 75, 76, and 77, or e) SEQ ID NO: 78, 79, 80, 81, 82, and 83, or f) SEQ ID NO: 84, 85, 86, 87, 88, and 89, or g) SEQ ID NO: 84, 85, 86, 90, 91, and 92, or h) SEQ ID NO: 93, 94, 95, 96, 97, and 98, or i) SEQ ID NO: 99, 100, 101, 75, 76, and 77, or j) SEQ ID NO: 72, 73, 74, 102, 103, and 104, or k) SEQ ID NO: 63, 64, 65, 66, 67, and 112, or l) SEQ ID NO: 63, 64, 65, 66, 67, and 113, or m) SEQ ID NO: 63, 64, 65, 66, 67, and 114.

[0014] In one embodiment, the heavy-chain variable region comprises an amino acid sequence selected from any of SEQ ID NO: 30, 32, 34, 35, 37, 39, 42, 44, 46, 49, or 51, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 30, 32, 34, 35, 37, 39, 42, 44, 46, 49, or 51.

[0015] In one embodiment, the light chain variable region comprises an amino acid sequence selected from any of SEQ ID NOs: 31, 33, 36, 38, 40, 41, 43, 45, 47, 48, 50, 52, 53, 54, 55, or 56, or comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NOs: 31, 33, 36, 38, 40, 41, 43, 45, 47, 48, 50, 52, 53, 54, 55, or 56.

[0016] In one embodiment, the heavy chain variable region and the light chain variable region each comprise a sequence selected from the following groups. 1) SEQ ID NOs: 30 and 31, or 2) SEQ ID NOs: 32 and 33, or 3) SEQ ID NOs: 34 and 33, or 4) SEQ ID NOs: 35 and 36, or 5) SEQ ID NOs: 37 and 38, or 6) SEQ ID NOs: 39 and 40, or 7) SEQ ID NOs: 39 and 41, or 8) SEQ ID NOs: 42 and 43, or 9) SEQ ID NOs: 44 and 36, or 10) SEQ ID NOs: 35 and 45, or 11) SEQ ID NOs: 46 and 47, or 12) SEQ ID NOs: 46 and 48, or 13) SEQ ID NOs: 49 and 50, or 14) SEQ ID NOs: 51 and 52, or 15) SEQ ID NOs: 51 and 53, or 16) SEQ ID NOs: 46 and 54, or 17) SEQ ID NOs: 46 and 55, or 18) SEQ ID NOs: 46 and 56.

[0017] A molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof further comprises a heavy-chain constant region and / or a light-chain constant region. Preferably, the heavy-chain constant region contains Fc. More preferably, Fc is derived from a mouse or a human. Even more preferably, the sequence of Fc is natural or modified.

[0018] A molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof may be a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a humanized antibody, a chimeric antibody, a reformatted antibody, a fully human antibody, a full-length antibody, a heavy-chain antibody, a nanobody, a Fab, an Fv, an scFv, an F(ab’)2, a linear antibody, or a single-domain antibody.

[0019] A molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof may be in the form of IgG1, IgG2, IgG3, or IgG4.

[0020] The present disclosure further provides a complex formed by coupling a molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof to a capture marker or a detection marker. Preferably, the detection marker includes a radionuclide, a luminescent substance, a colored substance, or an enzyme.

[0021] The present disclosure further provides an antibody-drug conjugate (ADC) formed by coupling a molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof to another biologically active molecule. Preferably, the other biologically active molecule is a small molecule drug. Preferably, the molecule that binds to DLL3 or an antigen-binding fragment thereof and the other biologically active molecule are connected by a linker.

[0022] The present disclosure further provides a nucleic acid encoding a molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof, a recombinant vector containing the nucleic acid, and a host cell containing the nucleic acid or the vector. Preferably, the host cell is a prokaryotic cell (preferably Escherichia coli), or a eukaryotic cell (preferably a mammalian cell or yeast, more preferably, the mammalian cell is a CHO cell or a HEK293 cell).

[0023] The present disclosure further provides a method for producing a molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof, which includes culturing the host cell under appropriate conditions and purifying the expression product from the cell.

[0024] The present disclosure further provides the use of a molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof in the preparation of a drug for treating or alleviating tumors.

[0025] In one embodiment, the drug targets tumor cells that abnormally express DLL3.

[0026] In one embodiment, the tumor is selected from small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer, and metastatic cancers of these tumors.

[0027] The present disclosure further provides the use of a molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof in the preparation of a detection reagent or a diagnostic reagent.

[0028] In one embodiment, the detection reagent is used to detect the expression of DLL3, and the diagnostic reagent is used to diagnose tumors. Preferably, the tumor is selected from small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer, and metastatic cancers of these tumors.

[0029] The present disclosure further provides a method for detecting the expression of DLL3 in a sample, which includes (1) contacting the sample with a molecule that binds to DLL3 of the present disclosure or an antigen-binding fragment thereof, and (2) detecting the formation of a complex of the molecule that binds to DLL3 or an antigen-binding fragment thereof and DLL3. Optionally, the molecule that binds to DLL3 or an antigen-binding fragment thereof is detectably labeled.

[0030] The present disclosure further provides a pharmaceutical composition comprising a molecule or an antigen-binding fragment thereof that binds to DLL3 of the present disclosure in an effective amount, or comprising an antibody-drug conjugate of the present disclosure in an effective amount, or comprising a nucleic acid of the present disclosure in an effective amount, or comprising a recombinant vector of the present disclosure in an effective amount, or comprising a host cell of the present disclosure in an effective amount.

[0031] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0032] Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0033] The present disclosure further provides a cartridge or kit comprising a container and the pharmaceutical composition of the present disclosure disposed within the container.

[0034] The present disclosure further provides a method for inducing death of DLL3-expressing cells, the method comprising contacting the cells with the pharmaceutical composition of the present disclosure, wherein the DLL3-expressing cells are tumor cells.

[0035] In one embodiment, the tumor cells are selected from cells of small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer, and metastatic cancers of these tumors.

[0036] The present disclosure further provides a method for treating a disease associated with the expression of DLL3 in a subject, the method comprising administering to a subject in need thereof the pharmaceutical composition of the present disclosure, or the cartridge or kit.

[0037] In one embodiment, the disease is a tumor, small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer, and metastatic cancers of these tumors.

[0038] In one embodiment, the method further comprises administering an additional therapeutic agent to the subject.

Advantages of the Invention

[0039] The technical solution of the present disclosure has the following beneficial effects. The molecule binding to DLL3 of the present disclosure and its antigen-binding fragment can specifically bind to DLL3 and do not non-specifically bind to the proteins DLL1 and DLL4 of the same family.

Brief Description of the Drawings

[0040] The drawings further illustrate the new features disclosed in this specification. Referring to these drawings, the characteristics and advantages disclosed in this specification can be better understood. However, these drawings are only used to illustrate the specific embodiments of the principles disclosed in this specification and are not intended to limit the scope of the appended claims.

