DLL3-targeting antibodies and uses thereof

Abstract

To provide an immunoglobulin-related composition that can bind to delta-like protein 3 (DLL3).SOLUTION: The present disclosure relates generally to immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof) that can bind to delta-like protein 3 (DLL3). The antibodies of the present technology are useful in methods for detecting and treating a DLL3-associated cancer in a subject in need thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 872,915, filed July 11, 2019, the entire disclosure of which is incorporated herein by reference. The present technology generally relates to the preparation and use of immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to Delta-like protein 3 (DLL3). In particular, the present technology relates to the preparation of DLL3-binding antibodies and their use in the detection and treatment of DLL3-associated cancers, including small cell lung cancer (SCLC), extrapulmonary neuroendocrine carcinoma, and large cell neuroendocrine carcinoma (LCNEC).

[0002] Statement of government support This invention was made with government support under CA213448 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] The following description of the background of the present technology is provided merely as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology. Neuroendocrine tumors of the lung (Lu-NETs) encompass a heterogeneous family of neoplasms classified into four histologic variants: typical carcinoid (TC), atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell lung cancer (SCLC). Both SCLC and pulmonary LCNEC are high-grade tumors with poor prognosis and a high incidence among smokers. Similar to SCLC, pulmonary LCNEC exhibits biologically aggressive behavior. At different stages, the survival curves for pulmonary LCNEC and SCLC overlap, and furthermore, the survival rates are lower than those of other NSCLCs. Even patients with resectable stage I lung cancer have a poor prognosis, with 5-year survival rates ranging from 27% to 67%. See Iyoda A. et al., J Thorac Cardiovasc Surg. 138:446-453 (2009). Summary of the Invention

[0004] In one aspect, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) V H is selected from the group consisting of: (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (iv) SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively. H -CDR1 sequence, V H -CDR2 sequence, and V H - (b) a CDR3 sequence comprising V L is selected from the group consisting of: (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. L -CDR1 sequence, V L -CDR2 sequence, and V L -CDR3 sequences. In certain embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the following features: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 7, 17, 27, or 37; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 12, 22, or 32.

[0005] In another aspect, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, and SEQ ID NO:32; and / or (b) V L provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, and SEQ ID NO: 37. In some embodiments of the antibody or antigen-binding fragment, V H Amino acid sequence and V L The amino acid sequences are selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:7 (7-I1-B), respectively; SEQ ID NO:12 and SEQ ID NO:17 (2-C8-A), respectively; SEQ ID NO:22 and SEQ ID NO:27 (10-O18-A), respectively; SEQ ID NO:32 and SEQ ID NO:37 (6-G23-F), respectively. Additionally or alternatively, in certain embodiments, the antibody or antigen-binding fragment of the present technology binds to an epitope present in a mammalian DLL3 polypeptide. The epitope may be a conformational or nonconformational epitope. Additionally or alternatively, in some embodiments, the mammalian DLL3 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54. In some embodiments, the mammalian DLL3 polypeptide has an amino acid sequence comprising amino acid residues 27-492 of SEQ ID NO: 50 or SEQ ID NO: 51 (e.g., the mammalian DLL3 polypeptide may comprise the extracellular domain of human DLL3). Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment undergoes internalization into the cell when bound to a DLL3 polypeptide expressed on the cell surface (e.g., tumor cell surface).

[0006] Additionally or alternatively, in any of the embodiments disclosed herein, the antibody or antigen-binding fragment further comprises an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. Additionally or alternatively, in any of the embodiments disclosed herein, the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', scFv, and Fv. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment of the present technology is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody.

[0007] In one aspect, the present disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies or antigen-binding fragments described herein. In some embodiments, the recombinant nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, and 36. In another aspect, the present disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences disclosed herein. In one aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment of the present technology and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to a substance selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0008] In one aspect, the present disclosure provides a method for treating DLL3-related cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment of the present technology, wherein the antibody or antigen-binding fragment is conjugated to at least one additional therapeutic agent. Examples of such additional therapeutic agents include, but are not limited to, isotopes, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof. The DLL3-related cancer may be small cell lung cancer, large cell neuroendocrine carcinoma, pulmonary neuroendocrine cell carcinoma, extrapulmonary neuroendocrine cell carcinoma, or melanoma.

[0009] In another aspect, the disclosure provides a method for detecting DLL3 protein levels in a biological sample, the method comprising contacting the biological sample with an antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment is conjugated to a detectable label, and detecting a signal generated by the detectable label in the biological sample.

[0010] Also disclosed herein is a kit for detecting and / or treating DLL3-related cancer, comprising at least one immunoglobulin-related composition of the present technology (for example, any antibody or antigen-binding fragment described herein) or its functional variant (for example, substitution variant) and instructions for use.In certain embodiments, the immunoglobulin-related composition is coupled with one or more detectable labels.In one embodiment, one or more detectable labels include radioactive labels, fluorescent labels or chromogenic labels. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the anti-DLL3 immunoglobulin-related compositions described herein, hi some embodiments, the secondary antibody is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the results of a Fab ZAP assay, a cytotoxicity-based internalization assay performed to assess DLL3 internalization by the indicated antibodies. Reference DLL3 monoclonal antibody SC16 was used as a positive control. See WO2015127407. All anti-DLL3 antibodies tested exhibited killing activity comparable to that of the reference monoclonal antibody. At higher concentrations of anti-DLL3 antibody, a hook effect was observed, as free anti-DLL3 competed with cell-bound anti-DLL3 for Fab ZAP. [Figure 2A] Figures 2A–2D show the binding curves for antibodies 7-I1-B (Figure 2A), 6-G23-F (Figure 2B), 10-O18-A (Figure 2C), and 2-C8-A (Figure 2D). The binding curves were measured at 25 °C via Octet HTX using 0.1% BSA, 0.02% Tween 20 in PBS as the binding buffer and 10 mM Glycine, pH 1.7 as the regeneration buffer. Monoclonal antibodies (5 μg / mL each) were loaded onto anti-mouse Fc sensors, and the loaded sensors were immersed in the indicated dilutions of Recombinant Human DLL3 Protein (amino acids Ala27-Ala479, catalog number 9749-DL, R&D Systems) at a starting concentration of 200 nM, diluted 1:3 seven times. The actual measurements and curve fits are shown for each DLL3 dilution. [Figure 2B]Figures 2A–2D show the binding curves for antibodies 7-I1-B (Figure 2A), 6-G23-F (Figure 2B), 10-O18-A (Figure 2C), and 2-C8-A (Figure 2D). The binding curves were measured at 25 °C via Octet HTX using 0.1% BSA, 0.02% Tween 20 in PBS as the binding buffer and 10 mM Glycine, pH 1.7 as the regeneration buffer. Monoclonal antibodies (5 μg / mL each) were loaded onto anti-mouse Fc sensors, and the loaded sensors were immersed in the indicated dilutions of Recombinant Human DLL3 Protein (amino acids Ala27-Ala479, catalog number 9749-DL, R&D Systems) at a starting concentration of 200 nM, diluted 1:3 seven times. The actual measurements and curve fits are shown for each DLL3 dilution. [Figure 2C] Figures 2A–2D show the binding curves for antibodies 7-I1-B (Figure 2A), 6-G23-F (Figure 2B), 10-O18-A (Figure 2C), and 2-C8-A (Figure 2D). The binding curves were measured at 25 °C via Octet HTX using 0.1% BSA, 0.02% Tween 20 in PBS as the binding buffer and 10 mM Glycine, pH 1.7 as the regeneration buffer. Monoclonal antibodies (5 μg / mL each) were loaded onto anti-mouse Fc sensors, and the loaded sensors were immersed in the indicated dilutions of Recombinant Human DLL3 Protein (amino acids Ala27-Ala479, catalog number 9749-DL, R&D Systems) at a starting concentration of 200 nM, diluted 1:3 seven times. The actual measurements and curve fits are shown for each DLL3 dilution. [Figure 2D]Figures 2A–2D show the binding curves for antibodies 7-I1-B (Figure 2A), 6-G23-F (Figure 2B), 10-O18-A (Figure 2C), and 2-C8-A (Figure 2D). The binding curves were measured at 25 °C via Octet HTX using 0.1% BSA, 0.02% Tween 20 in PBS as the binding buffer and 10 mM Glycine, pH 1.7 as the regeneration buffer. Monoclonal antibodies (5 μg / mL each) were loaded onto anti-mouse Fc sensors, and the loaded sensors were immersed in the indicated dilutions of Recombinant Human DLL3 Protein (amino acids Ala27-Ala479, catalog number 9749-DL, R&D Systems) at a starting concentration of 200 nM, diluted 1:3 seven times. The actual measurements and curve fits are shown for each DLL3 dilution. [Figure 2E] Figure 2E shows the dissociation constant (KD) values ​​for the four monoclonal antibodies described herein (6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A), which were calculated using the binding curves shown in Figures 2A-2D and a monovalent (1:1) binding model. [Figure 3A] Figure 3A shows the nucleotide and amino acid sequences of the VH domain of antibody 7-I1-B, represented as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. VH CDR1 (SEQ ID NO: 3) is shown in bold, VH CDR2 (SEQ ID NO: 4) is underlined, and VH CDR3 (SEQ ID NO: 5) is italicized and underlined. [Figure 3B] 3B depicts the nucleotide and amino acid sequences of the VL domain of antibody 7-I1-B, represented as SEQ ID NO: 6 and SEQ ID NO: 7, respectively. VL CDR1 (SEQ ID NO: 8) is shown in bold, VL CDR2 (SEQ ID NO: 9) is underlined, and VL CDR3 (SEQ ID NO: 10) is italicized and underlined. [Figure 4A]4A depicts the nucleotide and amino acid sequences of the VH domain of antibody 2-C8-A, represented as SEQ ID NO: 11 and SEQ ID NO: 12, respectively. VH CDR1 (SEQ ID NO: 13) is shown in bold, VH CDR2 (SEQ ID NO: 14) is underlined, and VH CDR3 (SEQ ID NO: 15) is italicized and underlined. [Figure 4B] 4B depicts the nucleotide and amino acid sequences of the VL domain of antibody 2-C8-A, represented as SEQ ID NO: 16 and SEQ ID NO: 17, respectively. VL CDR1 (SEQ ID NO: 18) is shown in bold, VL CDR2 (SEQ ID NO: 19) is underlined, and VL CDR3 (SEQ ID NO: 20) is italicized and underlined. [Figure 5A] Figure 5A depicts the nucleotide and amino acid sequences of the VH domain of antibody 10-O18-A, represented as SEQ ID NO: 21 and SEQ ID NO: 22, respectively. VH CDR1 (SEQ ID NO: 23) is shown in bold, VH CDR2 (SEQ ID NO: 24) is underlined, and VH CDR3 (SEQ ID NO: 25) is italicized and underlined. [Figure 5B] Figure 5B shows the nucleotide and amino acid sequences of the VL domain of antibody 10-O18-A, represented as SEQ ID NO: 26 and SEQ ID NO: 27, respectively. VL CDR1 (SEQ ID NO: 28) is shown in bold, VL CDR2 (SEQ ID NO: 29) is underlined, and VL CDR3 (SEQ ID NO: 30) is italicized and underlined. [Figure 6A] Figure 6A shows the nucleotide and amino acid sequences of the VH domain of antibody 6-G23-F, represented as SEQ ID NO: 31 and SEQ ID NO: 32, respectively. VH CDR1 (SEQ ID NO: 33) is shown in bold, VH CDR2 (SEQ ID NO: 34) is underlined, and VH CDR3 (SEQ ID NO: 35) is italicized and underlined. [Figure 6B]6B depicts the nucleotide and amino acid sequences of the VL domain of antibody 6-G23-F, represented as SEQ ID NO: 36 and SEQ ID NO: 37, respectively. VL CDR1 (SEQ ID NO: 38) is shown in bold, VL CDR2 (SEQ ID NO: 39) is underlined, and VL CDR3 (SEQ ID NO: 40) is italicized and underlined. [Figure 7] Figure 7 shows that monoclonal antibodies (mAbs) 6-G23-F, 10-O18-A, and 2-C8-A selectively bind to DLL3 but not to DLL1 or DLL4. 7-I1-B mAb binds to both DLL3 and DLL4, but not to DLL1. [Figure 8A] FIG. 8A shows the nucleotide and amino acid sequences of human (Homo sapiens) Delta-like canonical Notch ligand 3 (DLL3) isoform 1, represented as SEQ ID NO:55 and SEQ ID NO:50, respectively. [Figure 8B] FIG. 8B depicts the nucleotide and amino acid sequences of human Delta-like canonical Notch ligand 3 (DLL3) isoform 2, represented as SEQ ID NO:56 and SEQ ID NO:51, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0012] It will be appreciated that certain aspects, modes, embodiments, variations and features of the technology are described below at varying levels of detail in order to provide a substantial understanding of the technology.