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Modes for Carrying Out the Invention

[0041] <Terms> All publications, patents, and patent applications described herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0042] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodologies, solutions, and reagents described herein, and that these can vary. It should also be understood that the terms used herein are for the purpose of describing embodiments for carrying out the invention only and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0043] Some embodiments disclosed in this specification include numerical ranges, and some aspects of the present invention can be described in a range format. Unless otherwise stated, it should be understood that the numerical range or the format described in a range is for convenience and brevity only, and should not be regarded as strictly limiting the scope of the present invention. Therefore, the description in a range format should be considered as specifically disclosing all possible sub-ranges and all specific numerical values that can be taken within the range, as already explicitly stated in this specification. The above principle applies equally regardless of the magnitude of the numerical values. When describing a range, the range includes the endpoints of the range.

[0044] When referring to measurable values such as amounts and time durations, the term "about" means a variation that includes ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0045] The three-letter symbols and one-letter symbols of amino acids used in this specification are described in J. Biol. Chem, 243, p3558 (1968).

[0046] The term "antibody" as used in this specification may include a complete antibody (e.g., a full-length monoclonal antibody) and any antigen-binding fragment thereof (i.e., the antigen-binding portion) or its single chain, or may also include a product having antigen-specific binding ability formed by modifying (e.g., connecting other peptide segments, recombining functional units, etc.) a complete antibody or its antigen-binding fragment or its single chain.

[0047] In one embodiment, the term "antibody" as used herein typically refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains (HC) and two light (L) polypeptide chains (LC) held together by interactions of covalent disulfide bonds and non-covalent bonds. Natural IgG antibodies have such a structure. Each light chain consists of one variable domain (VL) and one constant domain (CL). Each heavy chain contains one variable domain (VH) and one constant domain (CH).

[0048] In the art, five main classes of antibodies, IgA, IgD, IgE, IgG, and IgM, are known, and the corresponding heavy-chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies derived from any vertebrate species can be classified into one of two clearly different types, called κ and λ, based on the amino acid sequence of their constant domains.

[0049] In the case of IgG, IgA, and IgD antibodies, the constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, a variable-length section rich in proline and cysteine. Antibodies of each class further contain interchain and intrachain disulfide bonds formed by pairs of cysteine residues.

[0050] The term "variable region" or "variable domain" refers to a significant change in amino acid composition from one type of antibody to another and mainly plays a role in antigen recognition and binding. The variable regions of each light chain / heavy chain pair form the antibody binding site, and a complete IgG antibody has two binding sites (i.e., it is bivalent). The variable region of the heavy chain (VH) and the variable region of the light chain (VL) domains each contain three regions of extreme variability, called hypervariable regions (HVRs) or more commonly complementarity-determining regions (CDRs). VH and VL each have four framework regions, FRs, represented by FR1, FR2, FR3, and FR4, respectively. Thus, the CDR and FR sequences generally appear in the FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4 sequence in the heavy chain variable domain (VH) (or the light chain variable domain (VL)).

[0051] The term "Fc" is used to define the C-terminal region of the heavy chain of an immunoglobulin, and said region includes at least a part of the constant region. The term includes the Fc region of the native sequence and the Fc region of variants.

[0052] As used herein, the term "antibody" in a broad sense includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotype antibodies, synthetic antibodies (including mutant proteins and their variants), and the like.

[0053] The term "monoclonal antibody" (or "mAb" for short) refers to an antibody that is substantially homogeneous and specific for a particular antigen epitope, produced from a clone of a single cell. Monoclonal antibodies can be prepared using various techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, genetically engineered animals, synthetic technology, or combinations of the above.

[0054] The CDR and FR regions of the variable region of the monoclonal antibody of the present invention are specified based on Kabat. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies based on the mAb sequence of the present invention and containing one or more CDRs derived from any nomenclature system are clearly retained within the scope of the present invention.

[0055] The term "humanized antibody" refers to an antibody in which all or some of the amino acids other than the CDRs of a non-human antibody (e.g., a mouse antibody) are replaced with corresponding amino acids derived from a human immunoglobulin. Minor amino acid additions, deletions, insertions, substitutions, or modifications are permitted, provided that they do not abolish the binding ability of the antibody to a specific antigen. The "humanized" antibody retains the same antigen specificity as the original antibody.

[0056] The term "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0057] The term "antibody fragment" includes at least a portion of a full antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment that specifically binds or reacts with a selected antigen or an antigen epitope thereof in an immunoglobulin or antibody, or a fusion protein product further derived from this fragment, such as a single-chain antibody, the extracellular binding region of a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to, variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments.

[0058] The term "antigen" refers to a substance that is recognized by an antibody or an antibody-binding fragment and specifically binds to the antibody or antibody-binding fragment. Broadly speaking, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, or a complete extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides, and lipids, as well as portions and combinations thereof, can all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. An "antigen" may be a molecule that elicits an immune response. Any form of antigen or a cell or formulation containing the antigen can be used to generate an antibody specific for the antigenic determinant. The antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with a cell expressing at least a portion of the antigen on its surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the foregoing antigens may be used alone or in combination with one or more immunogenicity-enhancing adjuvants well known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform a cell that expresses the antigen therein, and the vectors include, but are not limited to, non-viral vectors such as adenovirus vectors, lentivirus vectors, plasmids, and cationic lipids.

[0059] The term "epitope" refers to the site in an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by adjacent amino acids or by amino acids that are juxtaposed but not adjacent due to the tertiary folding of the protein. Epitopes composed of adjacent amino acids are usually retained upon exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost upon treatment with a denaturing solvent. Epitopes typically contain at least 3 to 15 amino acids in a unique spatial conformation. Methods for determining the epitope to which a particular antibody binds are well known in the art and include immunoblotting and immunoprecipitation detection assays. Methods for determining the spatial conformation of an epitope include, for example, techniques in the art such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and the techniques described herein.

[0060] The terms "bispecific binding molecule" and "multispecific binding molecule" each refer to a binding molecule (e.g., a molecule comprising an antibody or an antibody fragment) that has specificity for two or more different antigens (or epitopes), preferably a bispecific antibody.

[0061] When preparing an antibody, binding molecule, bispecific binding molecule, or multispecific binding molecule using the variable regions described in this disclosure, the constant region is not particularly limited, and a constant region known to those skilled in the art or a self-obtained constant region may be used, or amino acid mutations may be introduced into a portion of the constant region (e.g., mutations that improve or reduce binding to an Fcγ receptor or FcRn).

[0062] The method for obtaining the binding molecule, antigen-binding fragment, antibody, bispecific binding molecule or multispecific binding molecule of the present disclosure is not particularly limited, and it can be obtained by any method. For example, refer to Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapters 5-8, 15. The binding molecule, antigen-binding fragment, antibody, bispecific binding molecule or multispecific binding molecule of the present invention can be prepared and purified by ordinary methods. For example, the cDNA sequences encoding the heavy and light chains may be cloned into an expression vector and subjected to recombination. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems cause highly conserved N-terminal glycosylation in the Fc region of antibodies. Stable clones are obtained by expressing antibodies specific for human antigens. The positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies. The culture broth secreting the antibodies can be purified and collected by ordinary techniques. Antibodies can be filtered and concentrated by ordinary methods. Soluble mixtures and polymers can be removed by ordinary methods such as molecular sieves and ion exchange.

[0063] The term "antibody-drug conjugate (ADC)" refers to an antibody formed by covalent coupling to a therapeutic agent or active pharmaceutical ingredient (API), whereby the therapeutic agent or active pharmaceutical ingredient (API) targets the binding target of the antibody and can exert its pharmacological function. The therapeutic agent or active pharmaceutical ingredient may be a cytotoxin capable of killing the target cells of the ADC, preferably malignant or cancer cells. The covalent bond of the therapeutic agent, active pharmaceutical ingredient or cytotoxin may be carried out in a non-site-specific manner using a standard chemical linker that binds the payload to lysine or cysteine residues, or, preferably, the bond is carried out in a site-specific manner, which allows complete control of the ratio of the drug to the antibody at the binding site and in the resulting ADC.

[0064] The term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its partner that binds thereto (e.g., an antigen). The term "KD" refers to the dissociation constant of a specific antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0065] The term "biological activity" refers to the ability of an antibody to bind to an antigen and bring about a measurable biological response, which can be measured in vitro or in vivo.

[0066] The pharmaceutical compositions of the present disclosure can be formulated by mixing them, if necessary, with suitable pharmaceutically acceptable carriers, media, etc. that are inert thereto. For example, physiological saline, sterile water, excipients, stabilizers, antioxidants (e.g., ascorbic acid, etc.), buffers, preservatives, surfactants, chelating agents (e.g., EDTA, etc.), or binders, etc. may be mentioned. In addition, it may contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulins, amino acids such as glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine, and lysine, saccharides and carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols such as mannitol and sorbitol. When making an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose, and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, may be mentioned, and appropriate solubilizing agents, such as alcohols (e.g., ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, polysorbate 20, poloxamer 188, HCO-50, etc.) may be used in combination. Also, by mixing hyaluronidase in the formulation, it is also possible to administer a larger volume subcutaneously.

[0067] The binding molecules or antigen-binding fragments of the present disclosure may be used in combination with other drugs, the active ingredients may be mixed to form a single dosage unit, or they may be used separately as independent dosage units.

[0068] The term "effective amount" refers to the dosage of a pharmaceutical formulation comprising a binding molecule or antigen-binding fragment of the present disclosure, which, after administration to a patient in a single or multiple doses, results in the expected effect in the treated patient. The effective amount can be readily determined by an attending physician who is skilled in the art, taking into account a plurality of factors such as, for example, differences in race, body weight, age and health status, the specific disease involved, the severity of the disease, the response of the individual patient, the specific antibody administered, the mode of administration, the bioavailability characteristics of the formulation administered, the dosing regimen selected, and the use of any accompanying therapy.

[0069] The term "cartridge" or "kit" includes a pharmaceutical composition of the present disclosure in one or more unit dosage forms of an effective amount. In some embodiments, the "cartridge" or "kit" can include a sterile container, which can be in the form of a box, ampoule, bottle, vial, tube, bag, blister pack or other suitable container known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding the drug. The cartridge further includes instructions for administering the pharmaceutical composition of the present disclosure to an individual. The instructions generally include a method of treating a disease using the pharmaceutical composition of the present invention.

[0070] As used herein, the term "individual" or "subject" refers to any animal, such as a mammal or marsupial. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus monkeys, rhesus monkeys or other species of macaques), mice, pigs, horses, donkeys, cows, sheep, rats and any kind of poultry.

[0071] As used herein, the terms "disease", "pathological condition", "disorder", etc. refer to any change or disorder that impairs or interferes with the normal function of cells, tissues, or organs. For example, the "disease" includes, but is not limited to, tumors, pathogen infections, autoimmune diseases, T cell dysfunctional diseases, or defects in immune tolerance (e.g., transplant rejection).

[0072] As used herein, the term "tumor" refers to a disease characterized by the pathological growth of cells or tissues, and subsequent migration or invasion into other tissues or organs. Tumor growth is generally uncontrolled, progressive, and does not induce or inhibit the growth of normal cells.

[0073] As used herein, the term "treatment" refers to a clinical intervention in the process of attempting to change a disease caused by an individual or treated cells, which may be preventive or clinically pathological. Therapeutic effects include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological results caused by any disease, prevention of metastasis, reduction of the progression rate of a disease, improvement or alleviation of the disease state, alleviation or improvement of the prognosis, etc.

[0074] (Examples) Hereinafter, the present invention will be further described with reference to specific examples. It should be noted that these examples are for explaining the present invention and do not limit the scope of the present invention. Experimental methods for which specific conditions are not specified in the following examples generally follow conventional conditions as described in Molecular Cloning Experiment Guide (edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002), or follow the conditions recommended by the manufacturer.

[0075] Example 1: Information on Human DLL3 and Cynomolgus Monkey DLL3 Antigens The human DLL3 used in the examples was purchased from Kactus Biosystems (Catalog number: DLL-HM103), and its full-length amino acid sequence (SEQ ID NO: 105) (Uniprot ID: Q9NYJ7) was as follows.

Chemical formula

[0076] The cynomolgus monkey DLL3 (cyno-DLL3) used in the examples was purchased from Kactus Biosystems (Catalog number: CM103), and its full-length amino acid sequence (SEQ ID NO: 106) (Uniprot ID: A0A2K5WSR4) was as follows.

Chemical formula

[0077] Example 2: Information on DLL1 and DLL4 antigens, which are members of the same family The human DLL1 used in the examples was purchased from Sino Biological (Catalog number: 11635-H08H), and its full-length amino acid sequence (SEQ ID NO: 107) (Uniprot ID: O00548) was as follows.

Chemical formula

[0078] The human DLL4 used in the examples was purchased from Sino Biological (Catalog number: 10171 - H08H), and its full-length amino acid sequence (SEQ ID NO: 108) (Uniprot ID: Q9NR61) was as shown below. [Chemical formula] Note: The double-underlined part is the signal peptide (1 - 26), the underlined part is the DLL3 extracellular region (27 - 529), the dotted line part is the transmembrane region (530 - 550), and the italicized part was the intracellular region (551 - 685).

[0079] Example 3: Immunization of camels and construction of an immune library of antibodies consisting of heavy chains Camels were immunized with the human DLL3 antigen (as described in Example 1). The immunization days were day 0, day 21, day 35, and day 49, for a total of 4 immunizations. Blood samples were collected on day 28, day 42, and day 73 respectively to separate serum, and the immune response status in the serum was detected by protein-level ELISA. When the serum titer was detected to exceed 1:128000, the immunization was terminated, and another 100 mL of blood sample was collected from the immunized camels. According to the manufacturer's instructions, Solarbio's lymphocyte separation solution was used to separate camel PBMC respectively. After extracting total RNA (OMEGA cell total RNA extraction kit), Takara PrimeScript TMUsing the II reverse transcription kit, cDNA was synthesized as a template, and nested PCR amplification was performed with designed specific primers to obtain a VHH gene fragment. After recovering the VHH fragment, it was digested with Sfi I and ligated to the pADL-23c phagemid vector, and a DLL3 camel immune library was constructed with TG1 electrocompetent cells (the capacity of the library was 1.12E8).