[0013] The practice of the present method employs many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; US Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring levels of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0014] DLL3 is selectively expressed in high-grade pulmonary neuroendocrine tumors, including SCLC and LCNEC. Increased DLL3 expression has been observed in xenograft tumors derived from SCLC and LCNEC patients and confirmed in primary tumors. See Saunders et al., Sci Translational Medicine 7(302): 302ra136 (2015). Increased DLL3 expression has also been observed in extrapulmonary neuroendocrine cell carcinomas, including prostate neuroendocrine carcinoma (Puca et al., Sci Transl Med 11(484): pii: eaav0891 (2019)). DLL3 is expressed on the surface of such tumor cells but not in normal tissues. The present disclosure provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that are internalized upon binding to DLL3 on tumor cells and are therefore useful for delivering toxic payloads to these tumor cells. The immunoglobulin-related compositions of the present technology are useful in methods for detecting or treating DLL3-associated cancers in subjects in need thereof. Therefore, various aspects of the present method relate to the preparation, characterization and manipulation of anti-DLL3 antibodies.The immunoglobulin-related compositions of the present technology are useful alone or in combination with other therapeutic agents for treating cancer.In some embodiments, the immunoglobulin-related compositions are humanized antibodies, chimeric antibodies or bispecific antibodies.

[0015] definition Definitions of certain terms used herein are provided below: Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. For example, a reference to a "cell" includes a combination of two or more cells, etc. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are well known and commonly used in the art.

[0016] As used herein, the term "about" in connection with a number is generally interpreted as including numbers within 1%, 5% or 10% in either direction (more or less) of the number, unless otherwise stated or clear from the context (except where such number is less than 0% or more than 100% of the possible value). As used herein, "administration" of an agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be performed by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally, or topically. Administration includes self-administration and administration by another.

[0017] "Adjuvant" refers to one or more substances that stimulate the immune system. In this context, adjuvants are used to enhance the immune response to one or more vaccine antigens or antibodies. Adjuvants can be administered to a subject before, in combination with, or after administration of a vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and costimulatory factors.

[0018] As used herein, the term "amino acid" refers to any organic molecule containing at least one amino group and at least one carboxyl group. Typically, at least one amino group is alpha-positioned relative to the carboxyl group. The term "amino acid" includes naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but maintain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid but function in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0019] As used herein, the term "antibody" collectively refers to immunoglobulin or immunoglobulin-like molecules, including, by way of example and not limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response in any vertebrate, e.g., mammals such as humans, goats, rabbits, and mice, as well as in non-mammalian species, such as shark immunoglobulins. As used herein, "antibody" (including intact immunoglobulins) and "antigen-binding fragments" specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to substantially exclude binding to other molecules (e.g., with a binding constant at least 10 times higher than the binding constant for other molecules in a biological sample). 3 M-1 Greater than, at least 10 4 M -1 Greater than or at least 10 5 M -1 (Antibodies and antibody fragments having a larger binding constant for a molecule of interest). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), etc. Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.);Kuby, J., Immunology,3 rd Ed., W. H. Freeman & Co., New York, 1997. See also

[0020] More specifically, an antibody refers to a polypeptide ligand that contains at least a light chain immunoglobulin variable region or a heavy chain immunoglobulin variable region and specifically recognizes and binds to an epitope of an antigen. An antibody is composed of a heavy chain and a light chain, each of which contains a heavy chain variable region (V H ) region and the light chain variable (V L ) region. H Area and V LThese regions are involved in binding to the antigen recognized by the antibody. Immunoglobulins typically have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to antigens. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, incorporated herein by reference). The Kabat database is currently maintained online. The sequences of the framework regions of various light or heavy chains are relatively conserved within a species. The framework regions of antibodies, which are the combined framework regions of the constituent light and heavy chains, primarily adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions act to form a scaffold that positions the CDRs in the correct orientation through interchain, non-covalent interactions.

[0021] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are usually identified by the chain in which the individual CDR is located. Thus, V H CDR3 is located in the variable domain of the antibody heavy chain in which it is found, V LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to the DLL3 protein have a specific V H Area and V L Each CDR has a specific CDR sequence and therefore a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although the CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs). As used herein, "immunoglobulin-related compositions" refer to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) as well as antibody fragments. An antibody or its antigen-binding fragment specifically binds to an antigen.

[0022] As used herein, the term "antibody-related polypeptide" refers to an antigen-binding antibody fragment, including a single-chain antibody, which may contain a variable region alone or in combination with all or part of the following polypeptide elements: hinge region, CH1, CH2, and CH3 domains of an antibody molecule. Also encompassed within the present technology are any combinations of variable regions and hinge regions, CH1, CH2, and CH3 domains. Antibody-related molecules useful in the present method include, but are not limited to, Fab, Fab', and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V H Fragments containing any of the domains, such as (i) Fab fragments, V L , V H , C L and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H(vi) dAb fragments consisting of domains (Ward et al., Nature 341: 544-546, 1989), and (vi) isolated complementarity-determining regions (CDRs). As such, an "antibody fragment" or "antigen-binding fragment" can contain a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0023] "Bispecific antibody" or "BsAb" as used herein refers to an antibody that can simultaneously bind to two targets with distinct structures, e.g., two different target antigens, two different epitopes on the same target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding moiety in a bispecific antibody is V H and / or V L In some such embodiments, the V H and / or V L The V region is one found in a particular monoclonal antibody. In some embodiments, a bispecific antibody contains two antigen-binding portions, each derived from a different monoclonal antibody. H and / or V L In some embodiments, a bispecific antibody contains two antigen-binding moieties, one of which contains a V region containing the CDRs from a first monoclonal antibody. H and / or V L The other antigen-binding portion comprises an immunoglobulin molecule having a V region containing CDRs derived from a second monoclonal antibody. H and / or V L These include antibody fragments having regions (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.).

[0024] As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art.Suitable types of association include chemical bonds and physical bonds.Chemical bonds include, for example, covalent bonds and coordinate bonds.Physical bonds include, for example, hydrogen bonds, dipolar interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions and aromatic stacking.

[0025] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which fragments contain a light chain variable domain (V L ) connected to the heavy chain variable domain (V H )Includes(V H V L (The term "diabodies" is used interchangeably with "diabodies" and is used interchangeably with "diabodies" in the context of "antibody" or "antibody-specific antibodies"). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and generate two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0026] As used herein, the term "single chain antibody" or "single chain Fv (scFv)" refers to an Fv fragment, V L and V H A single-chain antibody molecule may comprise a polymer having several individual molecules, such as a dimer, trimer, or other polymer. v Fragment, V L and V H The two domains are encoded by separate genes, but they are L and V H The domains pair to form a monovalent molecule (single-chain F v (scF vThe antibodies may be joined using recombinant methods by synthetic linkers that allow them to be produced as a single protein chain forming a single chain (known as a single chain antibody). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Such single-chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0027] Any of the above antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as are intact antibodies. As used herein, "antigen" refers to a molecule to which an antibody (or antigen-binding fragment thereof) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a polypeptide (e.g., a DLL3 polypeptide comprising the DLL3 extracellular domain). The antigen can be administered to an animal to generate an immune response in the animal. The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that contains the portion of the polypeptide that is involved in binding to the antigen. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2.

[0028] "Binding affinity" refers to the strength of the overall non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate easily from the antigen, whereas high affinity complexes generally contain antibodies that tend to remain bound to the antigen for extended periods of time. As used herein, the term "biological sample" refers to a sample material derived from living cells.Biological samples can include tissues, cells, cell protein or membrane extracts and biological fluids (such as ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present in a subject.Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostatic fluid, semen, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph and tears. Biological samples can also be obtained from biopsies of internal organs or cancer.Biological samples can be obtained from subjects for diagnosis or research, or can be obtained from non-diseased individuals as controls or for basic research.Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy.In certain embodiments, biological samples are sputum samples, or tissue samples obtained by needle biopsy or surgical biopsy.

[0029] As used herein, the term "CDR-grafted antibody" refers to an antibody in which at least one CDR of an "acceptor" antibody has been replaced by a CDR "graft" from a "donor" antibody with the desired antigen specificity. As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody derived from one species (e.g., a murine Fc constant region) has been replaced, using recombinant DNA technology, with the Fc constant region derived from an antibody of another species (e.g., a human Fc constant region). In general, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO86 / 01533; Cabilly et al. al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al. See Proc. Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988. As used herein, the term "consensus FR" refers to the framework (FR) antibody region in the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen.

[0030] As used herein, "control" refers to a substitute sample used in an experiment for comparison purposes.Control can be "positive" or "negative".For example, when the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a specific type of disease, a positive control (a compound or composition known to exhibit desired therapeutic effect) and a negative control (a subject or sample that does not receive therapy or receives a placebo) are usually used. As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention or reduction of a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In therapeutic or prophylactic applications, the amount of a composition administered to a subject will vary depending on the composition, the extent, type, and severity of the disease, and individual characteristics, such as general health, age, sex, weight, and tolerance to drugs. Those skilled in the art can determine the appropriate dosage depending on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, a therapeutic composition may be administered to a subject with one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition that ameliorates or eliminates the physiological effects of the disease or condition. A therapeutically effective amount may be administered in one or more administrations.

[0031] An "isolated" or "purified" polypeptide or peptide is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which it is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. For example, an isolated anti-DLL3 antibody or antigen-binding fragment of the present technology will be free of substances that interfere with the diagnostic or therapeutic use of the agent. Such interfering substances may include enzymes, hormones, and other proteinaceous and non-proteinaceous solutes.

[0032] As used herein, the term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not. In some embodiments, the "epitope" of a DLL3 protein refers to a region of the protein to which an anti-DLL3 antibody of the present technology specifically binds. In some embodiments, the epitope is a conformational or nonconformational epitope. To screen for anti-DLL3 antibodies that bind to an epitope, a conventional cross-blocking assay, such as that described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), may be performed. This assay can be used to determine whether an anti-DLL3 antibody binds to the same site or epitope as an anti-DLL3 antibody of the present technology. Alternatively, or in addition, epitope mapping can be carried out by methods known in the art.For example, antibody sequence can be mutated by alanine scanning or the like to identify contact residues.In a different method, peptides corresponding to different regions of DLL3 protein can be used in a competitive assay using test antibody, or using test antibody and antibody with characterized or known epitope.

[0033] As used herein, "expression" includes one or more of the following, as necessary for proper expression and function: transcription of a gene into precursor mRNA, splicing and other processing of the precursor mRNA to produce a mature mRNA, mRNA stability; translation of the mature mRNA into a protein (including codon usage and tRNA availability), and glycosylation and / or other modifications of the translation product. As used herein, the term "gene" refers to a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns and other untranslated regions that control expression.

[0034] As used herein, the term "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage of "sequence identity" to another sequence (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), meaning that that percentage of bases (or amino acids) are identical when the two sequences are aligned in a comparison. This alignment and percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. Notably, programs include BLASTN and BLASTP, using the following default parameters: genetic code=standard, filter=none, strand=both, cutoff=60, expect=10, matrix=BLOSUM62, description=50 sequences, sorted=HIGH SCORE, database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that share a specified percent homology and encode polypeptides with the same or similar biological activity. Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% identity or less than 25% identity with each other.

[0035] As used herein, "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which recipient hypervariable region residues are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance, such as binding affinity. Generally, a humanized antibody comprises substantially all of at least one, and usually two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are of human immunoglobulin consensus FR sequences, although the FR regions may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FRs is usually no more than six in the heavy chain and no more than three in the light chain. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014); Saxena & Wu, Frontiers in immunology 7:580 (2016).

[0036] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V L Approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in H Approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the H1 sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from the "hypervariable loops" (e.g., V L Residues 26–32 (L1), 50–52 (L2), and 91–96 (L3) in V H Among these, 26-32 (H1), 52A-55 (H2), and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)) are included.

[0037] As used herein, the terms "identical" or percent "identity," when used in reference to two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein)) when compared and aligned for maximum correspondence over a comparison window or designated region as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (e.g., the NCBI website). Such sequences are then said to be "substantially identical." The term can also refer to or apply to the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as those that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0038] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (herein abbreviated as LCVR or V L The light chain constant region consists of one domain, C L It consists of: V H and V LThe regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are separated by more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0039] As used herein, the terms "individual," "patient," or "subject" can refer to an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including, but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0040] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulation are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.). As used herein, the term "polyclonal antibody" refers to a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. Use of this term includes at least two (2) antibody preparations containing antibodies that specifically bind to different epitopes or regions of an antigen.

[0041] As used herein, the term "polynucleotide" or "nucleic acid" refers to any RNA or DNA, and may be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more usually, double-stranded or a mixture of single-stranded and double-stranded regions. Furthermore, polynucleotide refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons.

[0042] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer comprising two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.

[0043] As used herein, "prevention" or "preventing" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in a treated sample relative to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample. As used herein, the term "recombinant," for example, when used in reference to a cell or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0044] As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients simultaneously or substantially simultaneously by different routes. As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, and the administration routes are the same or different. More specifically, sequential use refers to the complete administration of one of the active ingredients before the start of the administration of another active ingredient(s). Thus, one of the active ingredients can be administered over several minutes, hours, or days, and then the other active ingredient(s) can be administered. In this case, there is no simultaneous treatment.

[0045] As used herein, the term "concurrent" therapeutic use refers to the administration of at least two active ingredients by the same route at the same time or substantially the same time. As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize or bind to other molecules. As used herein, the term "specific binding," "specifically binds to" or "is specific for" a particular molecule (e.g., a polypeptide or epitope on a polypeptide), refers to, for example, a binding affinity of about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 K for molecules that bind to M D The term "specifically binds" can also refer to binding by a molecule (e.g., an antibody or antigen-binding fragment thereof) to a particular polypeptide (e.g., a DLL3 polypeptide) or an epitope on a particular polypeptide without substantially binding to any other polypeptides or epitopes on polypeptides.

[0046] As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, provides a desired therapeutic effect in a subject in need thereof. "Treating" or "treatment," as used herein, refers to the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., halting its development, (ii) alleviating the disease or disorder, i.e., causing regression of the disorder, (iii) slowing the progression of the disorder, and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that the symptoms associated with the disease are, for example, alleviated, reduced, cured, or placed in remission.

[0047] It should also be understood that the various modes of treatment for disorders as described herein include not only complete treatment, but also less-than-complete treatment, and are intended to mean "substantial" in which some biologically or medically relevant result is achieved. Treatment can be continuous long-term treatment for chronic diseases or a single or two or three administrations for treating acute conditions.

[0048] Amino acid sequence modification(s) of the anti-DLL3 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of anti-DLL3 antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion from and / or insertion into and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to obtain the antibody of interest, as long as the resulting antibody possesses the desired properties. Modifications also include changes in the glycosylation pattern of the protein. Hypervariable regions are the most popular sites for substitutional mutagenesis, although FR changes are also contemplated. "Conservative substitutions" are shown in the table below.

[0049] [Table 1]

[0050] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis may be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants may be subjected to screening as described herein to select antibodies with similar or superior properties in one or more relevant assays for further development.

[0051] Delta-like 3 (DLL3) In Drosophila, Notch signaling is primarily mediated by Notch receptors. Delta is one of the Drosophila Notch ligands that activates signaling in neighboring cells. In humans, there are four known Notch receptors (NOTCH1-NOTCH4) and three Delta homologs (termed Delta-like ligands, DLL1, DLL3, and DLL4). Unlike DLL1 and DLL4, DLL3 has been reported to inhibit, rather than activate, Notch signaling.

[0052] DLL3 (also known as Delta-like 3 or SCDO1) is a member of the Delta-like family of Notch DSL ligands. Representative DLL3 protein orthologs include, but are not limited to, human (accession numbers NP_058637 (SEQ ID NO: 50) and NP_982353 (SEQ ID NO: 51)), chimpanzee (accession number XP_003316395 (SEQ ID NO: 52)), mouse (accession number NP_031892 (SEQ ID NO: 53)), and rat (accession number NP_446118 (SEQ ID NO: 54)). In humans, the DLL3 gene consists of eight exons spanning 9.5 kBp, located on chromosome 19q13. Alternative splicing at the last exon generates a 2389 bp transcript (accession number NM_016941 (SEQ ID NO: 55)) and a 2052 bp transcript (accession number NM_203486 (SEQ ID NO: 56)). The former transcript encodes a protein that is 618 amino acids long (Accession No. NP_058637 (SEQ ID NO: 50)), and the latter transcript encodes a protein that is 587 amino acids long (Accession No. NP_982353 (SEQ ID NO: 51)). See Figures 8A-8B. These two protein isoforms of DLL3 share 100% overall identity between the extracellular and transmembrane domains and differ only in that the longer isoform contains an extended cytoplasmic tail containing an additional 32 residues at the carboxy terminus of the protein.

[0053] Both isoforms can be detected in tumor cells. Indeed, aberrant DLL3 expression (genotype and / or phenotype) is associated with various subpopulations of neoplastic cells, such as cancer stem cells and tumor-initiating cells. Thus, the present disclosure provides DLL3 antibodies that may be particularly useful for targeting such cells (e.g., cancer stem cells, tumor-initiating cells, and cancers, such as small cell lung cancer, large cell neuroendocrine carcinoma, pulmonary neuroendocrine cell carcinoma, extrapulmonary neuroendocrine cell carcinoma, and melanoma), thereby facilitating the treatment, management, or prevention of neoplastic disorders.

[0054] Immunoglobulin-related compositions of the present technology The present technology describes methods and compositions for making and using anti-DLL3 immunoglobulin-related compositions (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof). The anti-DLL3 immunoglobulin-related compositions of the present disclosure may be useful for diagnosing or treating DLL3-related cancers (e.g., small cell lung cancer, large cell neuroendocrine carcinoma, pulmonary neuroendocrine cell carcinoma, extrapulmonary neuroendocrine cell carcinoma, and melanoma). Anti-DLL3 immunoglobulin-related compositions within the scope of the present technology include, but are not limited to, monoclonal, chimeric, humanized, bispecific antibodies and diabodies, homologs, derivatives, or fragments thereof, that specifically bind to target polypeptides. The present disclosure also provides antigen-binding fragments of any of the anti-DLL3 antibodies disclosed herein, including Fab, F(ab)'2, Fab', scF, and the like. v and F v is selected from the group consisting of: The present technology discloses anti-DLL3 antibodies that can promote internalization of DLL3-antibody complexes and are therefore useful for delivering toxic payloads to tumor cells.

[0055] Figures 3 to 6 show the V H and V L The nucleotide and amino acid sequences of V and CDR sequences of the antibodies disclosed herein are provided (SEQ ID NOs: 1-40). H and V L The amino acid sequences of the antibodies disclosed herein are provided below, as well as the CDR sequences of the antibodies disclosed herein. [Table 2] TIFF2025156278000004.tif227169

[0056] In one aspect, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) V His selected from the group consisting of: (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (iv) SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively. H -CDR1 sequence, V H -CDR2 sequence, and V H - (b) a CDR3 sequence comprising V L is selected from the group consisting of: (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. L -CDR1 sequence, V L -CDR2 sequence, and V L -CDR3 sequences. In some embodiments, the antibody further comprises an Fc domain of any isotype, for example, but not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM and IgY. Non-limiting examples of constant region sequences include: Human IgD constant region, Uniprot:P01880 (SEQ ID NO: 41) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0057] Human IgG1 constant region, Uniprot:P01857 (SEQ ID NO: 42) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0058] Human IgG2 constant region, Uniprot:P01859 (SEQ ID NO: 43) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0059] Human IgG3 constant region, Uniprot:P01860 (SEQ ID NO: 44) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0060] Human IgM constant region, Uniprot:P01871 (SEQ ID NO: 45) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0061] Human IgG4 constant region, Uniprot:P01861 (SEQ ID NO: 46) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0062] Human IgA1 constant region, Uniprot:P01876 (SEQ ID NO: 47) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0063] Human IgA2 constant region, Uniprot:P01877 (SEQ ID NO: 48) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 49) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0064] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs: 41-48. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 49. In some embodiments, the immunoglobulin-related compositions of the present technology bind to the extracellular domain of DLL3. In some embodiments, the epitope is a conformational epitope. In another aspect, the present disclosure provides a heavy chain immunoglobulin variable domain (V) comprising SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, or a variant thereof with one or more conservative amino acid substitutions. H ) amino acid sequence. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain immunoglobulin variable domain (V) comprising SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, or a variant thereof with one or more conservative amino acid substitutions. L ) amino acid sequence.

[0065] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain immunoglobulin variable domain (V) selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:7 (7-I1-B), SEQ ID NO:12 and SEQ ID NO:17 (2-C8-A), SEQ ID NO:22 and SEQ ID NO:27 (10-O18-A), and SEQ ID NO:32 and SEQ ID NO:37 (6-G23-F), respectively. H ) amino acid sequence and the light chain immunoglobulin variable domain (V L ) amino acid sequence. In any of the above embodiments of the immunoglobulin-related composition, the HC and LC immunoglobulin variable domain sequences form an antigen-binding site that binds to the extracellular domain of DLL3. In any of the above embodiments of the immunoglobulin-related composition, the HC and LC immunoglobulin variable domain sequences form an antigen-binding site that binds to DLL3 and promotes internalization of the immunoglobulin-related composition. In some embodiments, the epitope is a conformational epitope. In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0066] In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one DLL3 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one DLL3 polypeptide. -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Dissociation constant of M (K D ) binds to at least one DLL3 polypeptide. In certain embodiments, the immunoglobulin-related composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

[0067] In certain embodiments, the immunoglobulin-related compositions comprise one or more of the following features: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 7, 17, 27, or 37, and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 12, 22, or 32. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution may be a "conservative substitution" as defined herein.

[0068] In certain embodiments, the immunoglobulin-related composition contains an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition contains an IgG4 constant region comprising a S228P mutation.

[0069] In some embodiments, the anti-DLL3 immunoglobulin-related compositions described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some embodiments, the anti-DLL3 immunoglobulin-related compositions (e.g., antibodies) of the present technology may contain deletions in the CH2 constant heavy chain region to promote rapid binding and cellular uptake and / or slow release. In some embodiments, Fab fragments are used to promote rapid binding and cellular uptake and / or sustained release. In some embodiments, F(ab)'2 fragments are used to promote rapid binding and cellular uptake and / or sustained release. In one aspect, the present technology provides a nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein. Also disclosed herein is a recombinant nucleic acid sequence encoding any of the antibodies described herein. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31 and 36.

[0070] In another aspect, the technology provides host cells or expression vectors that express any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein. The immunoglobulin-related compositions (e.g., anti-DLL3 antibodies) of the present technology can be monospecific, bispecific, trispecific, or more multispecific. Multispecific antibodies can be specific for different epitopes of one or more DLL3 polypeptides, or can be specific for both DLL3 polypeptides and heterologous compositions, such as heterologous polypeptides or solid support materials. See, e.g., WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt et al., J. Immunol. 147: 60-69 (1991), U.S. Patent Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648, and 6,106,835, and Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related composition is chimeric. In certain embodiments, the immunoglobulin-related composition is humanized.