[0080] Example 4: Screening of Positive Clones of Anti-DLL3 Derived from Camels To obtain a positive antibody that can cross-react with human DLL3 and cynomolgus monkey DLL3, the library was amplified, M13K07 helper phage was added, and after assembling as phage, 1×10 12 pfu of the camel immune library phage was added, incubated with biotinylated human DLL3 protein (8 μg / mL) bound to magnetic beads at room temperature for 1 hour, and unbound phage was washed away with 0.05% PBST. Then, phage specifically bound to DLL3 was eluted with 100 mM triethylamine. After gradient dilution, it was infected into exponentially growing Escherichia coli SS320, coated on plates containing ampicillin, and cultured overnight at 37°C. Single colonies were picked up, induced for expression with IPTG, and the supernatant was used for ELISA detection. Human DLL3 or cynomolgus monkey DLL3 antigen was coated on each ELISA plate at 2 μg / mL and kept at 4°C overnight. After washing 3 times with 0.05% PBST, it was blocked with 5% skim milk at room temperature for 1 hour. After washing 3 times with 0.05% PBST, 30 μL of the induced supernatant was added to each well, and medium was added to the negative control wells. Incubated at room temperature for 1 hour, and finally detected with anti-Myc HRP (the VHH induced by IPTG was tagged with his and c-Myc). Clones with an OD450 value binding to human and cynomolgus monkey DLL3 obtained by ELISA detection exceeding 1.0 and a ratio to the ELISA OD450 of the negative control binding to the medium all exceeding 3 were sequenced to obtain the amino acid sequences of the five heavy chain antibody variable regions of the present disclosure, and the results are shown in Table 1.

Table 1

[0081] In the amino acid sequence, the CDRs and FRs of the antibody variable region are classified by the Kabat numbering system, and the sequence compositions of the three CDRs of each antibody are shown in Table 2.

Table 2

[0082] Example 5: Construction of a camel-derived anti-DLL3 chimeric antibody and its transient transfection expression in eukaryotic cells The heavy chain antibody variable region of the present disclosure and the human IgG1 constant region whose sequencing has been completed were spliced, and the generated target gene fragment was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid. The heavy chain antibody variable region may be connected to the human IgG1 constant region by a connecting short peptide, that is, a heavy chain antibody variable region - connecting short peptide - human IgG1 constant region is formed. The sequence of the connecting short peptide used in this example is GGGGS.

[0083] The sequence of the introduced human IgG1 constant region (SEQ ID NO: 109) was as follows.

Chemical formula

[0084] Example 6: Binding of the anti-DLL3 chimeric antibody derived from camel to DLL3-expressing cells HEK293 cells (purchased from the Chinese Academy of Sciences) were transfected with the full-length cyno-DLL3 antigen gene (for the sequence, refer to Example 1), the cells were cultured and passaged, single colonies were picked up, the expression level of DLL3 protein in the single colony cells was identified by flow cytometry, and then they were expanded in culture and cryopreserved for use.

[0085] SHP-77 and HEK293-cyno DLL3 cells were cultured. The medium for SHP-77 cells was RPMI1640 + 10% FBS, and the medium for HEK293-cyno DLL3 cells was DMEM + 10% FBS + 200 μg / mL hygromycin. T75 cell culture flasks were used and cultured in an incubator at 37 °C and 5% CO₂. When using the cells, they were washed with sterile DPBS, digested with 0.25% trypsin-EDTA for about 5 minutes, and then stopped with complete medium.

[0086] The digested cells were centrifuged at a rotation speed of 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended in 100 μL of 1% BSA (in PBS). The cells were counted, the cell density was adjusted to 1E6 / mL, inoculated into a 96-well plate (Corning 3799), centrifuged at a rotation speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and left standing at 4°C for use. The test antibody sample was diluted with 1% BSA (in PBS), the initial concentration was 100 nM, and diluted to seven concentrations from 10-fold. The diluted antibody (100 μL / well) was used to resuspend the cells and incubated at 4°C for 1 hour. Centrifuged at a rotation speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. Resuspended and washed with 160 μL of 1% BSA (in PBS), centrifuged at a rotation speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. According to the instructions, the secondary antibody (goat anti-human IgG Fc PE) was diluted 1:200 with 1% BSA (in PBS), the diluted secondary antibody (100 μL / well) was used to resuspend the cells and incubated at 4°C for 30 minutes. Centrifuged at a rotation speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. Resuspended and washed with 160 μL of 1% BSA (in PBS), centrifuged at a rotation speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA (in PBS), filtered through a 300-mesh gauze, and the mean fluorescence intensity of the PE channel was detected using a flow cytometer.

[0087] Export the FCS file from the flow cytometer, analyze the mean fluorescence intensity of the PE channel (hereinafter abbreviated as MFI) of each sample using the software FlowJo, import the mean fluorescence intensity obtained by the analysis into GraphPad, and analyze the half-maximal effective concentration (hereinafter abbreviated as EC 50 and abbreviated) and the highest mean fluorescence intensity (Top MFI) of the antibody against the cells, and the results are shown in Table 3 and Figure 1.

Table 3

[0088] Example 7: Humanization of the anti-DLL3 heavy chain antibody derived from camel As a framework template for VHH transplantation, a germline gene sequence with high homology to the heavy chain antibody candidate was selected by sequence alignment. After grafting the antibody CDR region candidates onto the selected human antibody variable region framework, individual amino acid back mutations were performed to obtain a humanized antibody, and the amino acid sequence of the humanized variable region is shown in Table 4. [Table 4]

[0089] Example 8: Preparation of the anti-DLL3 humanized antibody derived from camel Referring to the description of Example 5, the variable region of the humanized antibody and the human IgG1 constant region were spliced, and the generated target gene fragment was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0090] Expi293F TM cells (Thermo Fisher Scientific) were cultured, the cells were inoculated into a shaking flask (Corning Inc.), and cultured on a shaker in an environment of 37 °C and 8% CO2. The cell density was adjusted, the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed at an appropriate ratio, added to the cell culture shaking flask, and after culturing the cells for 6 days, the expression supernatant was collected, cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was rinsed with PBS until the measured value of A280 dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0 - 3.5 and neutralized with 1 M Tris-HCl (pH 8.0 - 9.0). After appropriately concentrating the eluted sample, it was exchanged with PBS and aliquoted for use. Finally, SDS-PAGE, HPLC purity analysis and A280 concentration measurement were performed on the purified humanized antibody.

[0091] Example 9: Demonstration of in vitro cell binding of the anti-DLL3 humanized antibody derived from camel SHP-77 and HEK293-cyno DLL3 cells were cultured. The medium for SHP-77 cells was RPMI1640 + 10% FBS, and the medium for HEK293-cyno DLL3 cells was DMEM + 10% FBS + 200 μg / mL of Hygromycin. T75 cell culture flasks were used and placed in an incubator at 37°C and 5% CO2 for culture. When using the cells, the cells were washed with sterile DPBS, digested with 0.25% trypsin-EDTA for about 5 minutes, and then stopped with complete medium.

[0092] The digested cells were centrifuged at a rotational speed of 1000 rpm at room temperature for 5 minutes, the supernatant was discarded, and the cells were resuspended in 100 μL of 1% BSA (in PBS). The cells were counted, the cell density was adjusted to 1E6 / mL, inoculated into a 96-well plate (corning 3799), centrifuged at a rotational speed of 1500 rpm at 4°C for 5 minutes, the supernatant was discarded, and left standing at 4°C for use. The test antibody sample was diluted with 1% BSA (in PBS), the initial concentration was 100 nM, and diluted to seven concentrations from 10-fold. The diluted antibody (100 μL / well) was used to resuspend the cells and incubated at 4°C for 1 hour. Centrifuged at a rotational speed of 1500 rpm at 4°C for 5 minutes, the supernatant was discarded. Resuspended and washed with 160 μL of 1% BSA (in PBS), centrifuged at a rotational speed of 1500 rpm at 4°C for 5 minutes, the supernatant was discarded. According to the instructions, the secondary antibody (goat anti human IgG Fc PE) was diluted 1:200 with 1% BSA (in PBS), the diluted secondary antibody (100 μL / well) was used to resuspend the cells and incubated at 4°C for 30 minutes. Centrifuged at a rotational speed of 1500 rpm at 4°C for 5 minutes, the supernatant was discarded. Resuspended and washed with 160 μL of 1% BSA (in PBS), centrifuged at a rotational speed of 1500 rpm at 4°C for 5 minutes, the supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA (in PBS), filtered through a 300-mesh gauze, and the mean fluorescence intensity of the PE channel was detected on a flow cytometer.

[0093] Export the FCS file from the flow cytometer, analyze the mean fluorescence intensity (hereinafter abbreviated as MFI) of the PE channel of each sample with the software FlowJo, import the mean fluorescence intensity obtained by the analysis into GraphPad, and determine the half-maximal effective concentration (hereinafter abbreviated as EC 50 ) and the highest mean fluorescence intensity (Top MFI) of the antibody against cells, and the results are shown in Table 5 and Figure 2.

Table 5

[0094] Example 10: Biacore Affinity Experiment of Humanized Anti-DLL3 Antibody Derived from Camel The affinity and pharmacokinetic properties of the anti-DLL3 humanized antibody against human DLL3 were analyzed with a Biacore 8K instrument. First, the CM5 chip was activated with EDC and NHS, then a mouse monoclonal antibody against human Fc was immobilized, and further blocked with ethanolamine.

[0095] To measure the affinity and pharmacokinetic properties against human DLL3, the DLL3 humanized antibody was diluted to 0.2 μg / mL with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer and captured for 45 seconds at a flow rate of 10 μL / min. Human DLL3 was serially diluted 2-fold to obtain a series of concentrations (100 nM - 0.39 nM), and bound for 90 seconds at a flow rate of 50 μL / min and dissociated for 600 seconds.

[0096] After each cycle of the experiment was completed, it was rinsed with a 3 M MgCl2 solution at a flow rate of 30 μL / min for 30 seconds to remove the captured antibody together with the antigen, and the regeneration of the chip was completed. The original data was analyzed with Biacore Insight Evaluation Software (3.0.12.15655) and fitted with a (1:1) Langmuir model, and the results are shown in Table 6.

Table 6

[0097] Example 11: Binding Experiment of a Protein DLL1 and DLL4 of the Same Family as the Anti-DLL3 Humanized Antibody Derived from Camel to the Antigen Human DLL1 (SinoBiological, Catalog No.: 11635-H08H) or human DLL4 (SinoBiological, Catalog No.: 10171-H08H), which are antigen proteins, were dissolved in 1×PBS so that the concentration became 1 μg / mL. Next, the antigen (100 μL / well) was added to a high-affinity ELISA plate (Biolegend, Catalog No.: 423501) and allowed to stand at 4°C overnight. The antigen was washed with PBST (300 μL / well) three times. The antigen was blocked with 1% BSA (in PBST) (200 μL / well) and incubated at 37°C for 1.5 hours.

[0098] The test antibody was diluted with 1% BSA (in PBS), the initial concentration was 100 nM, and it was diluted to seven concentrations in a 10-fold dilution series. The plate (300 μL / well) was washed with PBST three times. The diluted antibody (100 μL / well) was added to the ELISA plate and incubated at room temperature for 2 hours. The antibody was washed three times with PBST (300 μL / well). The secondary antibody was diluted with 1% BSA (in PBST) (goat anti-human IgG Fc (HRP) used for DLL4 was diluted 1:20000, and HRP goat anti-mouse IgG (H+L) used for DLL1 was diluted 1:10000). The diluted secondary antibody (100 μL / well) was added to the ELISA plate and incubated at room temperature for 1 hour. The plate was washed six times with PBST (300 μL / well). A chromogenic solution was prepared (TMA and TMB were mixed at a ratio of 1:1), and the chromogenic solution (100 μL / well) was added to the plate and incubated in the dark for 5 minutes. 50 μL of ELISA stop solution was added to the plate and shaken uniformly.

[0099] Read OD450 in Envision, plot it in GraphPad with OD450, and EC 50The value was determined. The results are shown in Figure 3, indicating that the detection antibody did not bind non-specifically to either of the proteins DLL1 and DLL4 in the same family.

[0100] Example 12: Preparation of Variants of a Humanized Anti-DLL3 Antibody Derived from Camel When post-translational modification (PTM) analysis of the heavy chain antibody of the present disclosure was performed, it was found that there was one deamidation site in the variable region of hDLL3-3-1. Site-specific mutations at a single site were performed on the amino acid at position 103, and two variants of hDLL3-3-1 were prepared. They were hDLL3-3-1-NA and hDLL3-3-1-QS, respectively. The amino acid sequences of the variable regions of the two variants are shown in Table 7.

Table 7

[0101] With reference to the description of Example 5, the two variants were prepared. After transient transfection expression in a mammalian cell line, affinity detection was performed using SHP-77 cells. The results are shown in Table 8 and Figure 4, indicating that the two variants can eliminate the risk of post-translational modification when the cell-level binding does not change significantly. Affinity detection was performed using human DLL3 antigen protein, and the results are shown in Table 9.