[0071] The immunoglobulin-related composition of the present technology can also be recombinantly fused to heterologous polypeptides at N-terminus or C-terminus, or chemically conjugated to polypeptides or other compositions (including covalent and non-covalent conjugation).For example, the immunoglobulin-related composition of the present technology can be recombinantly fused to or conjugated to molecules that are useful as labels and effector molecules, such as heterologous polypeptides, drugs or toxins, in detection assays.For example, see WO92 / 08495, WO91 / 14438, WO89 / 12624, U.S. Patent No. 5,314,995 and EP0396387.

[0072] In any of the above embodiments of the immunoglobulin-related composition of the present technology, the antibody or antigen-binding fragment may be conjugated to a substance selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment of the present technology may be combined with a pharmaceutically acceptable carrier. For chemical or physical binding, a functional group on the immunoglobulin-related composition typically associates with a functional group on the substance. Alternatively, a functional group on the substance associates with a functional group on the immunoglobulin-related composition.

[0073] The functional groups on the substance and the immunoglobulin-related composition can be directly associated. For example, a functional group (e.g., a sulfhydryl group) on the substance can associate with a functional group (e.g., a sulfhydryl group) on the immunoglobulin-related composition to form a disulfide. Alternatively, the functional groups can associate via a crosslinker (i.e., a linker). Some examples of crosslinkers are described below. The crosslinker can be attached to either the substance or the immunoglobulin-related composition. The number of substances or immunoglobulin-related compositions in the conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of substances that can be associated with the conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions that can be associated with the substance depends on the number of functional groups present on the substance.

[0074] In yet another embodiment, the conjugate comprises one immunoglobulin-related composition associated with one substance. In one embodiment, the conjugate comprises at least one substance chemically bound (e.g., conjugated) to at least one immunoglobulin-related composition. The substance can be chemically bound to the immunoglobulin-related composition by any method known to those skilled in the art. For example, a functional group on the substance can be directly attached to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate, and hydroxyl. Substances can also be chemically linked to the immunoglobulin-related composition by cross-linking agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc., Rockford, Illinois. The Pierce Biotechnology, Inc. website can be helpful. Additional cross-linking agents include the platinum cross-linkers described in U.S. Patent Nos. 5,580,990, 5,985,566, and 6,133,038 to Kreatech Biotechnology, BV, Amsterdam, The Netherlands.

[0075] Alternatively, the functional groups on the substance and the immunoglobulin-related composition may be identical. Homobifunctional crosslinkers are typically used to crosslink identical functional groups. Examples of homobifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (i.e., bis-maleimidohexane). Such homobifunctional crosslinkers are also available from Pierce Biotechnology, Inc.

[0076] In other cases, it may be beneficial to cleave the substance from the immunoglobulin-related composition. The Pierce Biotechnology, Inc. website mentioned above can also assist those skilled in the art in selecting a suitable crosslinker that can be cleaved by an enzyme, for example, in a cell. Thus, the substance can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl). In another embodiment, the conjugate comprises at least one substance physically bound to at least one immunoglobulin-related composition. Any method known to those skilled in the art can be used to physically bind a substance to the immunoglobulin-related composition. For example, the immunoglobulin-related composition and the substance can be mixed together by any method known to those skilled in the art. The order of mixing is not important. For example, the substance can be physically mixed with the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the substance can be placed in a container and agitated, for example, by shaking the container, to mix the immunoglobulin-related composition and the substance. The immunoglobulin-related compositions can be modified by any method known to those of skill in the art, for example, the immunoglobulin-related compositions can be modified with crosslinking agents or functional groups, as described above.

[0077] A. Methods of Preparing the Anti-DLL3 Antibodies of the Present Technology General Overview. First, a target polypeptide to which the antibody of the present technology can be generated is selected. For example, the antibody can be generated against the full-length DLL3 protein or a part of the extracellular domain of the DLL3 protein. Techniques for generating antibodies against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, display libraries, heterologous or human mice, hybridomas, etc. Target polypeptides within the scope of the present technology include any polypeptide derived from the DLL3 protein that contains an extracellular domain that can induce an immune response.

[0078] It should be understood that recombinantly engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, directed against the DLL3 protein and fragments thereof are suitable for use in accordance with the present disclosure. Anti-DLL3 antibodies that can be subjected to the techniques described herein include monoclonal and polyclonal antibodies and antibody fragments, such as Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods useful for the high-yield production of antibody Fv-containing polypeptides, such as Fab' and F(ab')2 antibody fragments, have been described. See U.S. Patent No. 5,648,237.

[0079] Generally, the antibody is obtained from the original species. More specifically, the nucleic acid or amino acid sequence of the variable part of the light chain, heavy chain or both of the original species antibody having specificity for the target polypeptide antigen is obtained. The original species can be any species that has been useful for producing the antibody or antibody library of the present technology, such as rat, mouse, rabbit, chicken, monkey, human, etc. Phage or phagemid display technology is a useful technique for deriving the antibody of the present technology.Techniques for producing and cloning monoclonal antibodies are well known to those skilled in the art.The expression of the sequence encoding the antibody of the present technology can be carried out in E. coli.

[0080] Due to the degeneracy of nucleic acid encoding sequences, other sequences encoding substantially the same amino acid sequence as that of a naturally occurring protein may be used in the practice of the present technology. These include, but are not limited to, nucleic acid sequences comprising all or part of the nucleic acid sequences encoding the above polypeptides, altered by the substitution of different codons encoding functionally equivalent amino acid residues within the sequence, thus resulting in silent changes. It is understood that the nucleotide sequences of the immunoglobulins of the present technology allow for up to 25% sequence homology variation, as calculated by standard methods (see "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.), as long as such variants form functional antibodies that recognize the DLL3 protein. For example, one or more amino acid residues within a polypeptide sequence may be substituted with another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent change. Substitutes for amino acids within the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar natural amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Proteins or fragments or derivatives thereof that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, or linkage to antibody molecules or other cellular ligands, are also within the scope of the present technology.Additionally, nucleic acid sequences encoding immunoglobulins can be mutated in vitro or in vivo to create and / or destroy translation, initiation and / or termination sequences to generate variations in the coding region and / or to create new restriction endonuclease sites or destroy existing ones, further facilitating in vitro modifications. Any technique for mutagenesis known in the art may be used, including, but not limited to, in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, the use of Tab linkers (Pharmacia), etc.

[0081] Preparation of polyclonal antisera and immunogens. The method for producing antibodies or antibody fragments of the present technology typically involves immunizing a subject (generally a non-human subject such as a mouse or rabbit) with purified DLL3 protein or a fragment thereof, or with cells expressing DLL3 protein or a fragment thereof. Suitable immunogenic preparations can contain, for example, recombinantly expressed DLL3 protein or chemically synthesized DLL3 peptides. The extracellular domain of the DLL3 protein, or a portion or fragment thereof, can be used as an immunogen to generate anti-DLL3 antibodies that bind to the DLL3 protein, or a portion or fragment thereof, using standard techniques for polyclonal and monoclonal antibody preparation.

[0082] The full-length DLL3 protein or fragments thereof are useful as immunogens. In some embodiments, the DLL3 fragment comprises the extracellular domain of DLL3, such that antibodies raised against the peptide form specific immune complexes with the DLL3 protein.

[0083] The extracellular domain of DLL3 is 466 amino acids long, spanning amino acids 27 to 492 of the full-length DLL3 protein. In some embodiments, antigenic DLL3 peptides contain at least 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, or 450 amino acid residues. Longer antigenic peptides may be more desirable than shorter antigenic peptides, depending on the application and according to methods well known to those skilled in the art. Multimers of a given epitope may be more effective than monomers.

[0084] If necessary, the immunogenicity of the DLL3 protein (or fragment thereof) can be increased by fusion or conjugation with a carrier protein such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such carrier proteins are known in the art. To enhance the subject's immune response to the polypeptide, the DLL3 protein can be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant. Various adjuvants used to enhance immunological responses include, but are not limited to, Freund's (complete and incomplete), inorganic gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptide or oil emulsions, dinitrophenol, etc.), human adjuvants such as bacillus Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0085] In describing the present technology, an immune response can be described as a "primary" or "secondary" immune response. A primary immune response, also referred to as a "protective" immune response, refers to an immune response generated in an individual as a result of several initial exposures (e.g., initial "immunizations") to a particular antigen, such as a DLL3 protein. In some embodiments, immunizations can result from vaccination of an individual with a vaccine containing an antigen. For example, the vaccine can be a DLL3 vaccine containing antigens derived from one or more DLL3 proteins. A primary immune response can weaken or become attenuated over time, or can even disappear, or at least become so attenuated that it cannot be detected. Thus, the present technology also relates to a "secondary" immune response, also referred to herein as a "memory immune response." The term secondary immune response refers to an immune response induced in an individual after a primary immune response has already been generated.

[0086] Thus, a secondary immune response can be elicited to boost a pre-existing immune response that has become weakened or attenuated, or to reconstitute a previous immune response that has disappeared or is no longer detectable. A secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs upon stimulation of memory B cells generated upon initial presentation of the antigen. A delayed-type hypersensitivity (DTH) response is initiated by CD4 + It is a type of cell-mediated secondary or memory immune response mediated by T cells. Initial exposure to an antigen primes the immune system, and further exposure results in DTH.

[0087] After appropriate immunization, anti-DLL3 antibodies can be prepared from the subject's serum. If desired, antibody molecules directed against the DLL3 protein can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography, to obtain the IgG fraction.

[0088] Monoclonal antibody. In one embodiment of the present technology, the antibody is an anti-DLL3 monoclonal antibody. For example, in some embodiments, the anti-DLL3 monoclonal antibody can be a human or mouse anti-DLL3 monoclonal antibody. To prepare a monoclonal antibody against a DLL3 protein or its derivatives, fragments, analogs, or homologs, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, hybridoma technology (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497), trioma technology, human B cell hybridoma technology (see, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72), and EBV hybridoma technology for producing human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). In practicing the present technology, human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding regions of an antibody may be isolated. PCR may be used to amplify sequences encoding portions of antibodies from the population, using primers derived from sequences encoding conserved regions of the antibody, and DNA encoding the antibody or a fragment thereof, e.g., the variable domain, is then reconstructed from the amplified sequences.Such amplified sequences can also be fused to DNA encoding other proteins, such as bacteriophage coat or bacterial cell surface proteins, for expression and display of the fusion polypeptide in phage or bacteria. The amplified sequences can then be expressed and further selected or isolated, for example, based on the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on the DLL3 protein. Alternatively, hybridomas expressing anti-DLL3 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using routine methods. See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using standard methods yields monoclonal antibodies of varying specificity (i.e., against various epitopes) and affinity. Selected monoclonal antibodies with desired properties, such as DLL3 binding, can be used as expressed by hybridomas or conjugated to molecules such as polyethylene glycol (PEG) to alter their properties, or the cDNA encoding them can be isolated, sequenced, and manipulated in various ways. To enhance the immunogenicity of DLL3 proteins, synthetic dendritic trees can be added to reactive amino acid side chains, such as lysine. CPG dinucleotide technology can also be used to enhance the immunogenicity of DLL3 proteins. Other manipulations include substituting or deleting specific aminoacyl residues that contribute to antibody instability during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of antibodies for DLL3 proteins.

[0089] Hybridoma technology.In some embodiments, the antibody of the present technology is an anti-DLL3 monoclonal antibody produced by hybridoma, which comprises B cells obtained from a transgenic non-human animal, such as a transgenic mouse, whose genome comprises a human heavy chain transgene and a human light chain transgene, fused with immortalized cells.Hybridoma technology is known in the art and includes those taught in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981).Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0090] Phage display technology. As described above, the antibodies of this technology can be produced by applying recombinant DNA and phage display technology. For example, anti-DLL3 antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Phages with the desired binding properties are selected from repertoire or combinatorial antibody libraries (e.g., human or murine) by direct selection using antigens, usually antigens bound or captured to a solid surface or bead. The phages used in these methods are usually filamentous phages, including fd and M13, that have Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Furthermore, methods can be applied for the construction of Fab expression libraries to allow rapid and efficient identification of monoclonal Fab fragments having the desired specificity for a DLL3 polypeptide, e.g., a polypeptide or a derivative, fragment, analog, or homolog thereof (see, e.g., Huse, et al., Science 246: 1275-1281, 1989).Other examples of phage display methods that can be used to generate antibodies of the present technology include those described in Huston et al., Proc. Natl. Acad. Sci USA, 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994, PCT / GB91 / 01134, WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, WO96 / 06213, WO92 / 01047 (Medical Research Council al.), WO97 / 08320 (Morphosys), WO92 / 01047 (CAT / MRC), WO91 / 17271 (Affymax), and those disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, and 5,733,743. A method useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides through disulfide bonds is described by Lohning, US Pat. No. 6,753,136.As described in the above references, after phage selection, the antibody coding region obtained from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be used using methods known in the art, such as those disclosed in WO92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0091] Generally, hybrid antibodies or hybrid antibody fragments cloned into a display vector can be selected against an appropriate antigen to identify variants that maintain good binding activity, since the antibody or antibody fragment is presented on the surface of a phage or phagemid particle. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector formats can be used for this process, such as cloning an antibody fragment library into a lytic phage vector for selection and / or screening (modified T7 or lambda Zap systems).

[0092] Expression of Recombinant Anti-DLL3 Antibodies. As described above, the antibodies of the present technology can be produced by applying recombinant DNA technology. Recombinant polynucleotide constructs encoding the anti-DLL3 antibodies of the present technology typically contain expression control sequences operably linked to the coding sequences of the anti-DLL3 antibody chains, including naturally associated or heterologous promoter regions. As such, another embodiment of the present technology includes vectors containing one or more nucleic acid sequences encoding the anti-DLL3 antibodies of the present technology. For recombinant expression of one or more polypeptides of the present technology, nucleic acids containing all or part of the nucleotide sequence encoding the anti-DLL3 antibody are inserted into an appropriate cloning or expression vector (i.e., a vector containing the elements necessary for transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as described in detail below. Methods for producing a diverse collection of vectors are described by Lerner et al., U.S. Patent Nos. 6,291,160 and 6,680,192.

[0093] In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this disclosure, "plasmid" and "vector" can be used interchangeably, as plasmids are the most common form of vector. However, this technology is intended to include other forms of expression vectors that are not technically plasmids but serve equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses). Such viral vectors enable infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence encoding the anti-DLL3 antibody and for the collection and purification of the anti-DLL3 antibody, e.g., a cross-reactive anti-DLL3 antibody. See generally U.S. Patent Application Publication No. 2002 / 0199213. These expression vectors are typically replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selectable marker, such as ampicillin resistance or hygromycin resistance, to allow detection of cells transformed with the desired DNA sequence. The vector may also encode a signal peptide, such as pectate lyase, useful for directing the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0094] The recombinant expression vector of the present technology contains a nucleic acid encoding a protein with DLL3-binding properties in a form suitable for expression in a host cell, meaning that the recombinant expression vector contains one or more regulatory sequences selected based on the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in specific host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of an expression vector can vary depending on factors such as the choice of host cell to be transformed, the level of expression of the desired polypeptide, and the like. Typical regulatory sequences useful as promoters for recombinant polypeptide expression (e.g., anti-DLL3 antibodies) include, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization. In one embodiment, a polynucleotide encoding the anti-DLL3 antibody of the present technology is operably linked to the ara B promoter and can be expressed in a host cell. See U.S. Patent No. 5,028,530.The expression vectors of the present technology can be introduced into host cells to thereby produce polypeptides or peptides (such as, for example, anti-DLL3 antibodies) comprising fusion polypeptides encoded by nucleic acids as described herein.

[0095] Another aspect of the present technology relates to host cells expressing anti-DLL3 antibodies, containing nucleic acids encoding one or more anti-DLL3 antibodies. The recombinant expression vectors of the present technology can be designed for expression of anti-DLL3 antibodies in prokaryotic or eukaryotic cells. For example, anti-DLL3 antibodies can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, recombinant expression vectors can be transcribed and translated, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods useful for preparing and screening polypeptides with predetermined properties, such as anti-DLL3 antibodies, by expressing stochastically generated polynucleotide sequences have previously been described. See U.S. Patent Nos. 5,763,192, 5,723,323, 5,814,476, 5,817,483, 5,824,514, 5,976,862, 6,492,107, and 6,569,641.

[0096] Expression of polypeptides in prokaryotes is most often carried out using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides in E. coli. Fusion vectors add several amino acids to the polypeptide encoded herein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to increase expression of the recombinant polypeptide, (ii) to increase the solubility of the recombinant polypeptide, and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Fusion expression vectors often incorporate a proteolytic cleavage site at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Such enzymes and their cognate recognition sequences include factor X, thrombin, and enterokinase. Common fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0097] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). A method for targeted assembly of separate active peptides or protein domains to obtain multifunctional polypeptides by polypeptide fusion has been described by Pack et al., U.S. Pat. Nos. 6,294,353 and 6,692,935. One strategy for maximizing recombinant polypeptide expression in E. coli, e.g., anti-DLL3 antibodies, is to express the polypeptide in a host bacterium with an impaired ability to proteolytically cleave the recombinant polypeptide. For example, see Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into expression vector so that each individual codon of each amino acid is preferentially used in expression host, for example, E. coli (see, for example, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). This modification of the nucleic acid sequence of this technology can be carried out by standard DNA synthesis technology.

[0098] In another embodiment, the anti-DLL3 antibody expression vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (Invitrogen Corp, San Diego, CA). Alternatively, anti-DLL3 antibodies may be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expressing polypeptides, such as anti-DLL3 antibodies, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0099] In yet another embodiment, the nucleic acid encoding the anti-DLL3 antibody of the present technology is expressed in mammalian cells using mammalian expression vector.Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987).When used in mammalian cells, the control function of expression vectors is often provided by viral regulatory elements.For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells suitable for expressing the anti-DLL3 antibodies of the present technology, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0100] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1: 268-277, 1987), lymphocyte-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), promoters of T-cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1988). 1989), pancreatic-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., whey promoters; U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally regulated promoters are also included, such as mouse hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989).

[0101] Another aspect of the present method relates to a host cell into which the recombinant expression vector of the present technology is introduced.The terms "host cell" and "recombinant host cell" are used interchangeably herein.It is understood that these terms refer not only to the specific target cell, but also to the progeny or potential progeny of such a cell.Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term herein.

[0102] Host cells can be any prokaryotic or eukaryotic cell. For example, anti-DLL3 antibodies can be expressed in bacterial cells such as E. coli, insect cells, yeast, or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Exemplary expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. Co et al., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[0103] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics, or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., Molecular Cloning). Suitable methods for transducing or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[0104] For stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, a gene encoding a selection marker (e.g., resistance to antibiotics) is generally introduced into host cells together with the gene of interest. Various selection markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selection marker can be introduced into host cells on the same vector as that encoding the anti-DLL3 antibody, or on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have integrated the selection marker gene survive, while other cells die).

[0105] Host cells containing the anti-DLL3 antibody of the present technology, such as prokaryotic or eukaryotic host cells in culture, can be used to produce (i.e., express) recombinant anti-DLL3 antibodies. In one embodiment, the method includes culturing the host cells (into which a recombinant expression vector encoding the anti-DLL3 antibody has been introduced) in a suitable medium such that the anti-DLL3 antibody is produced. In another embodiment, the method further includes isolating the anti-DLL3 antibody from the medium or the host cells. Once expressed, the anti-DLL3 antibody, e.g., a collection of anti-DLL3 antibodies or anti-DLL3 antibody-related polypeptides, is purified from the culture medium and host cells. The anti-DLL3 antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like. In one embodiment, the anti-DLL3 antibody is produced in a host organism by the method of Boss et al., U.S. Pat. No. 4,816,397. Typically, the anti-DLL3 antibody chain is expressed with a signal sequence, thus releasing it into the culture medium. However, if the anti-DLL3 antibody chains are not naturally secreted by the host cells, they can be released by treatment with mild detergents. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0106] A polynucleotide encoding an anti-DLL3 antibody, for example, an anti-DLL3 antibody coding sequence, can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal.See, for example, U.S. Patent Nos. 5,741,957, 5,304,489 and 5,849,992.Suitable transgenes include light and / or heavy chain coding sequences operably linked to the promoter and enhancer from a mammary gland-specific gene, for example, casein or β-lactoglobulin.To produce transgenic animals, transgenes can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells, and the nucleus of such cells is transferred into enucleated oocytes.

[0107] Single-chain antibody. In one embodiment, the anti-DLL3 antibody of the present technology is a single-chain anti-DLL3 antibody. According to the present technology, technology can be adopted to produce single-chain antibodies specific to DLL3 protein (see, for example, U.S. Patent No. 4,946,778). Examples of the technology that can be used to produce single-chain Fv and antibodies of the present technology include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[0108] Chimeric and humanized antibodies. In one embodiment, the anti-DLL3 antibody of the present technology is a chimeric anti-DLL3 antibody. In one embodiment, the anti-DLL3 antibody of the present technology is a humanized anti-DLL3 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies derived from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), and in this case, the resulting CDR-grafted antibody is called a "humanized" antibody.

[0109] Recombinant anti-DLL3 antibodies, such as chimeric and humanized monoclonal antibodies containing both human and non-human portions, can be produced using standard recombinant DNA technology and are within the scope of the present technology. For some uses, including in vivo use of the anti-DLL3 antibodies of the present technology in humans and use of these materials in in vitro detection assays, chimeric or humanized anti-DLL3 antibodies may be used. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA technology known in the art. Such useful methods include, but are not limited to, those described in International Application No. PCT / US86 / 02269, U.S. Pat. No. 5,225,539, European Patent No. 184187, European Patent No. 171496, European Patent No. 173494, PCT International Publication No. WO86 / 01533, U.S. Pat. Nos. 4,816,567, 5,225,539, European Patent No. 125023, Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987. Cancer Res. 47: 999-1005; Wood, et al., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202-1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321: 552-525; Verhoeyan, et al., 1988.For example, antibodies can be humanized using a variety of techniques, including CDR grafting (EP 0239400, WO 91 / 09967, U.S. Pat. Nos. 5,530,101, 5,585,089, 5,859,205, 6,248,516, EP 460167), veneering or resurfacing (EP 0592106, EP 0519596, Padlan EA, Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332).In one embodiment, cDNA encoding a murine anti-DLL3 monoclonal antibody is digested with restriction enzymes specifically selected to remove sequences encoding the Fc constant region, and the equivalent portion of cDNA encoding a human Fc constant region is substituted (Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; U.S. Patent No. 6,180,370; U.S. Patent No. 6,300,064;

[0110] In one embodiment, the present technology provides for the construction of humanized anti-DLL3 antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response while still possessing effective antibody effector functions. As used herein, the terms "human" and "humanized" in relation to antibodies refer to any antibody that is predicted to induce a therapeutically acceptable, weak immunogenic response in human subjects. In one embodiment, the present technology provides humanized anti-DLL3 antibodies, heavy and light chain immunoglobulins.

[0111] CDR antibody. In some embodiments, the anti-DLL3 antibody of the present technology is an anti-DLL3 CDR antibody. Generally, the donor and acceptor antibodies used to generate anti-DLL3 CDR antibodies are monoclonal antibodies derived from different species, and the acceptor antibody is usually a human antibody (to minimize antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody. Grafting involves the transfer of a single V of the acceptor antibody to a single V of the acceptor antibody. H or V L or V H and V L The CDRs may be from multiple CDRs (or portions thereof) in one or both of the variable domains. Only the number of CDRs required to allow the resulting CDR-grafted antibody to properly bind to the DLL3 protein needs to be replaced, but often all three CDRs in all variable domains of the acceptor antibody are replaced with the corresponding donor CDRs. Methods for producing CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; and Winter U.S. Patent No. 5,225,539; and EP0682040. H and V LMethods useful for preparing polypeptides are taught by Winter et al., U.S. Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP 0451216; and EP 0120694.