Table 8

Table 9

[0102] Example 13: Obtaining an Anti-DLL3 Antibody Derived from a Mouse Hybridoma The anti-DLL3 monoclonal antibody was generated by immunizing mice. Six-week-old female Swiss Webster white mice (Charles River) were used in the experiment. The breeding environment was SPF. After purchasing the mice, they were bred in the laboratory environment for one week, with a 12 / 12-hour light / dark cycle, a temperature of 20-25 °C, and a humidity of 40-60%. The immunizing antigen was human DLL3 recombinant protein with a His tag (huDLL3-His). Titermax (sigma, lot number: T2684) was used as an adjuvant. The ratio of the antigen to the adjuvant (titermax) was 1:1. After emulsifying the antigen, it was inoculated. The inoculation days were day 0, day 14, day 35, and day 56, and a booster inoculation was performed 3 days before the fusion of splenocytes. During this period, mouse sera were detected by ELISA and FACS methods to determine the antibody titers in the mouse sera. After the fifth immunization, mice with high antibody titers in the sera and a tendency towards a plateau in the titers were selected for splenocyte fusion. The spleen lymphocytes were fused with Sp2 / 0 cells (ATCC® CRL-8287 TM ) of myeloma cells using an optimized electrofusion step to obtain hybridoma cells.

[0103] After culturing the hybridoma cells after fusion for 7-14 days, the culture supernatant was taken out. Using the DLL3 recombinant protein, the hybridoma supernatant was screened for antibodies by ELISA experiments. For the obtained positive antibody strains, CHO-K1 cells stably expressing DLL3 were further used. Compared with blank CHO-K1 cells, non-specifically binding antibody hybridoma strains were removed, and screening was performed by flow cytometry. Hybridomas that bound to the recombinant protein and also to the cell-expressed antigen were selected. Logarithmically growing hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TMReverse Transcriptase, Takara #2680A). After PCR amplification of the cDNA obtained by reverse transcription using the mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503), sequencing was performed to obtain the amino acid sequences of the variable regions of the 10 monoclonal antibodies of the present disclosure, which are shown in Table 10A. [Table 10] [Table 11]

[0104] In the above amino acid sequences, the CDRs and FRs of the antibody variable regions were distinguished by the Kabat numbering system, and the compositions of the six CDR sequences of each antibody are shown in Table 10B. [Table 12]

[0105] Example 14: Construction of an anti-DLL3 chimeric antibody derived from a mouse hybridoma and its transient transfection expression in eukaryotic cells The heavy chain variable region and light chain variable region of the monoclonal antibody of the present disclosure whose sequencing was completed were respectively spliced into the IgG1 heavy chain constant region and κ light chain constant region, and the generated target gene fragment was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0106] In serum-free medium, Expi293F TMCells (Thermo Fisher Scientific) were cultured, inoculated into a shaking flask (Corning Inc.), and placed on a shaker for culture in an environment of 37 °C and 8% CO2. The cell density was adjusted, and a recombinant expression vector containing the target gene fragment and a PEI transfection reagent were mixed at an appropriate ratio, added to the cell culture shaking flask. Six days after cell culture, the expression supernatant was collected, cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was rinsed with PBS until the measured value of A280 dropped to the baseline. The target protein was eluted with an acidic eluent at pH 3.0 - 3.5 and neutralized with 1 M Tris-HCl (pH 8.0 - 9.0). After appropriately concentrating the eluted sample, it was exchanged with PBS and dispensed for use. Finally, SDS-PAGE, HPLC purity analysis, and A280 concentration measurement were performed on the purified chimeric antibody.

[0107] Example 15: Demonstration of in vitro cell binding of an anti-DLL3 chimeric antibody derived from a mouse hybridoma SHP-77 and HEK293-cyno DLL3 cells were cultured. The medium for SHP-77 cells was RPMI1640 + 10% FBS, and the medium for HEK293-cyno DLL3 cells was DMEM + 10% FBS + 200 μg / mL of Hygromycin. Using a T75 cell culture flask, they were placed in an incubator at 37 °C and 5% CO2 for culture. When using the cells, the cells were washed with sterile DPBS, digested with 0.25% trypsin-EDTA for about 5 minutes, and then stopped with complete medium.

[0108] The digested cells were centrifuged at a rotational speed of 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended in 100 μL of 1% BSA (in PBS). The cells were counted, the cell density was adjusted to 1E6 / mL, inoculated into a 96-well plate (corning 3799), centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and left standing at 4°C for use. The test antibody sample was diluted with 1% BSA (in PBS), the initial concentration was 100 nM, and diluted to seven concentrations with a 10-fold dilution. The diluted antibody (100 μL / well) was used to resuspend the cells, and incubated at 4°C for 1 hour. Centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. Resuspended and washed with 160 μL of 1% BSA (in PBS), centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. According to the instructions, the secondary antibody (goat anti human IgG Fc PE) was diluted 1:200 with 1% BSA (in PBS), the diluted secondary antibody (100 μL / well) was used to resuspend the cells, and incubated at 4°C for 30 minutes. Centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. Resuspended and washed with 160 μL of 1% BSA (in PBS), centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA (in PBS), filtered through a 300-mesh gauze, and the mean fluorescence intensity of the PE channel was detected on a flow cytometer.

[0109] Export the FCS file from the flow cytometer, analyze the mean fluorescence intensity (hereinafter abbreviated as MFI) of the PE channel of each sample with the software flowjo, import the mean fluorescence intensity obtained by the analysis into Graphpad, and analyze the half-maximal effective concentration (hereinafter abbreviated as EC 50 and abbreviated) and the highest mean fluorescence intensity (Top MFI) of the antibody against the cells, and the results are shown in Table 11 and Figure 5.

Table 13

[0110] Example 16: Humanization of an anti-DLL3 antibody derived from a mouse hybridoma Ten chimeric antibodies were subjected to expression purification tests and cell-level binding tests, etc., and then three clones were picked up for humanization design.

[0111] Humanization of the mouse anti-human DLL3 monoclonal antibody was performed as in the methods disclosed in many literatures in the art. Briefly, the constant domain of the parent (mouse antibody) was replaced with a human constant domain, and a human antibody sequence was selected based on the homology between the mouse antibody and the human antibody. In the typical structure of the obtained mouse antibody VH / VL CDR, the sequences of the heavy chain and light chain variable regions were compared with the human antibody germline database to obtain a highly homologous human germline template.

[0112] The CDR regions of the mouse antibody were transplanted into the selected corresponding humanized templates, the humanized variable regions were replaced, and recombination was performed with the IgG constant region (preferably, the heavy chain is IgG1 and the light chain is κ). Next, based on the three-dimensional structure of the mouse antibody, reverse mutations were made to the buried residues, the residues that directly interact with the CDR regions, and the residues that have an important influence on the three-dimensional structure of VL and VH, and antibodies combining the humanized light chain and heavy chain variable region sequences were designed and shown in Table 12. [Table 14]

[0113] Example 17: Demonstration of in vitro cell binding of a humanized anti-DLL3 antibody derived from a mouse hybridoma SHP-77 cells were cultured, and the medium for SHP-77 cells was RPMI1640 + 10% FBS. A T75 cell culture flask was used and placed in an incubator at 37 °C and 5% CO2 for culture. When using the cells, the cells were washed with sterile DPBS, digested with 0.25% trypsin-EDTA for about 5 minutes, and then stopped with complete medium.

[0114] The digested cells were centrifuged at a rotational speed of 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended in 100 μL of 1% BSA (in PBS). The cells were counted, the cell density was adjusted to 1E6 / mL, inoculated into a 96-well plate (Corning 3799), centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and left standing at 4°C for use. The test antibody sample was diluted with 1% BSA (in PBS), the initial concentration was 100 nM, and diluted to seven concentrations with a 10-fold dilution. The diluted antibody (100 μL / well) was used to resuspend the cells, and incubated at 4°C for 1 hour. Centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. Resuspended and washed with 160 μL of 1% BSA (in PBS), centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. According to the instruction manual, the secondary antibody (goat anti human IgG Fc PE) was diluted 1:200 with 1% BSA (in PBS), the diluted secondary antibody (100 μL / well) was used to resuspend the cells, and incubated at 4°C for 30 minutes. Centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. Resuspended and washed with 160 μL of 1% BSA (in PBS), centrifuged at a rotational speed of 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA (in PBS), filtered through a 300-mesh gauze, and the mean fluorescence intensity of the PE channel was detected on a flow cytometer.

[0115] Export the FCS file from the flow cytometer, analyze the mean fluorescence intensity (hereinafter abbreviated as MFI) of the PE channel of each sample with the software FlowJo, import the mean fluorescence intensity obtained by the analysis into GraphPad, and analyze the half-maximal effective concentration (hereinafter abbreviated as EC 50 and abbreviated) and the maximum mean fluorescence intensity (Top MFI) of the antibody against the cells, and the results are shown in Table 13 and Figure 6.

Table 15

[0116] Example 18: Biacore Affinity Experiment of Anti-DLL3 Humanized Antibody Derived from Mouse Hybridoma The affinity and pharmacokinetic properties of the anti-DLL3 humanized antibody against human DLL3 were analyzed using a Biacore 8K instrument. First, the CM5 chip was activated with EDC and NHS, then a mouse monoclonal antibody against human Fc was immobilized, and further blocked with ethanolamine.

[0117] To measure the affinity and pharmacokinetic properties against human DLL3, the DLL3 humanized antibody was diluted with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer to a concentration of 0.2 μg / mL and captured at a flow rate of 10 μL / min for 45 seconds. Human DLL3 was serially diluted two-fold to obtain a series of concentrations (100 nM - 0.39 nM), and was allowed to bind at a flow rate of 50 μL / min for 90 seconds and dissociate for 600 seconds.

[0118] After each cycle of the experiment was completed, the chip was rinsed with 3M MgCl2 solution at a flow rate of 30 μL / min for 30 seconds to remove the captured antibody together with the antigen, and the regeneration of the chip was completed. The initial data was analyzed using the software Biacore Insight Evaluation Software (3.0.12.15655) and fitted with the (1:1) Langmuir model, and the results are shown in Table 14.

Table 16

[0119] Example 19: Binding Experiment of Anti-DLL3 Humanized Antibody Derived from Mouse Hybridoma against Proteins DLL1 and DLL4 of the Same Family The human DLL1 (SinoBiological, catalog number 11635-H08H) or human DLL4 (SinoBiological, catalog number 10171-H08H), which are antigen proteins, were dissolved in 1×PBS6 to a concentration of 1 μg / mL. Next, the antigen (100 μL / well) was added to a high-affinity ELISA plate (Biolegend, catalog number 423501) and left standing overnight at 4°C. The antigen was washed three times with PBST (300 μL / well). The antigen was blocked with 1% BSA (in PBST) (200 μL / well) and incubated at 37°C for 1.5 hours.

[0120] The test antibody was diluted with 1% BSA (in PBS) at an initial concentration of 100 nM and diluted to seven concentrations in 10-fold increments. The plate was washed three times with PBST (300 μL / well). The diluted antibody (100 μL / well) was added to the ELISA plate and incubated at room temperature for 2 hours. The antibody was washed three times with PBST (300 μL / well). The secondary antibody was diluted with 1% BSA (in PBST) (goat anti-human IgG Fc (HRP) used for DLL4 was diluted 1:20000, and HRP goat anti-mouse IgG (H+L) used for DLL1 was diluted 1:10000). The diluted secondary antibody (100 μL / well) was added to the ELISA plate and incubated at room temperature for 1 hour. The plate was washed six times with PBST (300 μL / well). A chromogenic solution was prepared (TMA and TMB were mixed 1:1), and the chromogenic solution (100 μL / well) was added to the plate and incubated in the dark for 5 minutes. 50 μL of ELISA stop solution was added to the plate and shaken uniformly.

[0121] OD450 was read in envision, plotted in GraphPad with OD450, and the EC 50 value was determined. The results are shown in Figure 7, indicating that the antibody being tested did not bind nonspecifically to either of the proteins DLL1 and DLL4 of the same family.

[0122] Example 20: Preparation of Variants of a Humanized Anti-DLL3 Antibody Derived from a Mouse Hybridoma When post-translational modification (PTM) analysis was performed on the antibodies of the present disclosure, it was found that there was one deamidation site in the light chain variable region of H2-39E2D11. Single-site site-specific mutations were made to the amino acid at position 99, and three mutants of H2-39E2D11 were prepared. They were H2-39E2D11-NA, H2-39E2D11-QS, and H2-39E2D11-AS, respectively. The amino acid sequences of the variable regions of the three mutants are shown in Table 15.

Table 17

[0123] Referring to the description of Example 5, the three mutants were prepared. After transient transfection expression in a mammalian cell line, affinity detection was performed using SHP-77 cells, and the results are shown in Figure 8. It was shown that if the binding at the cell level did not change significantly for the mutants, the risk of post-translational modification could be eliminated. Affinity detection was performed using human DLL3 antigen protein, and the results are shown in Table 16.

Table 18

Table 19

[0124] The above-described forms of the present disclosure are merely exemplary, and those skilled in the art can recognize and determine countless equivalents of specific compounds, materials, and operations without the need to perform experiments different from the prior art. All of these equivalents are within the scope of the present invention and are included in the claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to DLL3, comprising a heavy-chain variable region (VH) and a light-chain variable region (VL), wherein the heavy-chain variable region comprises HCDR1, HCDR2, and HCDR3, the light-chain variable region comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are, respectively, as follows in a) to m): a) SEQ ID NOs: 63, 64, 65, 66, 67, and 112, or b) SEQ ID NOs: 57, 58, 59, 60, 61, and 62, or c) SEQ ID NOs: 63, 64, 65, 66, 67, and 68, or d) SEQ ID NOs: 69, 70, 71, 66, 67, and 68, or e) SEQ ID NOs: 72, 73, 74, 75, 76, and 77, or f) SEQ ID NOs: 78, 79, 80, 81, 82, and 83, or g) SEQ ID NOs: 84, 85, 86, 87, 88, and 89, or h) SEQ ID NOs: 84, 85, 86, 90, 91, and 92, or i) SEQ ID NOs: 93, 94, 95, 96, 97, and 98, or j) SEQ ID NOs: 99, 100, 101, 75, 76, and 77, or k) SEQ ID NOs: 72, 73, 74, 102, 103, and 104, or l) SEQ ID NOs: 63, 64, 65, 66, 67, and 113, or m) SEQ ID NOs: 63, 64, 65, 66, 67, and 114, An antibody or antigen-binding fragment thereof that binds to DLL3, which is selected from one of the above.