[0112] After selecting suitable framework region candidates from the same family and / or family members, either or both of the heavy and light chain variable regions are produced by grafting CDRs from the original species into the hybrid framework regions. For any of the above embodiments, assembly of a hybrid antibody or hybrid antibody fragment having a hybrid variable chain region can be achieved using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on CDRs from the target species and the original species) can be produced by oligonucleotide synthesis and / or PCR. Nucleic acids encoding CDR regions can also be isolated from the original species antibody using appropriate restriction enzymes and ligated into the target species framework by ligation using appropriate ligation enzymes. Alternatively, the framework regions of the variable chains of the original species antibody can be altered by site-directed mutagenesis.

[0113] Since hybrids are constructed by selecting from multiple candidates corresponding to each framework region, there are numerous combinations of sequences that are amenable to construction according to the principles described herein. Thus, a library of hybrids can be assembled, with members having different combinations of individual framework regions. Such a library can be an electronic database collection or a physical collection of hybrid sequences.

[0114] This process usually does not alter the FRs of the acceptor antibody that flank the grafted CDRs. However, one skilled in the art can sometimes improve the antigen-binding affinity of the resulting anti-DLL3 CDR-grafted antibody by replacing specific residues in a given FR to create a similar FR with the corresponding FR of the donor antibody. Suitable locations for substitution include amino acid residues adjacent to or capable of interacting with the CDR (see, e.g., U.S. Pat. No. 5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with the donor FR and modify it to be more similar to the acceptor FR or human consensus FR. Techniques for making these modifications are known in the art. In particular, if the resulting FR matches or is at least 90% or more identical to the human consensus FR at that position, doing so may not significantly increase the antigenicity of the resulting modified anti-DLL3 CDR-grafted antibody compared to the same antibody with fully human FRs.

[0115] Bispecific antibodies (BsAbs). Bispecific antibodies are antibodies that can simultaneously bind to two targets with distinct structures, e.g., two different target antigens or two different epitopes on the same target antigen. BsAbs can be generated, for example, by combining heavy and / or light chains that recognize different epitopes on the same or different antigens. In some embodiments, a bispecific binding agent functions by binding to one antigen (or epitope) on one of its two binding arms (one VH / VL pair) and to a different antigen (or epitope) on its second arm (a different VH / VL pair). By this definition, a bispecific binding agent has two distinct antigen-binding arms (both in terms of specificity and CDR sequence) and is monovalent for each antigen it binds to. The bispecific antibodies (BsAbs) and bispecific antibody fragments (BsFabs) of the present technology have, for example, at least one arm that specifically binds to DLL3 and at least one other arm that specifically binds to a second target antigen. In certain embodiments, the BsAbs are capable of binding to tumor cells that express the DLL3 antigen on their cell surface.

[0116] A variety of bispecific fusion proteins can be produced using molecular engineering. For example, BsAbs have been constructed that utilize either a complete immunoglobulin framework (e.g., IgG), a single-chain variable fragment (scFv), or a combination thereof. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and another scFv fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, e.g., comprising an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs, composed of two scFv units in tandem, have proven to be a clinically successful bispecific antibody format. In some embodiments, the BsAb comprises two single-chain variable fragments (scFvs) in tandem, designed such that an scFv that binds a tumor antigen (e.g., DLL3) is linked to an scFv that binds a different target antigen.

[0117] Recent methods for producing BsAbs include engineered recombinant monoclonal antibodies with additional cysteine ​​residues that cross-link more strongly than more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins by linking two or more different single-chain antibody or antibody fragment segments with the required dual specificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163 (1997). Using molecular engineering, a variety of bispecific fusion proteins can be produced.

[0118] Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce various fusion proteins. In certain embodiments, the BsAb of the present technology comprises an immunoglobulin comprising a heavy chain, a light chain, and an scFv. In certain embodiments, the scFv is linked to the C-terminus of the heavy chain of any DLL3 immunoglobulin disclosed herein. In certain embodiments, the scFv is linked to the C-terminus of the light chain of any DLL3 immunoglobulin disclosed herein. In various embodiments, the scFv is linked to the heavy or light chain via a linker sequence. The appropriate linker sequence required for in-frame connection of the heavy chain Fd to the scFv is synthesized by PCR reaction. L and V kappa The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and the V domain of the DLL3 antibody is expressed. H The resulting vector is ligated into a vector containing DNA sequences encoding the bispecific fusion protein. The resulting vector can be used to transfect an appropriate host cell, e.g., a mammalian cell, for expression of the bispecific fusion protein.

[0119] In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, the linker is characterized in that it does not adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., the first and / or second antigen-binding site). In some embodiments, linkers are used in the BsAbs described herein based on the specific properties they confer to the BsAb, such as increased stability. In some embodiments, the BsAbs of the present technology comprise a G4S linker. In some specific embodiments, the BsAb of the present technology is (G4S) n linker, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0120] Fc Modifications. In some embodiments, the anti-DLL3 antibodies of the present technology comprise a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region (or parent Fc region) such that the molecule has an altered affinity for an Fc receptor (e.g., FcγR), based on crystallographic and structural analyses of Fc-Fc receptor interactions, such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000), provided that the variant Fc region does not have substitutions at positions that make direct contact with the Fc receptor. Examples of positions within the Fc region that make direct contact with an Fc receptor, e.g., FcγR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G loop). In some embodiments, the anti-DLL3 antibodies of the present technology having a variant Fc region with one or more amino acid modifications have altered affinity for activating and / or inhibitory receptors, wherein the one or more amino acid modifications are an N297 substitution with alanine or a K322 substitution with alanine.

[0121] Glycosylation modifications. In some embodiments, the anti-DLL3 antibodies of the present technology have an Fc region with variant glycosylation compared to a parent Fc region. In some embodiments, the variant glycosylation comprises the absence of fucose, and in some embodiments, the variant glycosylation results from expression in GnT1-deficient CHO cells. In some embodiments, the antibodies of the present technology may have glycosylation sites that are modified relative to a suitable reference antibody that binds to an antigen of interest (e.g., DLL3) without altering the functionality of the antibody, e.g., its binding activity to the antigen. As used herein, a "glycosylation site" includes any particular amino acid sequence in an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) is specifically and covalently attached.

[0122] Oligosaccharide side chains are usually linked to the antibody backbone by either N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, such as serine or threonine. For example, an Fc-glycoform (hDLL3-IgGln) lacking certain oligosaccharides containing fucose and terminal N-acetylglucosamine can be produced in certain CHO cells and exhibit enhanced ADCC effector function. In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, for example, U.S. Patent No. 6,218,149, EP 0359096 B1, U.S. Patent Publication No. US 2002 / 0028486, International Patent Application Publication No. WO 03 / 035835, U.S. Patent Publication No. 2003 / 0115614, U.S. Patent No. 6,218,149, and U.S. Patent No. 6,472,511, all of which are incorporated herein by reference in their entireties. In some embodiments, the carbohydrate content of an antibody (or a relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In some specific embodiments, the present technology involves deleting a glycosylation site in the Fc region of an antibody by modifying position 297 from asparagine to alanine.

[0123] Engineered glycoforms can be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms can be made by any method known to those of skill in the art, for example, by using engineered or variant expression strains, by co-expression with one or more enzymes, such as N-acetylglucosaminyltransferase III (GnTIII), by expressing molecules comprising an Fc region in different organisms or cell lines derived from different organisms, or by modifying the carbohydrate(s) after the molecule comprising an Fc region has been expressed. Methods for producing engineered glycoforms are known in the art and include, but are not limited to, Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473, U.S. Patent No. 6,602,684, U.S. Patent Application No. 10 / 277,370, U.S. Patent Application No. 10 / 113,929, International Patent Application Publication Nos. WO00 / 61739A1, WO01 / 292246A1, WO02 / 311140A1, WO02 / 30954A1, POTILLEGENT™ technology (Biowa, Inc. Princeton, New Jersey), GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland), each of which is incorporated herein by reference in its entirety. See, for example, International Patent Application Publication No. WO 00 / 061739, U.S. Patent Application Publication No. 2003 / 0115614, Okazaki et al., 2004, JMB, 336: 1239-49.

[0124] Fusion Protein. In one embodiment, the anti-DLL3 antibody of the present technology is a fusion protein. When fused to a second protein, the anti-DLL3 antibody of the present technology can be used as an antigenic tag. Examples of domains that can be fused to a polypeptide include heterologous signal sequences as well as other heterologous functional regions. The fusion does not necessarily have to be direct, but can occur via a linker sequence. Furthermore, the fusion protein of the present technology can also be genetically engineered to improve the characteristics of the anti-DLL3 antibody. For example, additional amino acids, particularly a region of charged amino acids, can be added to the N-terminus of the anti-DLL3 antibody to improve stability and persistence during purification from host cells or subsequent handling and storage. Peptide moieties can also be added to the anti-DLL3 antibody to facilitate purification. Such regions can be removed prior to final preparation of the anti-DLL3 antibody. Addition of peptide moieties to facilitate handling of polypeptides is well known and routine in the art. The anti-DLL3 antibody of the present technology can also be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In selected embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in pQE vectors (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available. For example, hexahistidine provides convenient purification of the fusion protein, as described by Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

[0125] Thus, any of these above fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology. Also, in some embodiments, the fusion proteins described herein exhibit increased half-life in vivo.

[0126] Fusion proteins with disulfide-bonded dimeric structures (such as IgG) can be more effective at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995. Similarly, EP-AO 464533 (Canadian counterpart 2045869) discloses fusion proteins containing various portions of the constant region of immunoglobulin molecules together with another human protein or fragment thereof. In many cases, the Fc portion in the fusion protein is beneficial in therapy and diagnosis, and can therefore, for example, result in improved pharmacokinetic properties. See EP-A 0232262. Alternatively, it may be desirable to delete or modify the Fc portion after the fusion protein has been expressed, detected, and purified. For example, the Fc portion may interfere with therapy and diagnosis when the fusion protein is used as an antigen for immunization. In drug discovery, for example, human proteins such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify hIL-5 antagonists. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0127] Labeled anti-DLL3 antibody. In one embodiment, the anti-DLL3 antibody of the present technology is coupled to a labeling moiety, i.e., a detectable group. The particular label or detectable group conjugated to the anti-DLL3 antibody is not a critical aspect of the technology, as long as it does not significantly interfere with the specific binding of the anti-DLL3 antibody of the present technology to the DLL3 protein. The detectable group can be any material with detectable physical or chemical properties. Such detectable labels have been well developed in the fields of immunoassays and imaging. Generally, almost any label useful in such methods can be applied to the present technology. Thus, the label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the implementation of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radiolabels (e.g., 3 H, 14 C. 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other contrast agents such as microbubbles (for ultrasound imaging), 18 F, 11 C. 15 O. 89 Zr (for positron emission tomography), 99m Tc, 111In (for single photon emission computed tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference in its entirety for all purposes. Also, see Handbook of Fluorescent Probes and Research Chemicals (6 th See also Molecular Probes, Inc., Eugene, OR).

[0128] The label is coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on factors such as the sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal regulations.

[0129] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule that is inherently detectable or covalently bound to a signal system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Several ligands and anti-ligands can be used. When the ligand has a neutral anti-ligand, such as biotin, thyroxine, and cortisol, it can be used in conjunction with a labeled, naturally occurring anti-ligand. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, such as an anti-DLL3 antibody.

[0130] Molecules can also be directly conjugated to signal-generating compounds, for example, by conjugation with enzymes or fluorophores. Enzymes of interest as labels are primarily hydrolases, particularly phosphatases, esterases, and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties include, but are not limited to, luciferin and 2,3-dihydrophthalazinediones, such as luminol. For a summary of various labeling or signal-generating systems that can be used, see U.S. Patent No. 4,391,904.

[0131] Means for detecting labels are well known to those skilled in the art. Thus, for example, if the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. If the label is a fluorescent label, it can be detected by exciting the fluorescent dye with the appropriate wavelength of light and detecting the resulting fluorescence. Fluorescence can be detected visually by photographic film, by using an electronic detector such as a charge-coupled device (CCD) or a photomultiplier tube, etc. Similarly, enzymatic labels can be detected by providing an appropriate substance to the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels can be easily detected by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold appears pink, while various conjugated beads appear the color of the bead.

[0132] Some assay formats do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of labeled antibodies, such as anti-DLL3 antibodies. In this case, antigen-coated particles are agglutinated by a sample containing the target antibody. In this format, no components need to be labeled, and the presence of the labeled antibody is detected by simple visual inspection.

[0133] B. Identification and Characterization of Anti-DLL3 Antibodies of the Present Technology Methods for identifying and / or screening anti-DLL3 antibodies of the present technology. Methods useful for identifying and screening antibodies against DLL3 polypeptides for those with the desired specificity for the DLL3 protein (e.g., those that bind to the extracellular domain of DLL3) include any immunologically mediated technique known in the art. Components of the immune response can be detected in vitro by a variety of methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes are incubated with radioactively labeled target cells, and the lysis of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes are incubated with antigen and antigen-presenting cells, and cytokine synthesis and secretion can be measured by standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen-presenting cells are incubated with whole protein antigen, and the presentation of that antigen on MHC can be detected by either T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells are incubated with a reagent that crosslinks their Fc-epsilon receptors, and histamine release can be measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983) and (5) enzyme-linked immunosorbent assay (ELISA).

[0134] Similarly, the products of an immune response in either a model organism (e.g., a mouse) or a human subject can also be detected by a variety of methods well known to those skilled in the art. For example, (1) the production of antibodies in response to vaccination can be easily detected by standard methods currently used in clinical laboratories, such as ELISA; (2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of the skin and placing a sterile container over the scratch site to capture the migrating cells (Peters et al., Blood, 72:1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactants can be detected by immunohistochemistry. 3 (4) phagocytosis of granulocytes, macrophages and other phagocytes in PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and (5) differentiation of immune system cells can be measured by labeling PBMCs with antibodies against CD molecules such as CD4 and CD8 and measuring the fraction of PBMCs that express these markers.

[0135] In one embodiment, the anti-DLL3 antibody of the present technology is selected by displaying DLL3 peptide on the surface of replicable genetic packaging.For example, see U.S. Patent Nos. 5,514,548, 5,837,500, 5,871,907, 5,885,793, 5,969,108, 6,225,447, 6,291,650, 6,492,160, EP585287, EP605522, EP616640, EP1024191, EP589877, EP774511, EP844306.Methods useful for producing / selecting filamentous bacteriophage particles containing phagemid genomes encoding binding molecules with desired specificity have been described. See, for example, EP 774511, US 5,871,907, US 5,969,108, US 6,225,447, US 6,291,650, US 6,492,160. In some embodiments, the anti-DLL3 antibodies of the present technology are selected using display of DLL3 peptides on the surface of yeast host cells. A method useful for isolating Fv polypeptides by yeast surface display is described by Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10.

[0136] In some embodiments, the anti-DLL3 antibody of the present technology is selected using ribosome display.The method of identifying ligands in peptide libraries using ribosome display is described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994 and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997. In certain embodiments, the anti-DLL3 antibody of the present technology is selected using tRNA display of DLL3 peptide. A useful method for in vitro selection of ligands using tRNA display is described by Merryman et al., Chem. Biol., 9: 741-46, 2002.

[0137] In one embodiment, the anti-DLL3 antibody of the present technology is selected using RNA display.The method useful for selecting peptides and proteins using RNA display library is described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997, and Nemoto et al., FEBS Lett., 414: 405-8, 1997.The method useful for selecting peptides and proteins using non-natural RNA display library is described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003. In some embodiments, the anti-DLL3 antibodies of the present technology are expressed in the periplasm of Gram-negative bacteria and mixed with labeled DLL3 protein. See WO02 / 34886. Clones expressing recombinant polypeptides with affinity to DLL3 protein will have an enriched concentration of labeled DLL3 protein bound to the anti-DLL3 antibody, allowing the cells to be isolated from the rest of the library, as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Patent Publication No. 2004 / 0058403.

[0138] It is contemplated that after selection of a desired anti-DLL3 antibody, the antibody can be produced in large quantities by any technique known to those of skill in the art, such as, for example, prokaryotic or eukaryotic expression. For example, but not limited to, an anti-DLL3 antibody that is an anti-DLL3 hybrid antibody or fragment may be produced using conventional techniques to construct an expression vector encoding an antibody heavy chain, in which the CDRs and, optionally, the minimum portion of the variable region framework necessary to retain the original species' antibody binding specificity (as genetically engineered according to the techniques described herein) are derived from the original species' antibody, and the remainder of the antibody is derived from a target species immunoglobulin engineered as described herein, thereby generating a vector for expression of the hybrid antibody heavy chain.

[0139] Measurement of DLL3 binding. In some embodiments, a DLL3 binding assay refers to an assay format in which a DLL3 protein and an anti-DLL3 antibody are mixed under conditions suitable for binding between the DLL3 protein and the anti-DLL3 antibody, and the amount of binding between the DLL3 protein and the anti-DLL3 antibody is assessed. The amount of binding is compared to a suitable control, which can be the amount of binding in the absence of the DLL3 protein, the amount of binding in the presence of a nonspecific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assays include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for direct measurement of DLL3 protein binding with an anti-DLL3 antibody include, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), biolayer interferometry, etc. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of the candidate anti-DLL3 antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-DLL3 antibody, then the candidate anti-DLL3 antibody is useful as an anti-DLL3 antibody of the present technology.

[0140] Use of the anti-DLL3 antibody of the present technology General. The antibodies of the present technology are useful in methods known in the art for localizing and / or quantitating DLL3 polypeptides (e.g., for use in measuring the level of DLL3 protein in an appropriate physiological sample, for use in diagnostic methods, for use in imaging of the polypeptide, etc.). The anti-DLL3 antibodies of the present technology are useful for isolating DLL3 polypeptides by standard techniques such as affinity chromatography or immunoprecipitation. The anti-DLL3 antibodies of the present technology can facilitate the purification of natural immunoreactive DLL3 protein from biological samples, such as mammalian serum or cells, as well as the purification of recombinantly produced immunoreactive DLL3 protein expressed in a host system. Furthermore, the anti-DLL3 antibodies of the present technology can be used to detect immunoreactive DLL3 protein (e.g., in plasma, cell lysates, or cell supernatants) to assess the amount and pattern of expression of the immunoreactive polypeptide. The anti-DLL3 antibodies of the present technology can be used diagnostically to monitor immunoreactive DLL3 protein levels in tissues as part of a clinical trial procedure, for example, to determine the effectiveness of a given treatment regimen. As described above, detection can be facilitated by coupling (ie, physically linking) the anti-DLL3 antibodies of the present technology to a detectable substance.

[0141] Detection of DLL3 protein. An exemplary method for detecting the presence or absence of immunoreactive DLL3 protein in a biological sample includes obtaining a biological sample from a test subject and contacting the biological sample with an anti-DLL3 antibody of the present technology that can detect immunoreactive DLL3 protein, thereby detecting the presence of immunoreactive DLL3 protein in the biological sample. Detection can be achieved by a detectable label attached to the antibody. The term "labeled," with respect to anti-DLL3 antibodies, is intended to encompass direct labeling of an antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of an antibody by reactivity with another compound, such as a secondary antibody, which is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected using fluorescently labeled streptavidin.

[0142] In some embodiments, the anti-DLL3 antibodies disclosed herein are conjugated to one or more detectable labels. For such use, the anti-DLL3 antibodies can be detectably labeled by covalent or non-covalent attachment of chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance imaging agent, or other labels. Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6 phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0143] Examples of suitable radioisotope labels include: 3 H, 111 In, 125 I, 131 I, 32 P, 35 S, 14 C. 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 EU, 90 Y,67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Examples include Pd. 111 In is due to the liver 125 I or 131 I-labeled DLL3-binding antibodies avoid the problem of dehalogenation, making this isotope an exemplary isotope when in vivo imaging is used. Furthermore, this isotope has a more convenient gamma-emission energy for imaging (Perkins et al., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, I-labeled DLL3-binding antibodies coupled to monoclonal antibodies using 1-(P-isothiocyanatobenzyl)-DPTA are useful. 111 In shows little uptake in non-tumor tissues, especially the liver, enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870(1987)). Examples of suitable non-radioactive isotope labels include: 157 Gd, 55 Mn, 162 Dy, 52 Tr and 56 Fe is one example.

[0144] Examples of suitable fluorescent labels include: 152 Examples of suitable toxin labels include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

[0145] Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn, and iron. The detection method of the present technology can be used to detect immunoreactive DLL3 protein in biological samples in vitro and in vivo. In vitro techniques for detecting immunoreactive DLL3 protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay, and immunofluorescence. Furthermore, in vivo techniques for detecting immunoreactive DLL3 protein include introducing a labeled anti-DLL3 antibody into a subject. For example, the anti-DLL3 antibody may be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains DLL3 protein molecules obtained from the test subject.

[0146] Immunoassays and imaging. The anti-DLL3 antibodies of the present technology can be used to assay immunoreactive DLL3 protein levels in biological samples (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be studied using classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase and radioisotopes or other radioactive substances, such as iodine ( 125 I, 121 I,131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 111 In) and technetium ( 99 mTc) and fluorescent labels such as fluorescein, rhodamine and green fluorescent protein (GFP) and biotin.

[0147] In addition to assaying the immunoreactive DLL3 protein level in biological samples, the anti-DLL3 antibody of the present technology can be used for in vivo imaging of DLL3.Antibodies useful for this method include those that can be detected by X-ray imaging, NMR or ESR.For X-ray imaging, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not obviously harmful to subjects.Suitable markers for NMR and ESR include those with detectable characteristic spins, such as deuterium, which can be incorporated into anti-DLL3 antibodies by labeling the nutrients of related scFv clones.

[0148] Suitable detectable imaging moieties, such as radioisotopes (e.g., 131 I, 111 In, 99 mTc, 18 F, 89 An anti-DLL3 antibody of the present technology labeled with a radiopaque substance or a material detectable by nuclear magnetic resonance (NMR) is introduced into a subject (e.g., parenterally, subcutaneously, or intraperitoneally). It will be understood in the art that the size of the subject and the imaging system used will dictate the amount of imaging moiety required to generate a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is typically about 5-20 millicuries. 99The range of mTc is reached.Labeled anti-DLL3 antibody then accumulates at the location of the cells that contain specific target polypeptide.For example, labeled anti-DLL3 antibody of the present technology accumulates in the cells and tissues in which DLL3 protein is localized in the subject. Thus, the present technology provides a method for diagnosing a medical condition, comprising (a) assaying the expression of immunoreactive DLL3 protein in an individual's cells or body fluids by measuring binding of an anti-DLL3 antibody of the present technology, and (b) comparing the amount of immunoreactive DLL3 protein present in the sample with a standard reference, wherein an increase or decrease in the level of immunoreactive DLL3 protein compared to the standard indicates the medical condition.

[0149] Affinity purification. The anti-DLL3 antibody of the present technology can be used to purify immunoreactive DLL3 protein from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose, and acrylic resins such as polyacrylamide and latex beads. The technology of coupling antibodies to such solid supports is well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, NY (1974)). The simplest method for binding an antigen to an antibody-support matrix is ​​to collect beads in a column and allow the antigen solution to flow through the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using a slow flow rate. The immobilized antibody captures the antigen as it flows past. Alternatively, the antigen can be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or shaking the slurry, which allows for maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed through the column to recover the beads. The beads are washed using an appropriate wash buffer, and then the pure or substantially pure antigen is eluted.

[0150] The target antibody or polypeptide can be conjugated to a solid support such as beads.In addition, the first solid support such as beads can also be conjugated to a second solid support, which can be a second bead or other support, if necessary, by any suitable means, including those disclosed herein for the conjugation of polypeptide with support.Therefore, any of the conjugation methods and means disclosed herein for the conjugation of polypeptide with solid support can be applied to the conjugation of the first support with the second support, and the first and second solid support can be the same or different.