2. The heavy-chain variable region comprises any amino acid sequence selected from SEQ ID NOs: 46, 30, 32, 34, 35, 37, 39, 42, 44, 49, or 51, or comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NOs: 46, 30, 32, 34, 35, 37, 39, 42, 44, 49, or 51, and / or The light chain variable region contains any amino acid sequence selected from SEQ ID NO: 54, 31, 33, 36, 38, 40, 41, 43, 45, 47, 48, 50, 52, 53, 55 or 56, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any of SEQ ID NO: 54, 31, 33, 36, 38, 40, 41, 43, 45, 47, 48, 50, 52, 53, 55 or 56, and the antibody or antigen-binding fragment thereof that binds to DLL3 according to claim 1.

3. The heavy chain variable region and the light chain variable region each 1) SEQ ID NO: 46 and 54, or 2) SEQ ID NO: 30 and 31, or 3) SEQ ID NO: 32 and 33, or 4) SEQ ID NO: 34 and 33, or 5) SEQ ID NO: 35 and 36, or 6) SEQ ID NO: 37 and 38, or 7) SEQ ID NO: 39 and 40, or 8) SEQ ID NO: 39 and 41, or 9) SEQ ID NO: 42 and 43, or 10) SEQ ID NO: 44 and 36, or 11) SEQ ID NO: 35 and 45, or 12) SEQ ID NO: 46 and 47, or 13) SEQ ID NO: 46 and 48, or 14) SEQ ID NO: 49 and 50, or 15) SEQ ID NO: 51 and 52, or 16) SEQ ID NO: 51 and 53, or 17) SEQ ID NO: 46 and 55, or 18) SEQ ID NO: 46 and 56 and the antibody or antigen-binding fragment thereof that binds to DLL3 according to claim 1.

4. i) further comprises a heavy chain constant region and / or a light chain constant region, ii) The antibody or antigen-binding fragment thereof that binds to the DLL3 is a monoclonal antibody, bispecific binding molecule, multispecific binding molecule, humanized antibody, chimeric antibody, full-length antibody, Fab, Fv, scFv, F(ab')[ ２ [ , or linear antibody, and / or[ iii) characterized by comprising one or more selected from the forms of IgG1, IgG2, IgG3 or IgG4, and the antibody or antigen-binding fragment thereof that binds to DLL3 according to any one of claims 1 to 3.

5. The heavy chain constant region contains Fc, and the antibody or antigen-binding fragment thereof that binds to DLL3 according to claim 4.

6. The Fc is derived from mouse or human, and the antibody or antigen-binding fragment thereof that binds to DLL3 according to claim 5.

7. A complex characterized by being formed by coupling the antibody or antigen-binding fragment thereof that binds to DLL3 according to any one of claims 1 to 3 to a capture marker or a detection marker.

8. The detection marker contains a radionuclide, a luminescent substance, a colored substance or an enzyme, and the complex according to claim 7.

9. An antibody-drug conjugate formed by conjugating a biologically active molecule to an antibody or an antigen-binding fragment thereof that binds to DLL3 according to claim 1.

10. The antibody-drug conjugate according to claim 9, wherein the biologically active molecule is a small molecule drug.

11. The antibody-drug conjugate according to claim 9, wherein the antibody or an antigen-binding fragment thereof that binds to DLL3 is connected to the biologically active molecule by a linker.

12. A nucleic acid encoding an antibody or an antigen-binding fragment thereof that binds to DLL3 according to claim 1, or a recombinant vector containing the nucleic acid, or a host cell containing the nucleic acid or the recombinant vector.

13. The host cell according to claim 12, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

14. The host cell according to claim 13, wherein the prokaryotic cell is Escherichia coli.

15. The host cell according to claim 13, wherein the eukaryotic cell is a mammalian cell or yeast.

16. The host cell according to claim 15, wherein the mammalian cell is a CHO cell or a HEK293 cell.

17. A method for producing an antibody or an antigen-binding fragment thereof that binds to DLL3 according to any one of claims 1 to 3, the method comprising culturing the host cell according to claim 12 and purifying the expression product from the cells.

18. An antibody or an antigen-binding fragment thereof that binds to DLL3 according to any one of claims 1 to 3, for use in the preparation of a drug for the treatment or remission of a tumor.

19. The antibody or an antigen-binding fragment thereof that binds to DLL3 according to claim 18, wherein the drug targets tumor cells that abnormally express DLL3.

20. The antibody or an antigen-binding fragment thereof that binds to DLL3 according to claim 18, wherein the tumor is selected from small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer, and metastatic cancers of the above tumors.

21. An antibody or an antigen-binding fragment thereof that binds to DLL3 according to any one of claims 1 to 3, for use in the detection of DLL3 expression or the diagnosis of a tumor, wherein the tumor is selected from small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer, and metastatic cancers of the above tumors, and the antibody or an antigen-binding fragment thereof that binds to DLL3 according to any one of claims 1 to 3.

22. A method for detecting the expression of DLL3 in a sample, comprising: (1) contacting the sample with an antibody or an antigen-binding fragment thereof that binds to DLL3 according to any one of claims 1 to 3, and (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof that binds to DLL3 and DLL3, wherein the antibody or antigen-binding fragment thereof that binds to DLL3 is detectably labeled.

23. A pharmaceutical composition comprising an effective amount of an antibody or an antigen-binding fragment thereof that binds to DLL3 according to claim 1, or an effective amount of an antibody-drug conjugate according to claim 9, or an effective amount of a nucleic acid, recombinant vector or host cell according to claim 12.

24. The pharmaceutical composition according to claim 23, further comprising a pharmaceutically acceptable carrier.

25. The pharmaceutical composition according to claim 23, further comprising one or more additional therapeutic agents.

26. A cartridge or kit comprising a container and the pharmaceutical composition according to claim 23 disposed within the container.

27. The pharmaceutical composition according to claim 23, for use in inducing the death of DLL3-expressing cells, wherein the DLL3-expressing cells are tumor cells.

28. The pharmaceutical composition according to claim 27, wherein the tumor cells are selected from cells of small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer and metastatic cancers of the above tumors.

29. The pharmaceutical composition according to claim 23, for use in the treatment of a disease associated with the expression of DLL3 in a subject.

30. The pharmaceutical composition according to claim 29, wherein the disease is a tumor.

31. The pharmaceutical composition according to claim 30, wherein the tumor is selected from small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer and metastatic cancers of the above tumors.

32. The pharmaceutical composition according to claim 29, further comprising administering an additional therapeutic agent to the subject.

33. The cartridge or kit according to claim 26, for use in the treatment of a disease associated with the expression of DLL3 in a subject.

34. The cartridge or kit according to claim 33, wherein the disease is a tumor.

35. The cartridge or kit according to claim 34, wherein the tumor is selected from small cell lung cancer, glioblastoma, neuroendocrine cancer, melanoma, pancreatic cancer, rectal cancer, and metastatic cancer of the tumor.

36. The cartridge or kit according to claim 33, further comprising administering an additional therapeutic agent to the subject.

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