[0151] Suitable linkers that can be used as cross-linking agents to conjugate polypeptides to solid supports include various substances that can react with functional groups present on the surface of the support, with the polypeptide, or with both. Reagents useful as cross-linking agents include homobifunctional and, in particular, heterobifunctional reagents. Useful bifunctional cross-linking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. Cross-linking agents can be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, photolabile cross-linking agents, such as 3-amino-(2-nitrophenyl)propionic acid, can be used as a means to cleave the polypeptide from the solid support (Brown et al., Mol. Divers, pp. 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996) and U.S. Patent No. 5,643,722). Other cross-linking reagents are well known in the art (see, eg, Wong (1991), supra; and Hermanson (1996), supra).

[0152] Antibodies or polypeptides can be immobilized on solid supports, such as beads, via a covalent amide bond formed between a carboxyl-functionalized bead and the amino terminus of the polypeptide, or conversely, via a covalent amide bond formed between an amino-functionalized bead and the carboxyl terminus of the polypeptide. Furthermore, a bifunctional trityl linker can be attached to a support, such as a 4-nitrophenyl active ester on a resin, such as a Wang resin, via the amino or carboxyl groups on the resin. Using the bifunctional trityl approach, the solid support may require treatment with a volatile acid, such as formic acid or trifluoroacetic acid, to ensure that the polypeptide can be cleaved and removed. In such cases, the polypeptide can be deposited on the bottom of a well on the solid support or as a bead-free patch on the flat surface of the solid support. After adding a matrix solution, the polypeptide can be released into MS.

[0153] Hydrophobic trityl linkers can also be used as acid-labile linkers by using a volatile acid or an appropriate matrix solution, such as a matrix solution containing 3-HPA, to cleave the amino-linked trityl group from the polypeptide. Acid lability can also be modified. For example, trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl can be modified with an appropriate p-substituted or more acid-labile tritylamine derivative of the polypeptide, i.e., trityl ether and tritylamine bonds are formed on the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker by, for example, disrupting the hydrophobic attraction, or, if necessary, by cleaving the trityl ether or tritylamine bond under acidic conditions, including under typical MS conditions where a matrix such as 3-HPA acts as an acid.

[0154] Orthogonally cleavable linkers can also be useful for binding a first solid support, such as beads, to a second solid support, or for binding a target polypeptide to a solid support.Using such linkers, the first solid support, such as beads, can be selectively cleaved from the second solid support without cleaving the polypeptide from the support, and then the polypeptide can be cleaved from the beads at a later time.For example, a disulfide linker that can be cleaved using a reducing agent such as DTT can be used to bind beads to the second solid support, and an acid-cleavable bifunctional trityl group can be used to immobilize the polypeptide to the support.Optionally, for example, the link between the first and second supports can remain intact, and the link between the polypeptide and the solid support can be cleaved first.The trityl linker can provide covalent or hydrophobic conjugation, but regardless of the nature of the conjugation, the trityl group is easily cleaved under acidic conditions. For example, beads can be attached to a second support by a linking group that can be selected to have a length and chemical properties that promote high-density binding of the beads to the solid support or high-density binding of the polypeptide to the beads. Such linking groups can have, for example, a "tree-like" structure, thereby providing a variety of functional groups per attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, pentaerythrol, and tris-hydroxy-aminomethane. Non-covalent association. By non-covalent interaction, an antibody or polypeptide can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support. For example, magnetic beads made of ferromagnetic materials that can be magnetized can be attracted to a magnetic solid support and can be released from the support by removing the magnetic field. Alternatively, the solid support can be provided with ionic or hydrophobic moieties, which can respectively allow the ionic or hydrophobic moieties to interact with polypeptides, such as polypeptides containing attached trityl groups, or with a second solid support having hydrophobic properties.

[0155] The solid support may also be provided with a member of a specific binding pair and thus be conjugated to a polypeptide or a second solid support containing a complementary binding moiety. For example, avidin- or streptavidin-coated beads can be bound to a polypeptide incorporating a biotin moiety, or to a second solid support coated with biotin or a biotin derivative, such as iminobiotin.

[0156] It should be understood that any of the binding members disclosed herein or otherwise known in the art may be reversed. Thus, biotin, for example, may be incorporated into either the polypeptide or the solid support, and conversely, avidin or other biotin-binding moiety is incorporated into the support or polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and specifically interacting antigens, and other such pairs known to those of skill in the art.

[0157] A. Diagnostic Uses of the Anti-DLL3 Antibodies of the Present Technology General. The anti-DLL3 antibodies of the present technology are useful in diagnostic methods. As such, the present technology provides methods for using antibodies in diagnosing DLL3 activity in a subject. The anti-DLL3 antibodies of the present technology can be selected to have any level of epitope binding specificity and extremely high binding affinity for DLL3 polypeptides. Generally, the higher the binding affinity of the antibody, the more stringent washing conditions can be implemented to remove nonspecifically bound materials in immunoassays without removing the target polypeptide. Thus, the anti-DLL3 antibodies of the present technology useful in diagnostic assays typically have a binding affinity of about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1or 10 12 M -1 Additionally, it is desirable for anti-DLL3 antibodies used as diagnostic reagents to have a sufficient kinetic on-rate to reach equilibrium under standard conditions in at least 12 hours, at least five (5) hours, or at least one (1) hour.

[0158] Anti-DLL3 antibodies can be used to detect immunoreactive DLL3 protein in a variety of standard assay formats, including immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, NY) See U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,879,262, 4,034,074, 3,791,932, 3,817,837, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, and 4,098,876. Biological sample can be obtained from any tissue or body fluid of subject.In certain embodiments, subject is in the early stage of cancer.In one embodiment, the early stage of cancer is determined by the level or expression pattern of DLL3 protein in the sample obtained from subject.In certain embodiments, sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF) and biopsied body tissue.

[0159] Immunoassays or sandwich assays are one format for the diagnostic method of this technology. See U.S. Patent Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use one antibody, for example, an anti-DLL3 antibody or a population of anti-DLL3 antibodies immobilized on a solid phase, and another anti-DLL3 antibody or a population of anti-DLL3 antibodies in solution. Typically, the solution anti-DLL3 antibody or population of anti-DLL3 antibodies is labeled. When an antibody population is used, the population may contain antibodies that bind to different epitope specificities within the target polypeptide. Thus, the same population can be used for both the solid phase and the solution antibody. When an anti-DLL3 monoclonal antibody is used, first and second DLL3 monoclonal antibodies with different binding specificities are used for the solid and solution phases. The solid phase (also called "capture") and solution (also called "detection") antibodies can be contacted with the target antigen in either order or simultaneously. If the solid-phase antibody is contacted first, the assay is called a forward assay. Conversely, if the solution antibody is contacted first, the assay is called a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is called a simultaneous assay. After contacting the DLL3 protein with the anti-DLL3 antibody, the sample is usually incubated for a period varying from about 10 minutes to about 24 hours, usually about 1 hour. A wash step is then performed to remove sample components that do not specifically bind to the anti-DLL3 antibody used as a diagnostic reagent. If the solid-phase and solution antibodies are bound in separate steps, washing may be performed after either or both binding steps. After washing, binding is usually quantified by detecting a label linked to the solid phase by binding of the labeled solution antibody. Typically, for a given pair of antibodies or antibody populations and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of target antigen. The concentration of immunoreactive DLL3 protein in the sample being tested is then read from interpolation from the calibration curve (ie, standard curve).The analyte can be determined from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of the bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of DLL3 protein in the sample.

[0160] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, as well as particles such as agarose, dextran-based gels, dipsticks, microparticles, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway, NJ), etc. Immobilization can be by absorption or covalent attachment. Anti-DLL3 antibodies can be tethered to a linker molecule such as biotin for attachment to a surface-bound linker such as avidin.

[0161] In some embodiments, the present disclosure provides an anti-DLL3 antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent may include a radioactive or non-radioactive label, an imaging agent (e.g., for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label may be a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope. The diagnostic agent is an antibody moiety, i.e., a molecule conjugated to an antibody or antibody fragment or subfragment and administered, and is useful in diagnosing or detecting disease by determining the location of cells containing the antigen.

[0162] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (e.g., using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to MRI enhancement agents, and is incorporated herein by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancement agents for use in magnetic resonance imaging, and fluorescent compounds. To load an antibody component with a radiometal or paramagnetic ion, it may be necessary to react it with a reagent having a long tail to which multiple chelating groups are attached to bind the ion. Such tails can be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains bearing pendant groups to which chelating groups, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose, can be attached. Chelates can be coupled to the antibodies of the present technology using standard chemistry. Chelates are usually linked to antibodies by groups that allow for the formation of bonds with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal crosslinking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. A particularly useful metal-chelate combination includes 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, which are used with diagnostic isotopes for radioimaging. The same chelates, when complexed with non-radioactive metals such as manganese, iron, and gadolinium, are useful for MRI when used with the anti-DLL3 antibodies of the present technology.

[0163] B. Therapeutic Uses of Anti-DLL3 Antibodies of the Present Technology The immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) of the present technology are useful for treating DLL3-associated cancers. Such treatment can be used in patients identified as having pathologically high levels of DLL3 (e.g., those diagnosed by the methods described herein) or in patients diagnosed with a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method for treating DLL3-associated cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology. Examples of cancers that can be treated by the antibodies of the present technology include, but are not limited to, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), pulmonary neuroendocrine cell carcinoma, extrapulmonary neuroendocrine cell carcinoma, and melanoma.

[0164] The immunoglobulin-related compositions of the present technology can be used with other therapeutic agents useful in the treatment of DLL3-associated cancers. For example, the antibodies of the present technology can be administered separately, sequentially, or simultaneously with at least one additional therapeutic agent, or can be conjugated to at least one additional therapeutic agent, wherein at least one additional therapeutic agent is a marine-derived compound (e.g., dolastatin 10, auristatin, tacidotin, dolastatin 15 or a variant thereof, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF)) (Newman & Cragg, Mar Drugs. 2017 Apr;15(4): 99), vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutics, targeted biological therapeutics (e.g., therapeutic peptides described in US6306832, WO2012007137, WO2005000889, WO2010096603, etc.), alkylating agents, alkyl sulfonates, amanitin, aziridine, Ethylenimine and methylamelamine, acetogenins, camptothecins, bryostatins, kallistatins, CC-1065, cryptophycins, dolastatins, duocarmycins, eleutherobin, pancratistatins, sarcodictyins, spongistatins, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicins, bisphosphonates, esperamicins, cloricin, cyclohexyl methylam ... Antiobiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, AD RIAMYCIN® doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;Antimetabolites, folic acid analogs, purine analogs, androgens, anti-adrenal steroids, folic acid replenishers such as flolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine famine, demecolcine, diaziquone, eflornithine, elliptinurm acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitracrine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK® polysaccharide complex (JHS natural products, Eugene, OR), razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidin); urethane; vindesine; vemurafenib; dacarbazine; mannomustine; Mitobronitrol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, chloranbucil; GEMZAR® gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine;The at least one additional therapeutic agent is selected from the group consisting of: NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11), the topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; inhibitors of PKC alpha, Raf, H-Ras, EGFR, and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, solvates, acids, or derivatives of any of the above. In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapy agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastomoses, fluticasone, fluoxetine, fluoxetine-10 ... Trozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, Tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, auristatins, maytansinoids, tubulisin, calicheamicin, duocarmycin, benzodiazepines, camptothecin, or combinations thereof;

[0165] Other suitable anti-cancer agents include commercially or clinically available compounds, such as erlotinib (TARCEVA®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, CAS number 51-21-8), PD-0325901 (CAS number 391210-10-9, Pfizer), cisplatin (cis-diamminedichloroplatinum(II), CAS number 15663-27-1), carboplatin (CAS number 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb), and others. Oncology, Princeton, NJ), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo 2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS number 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(l,2-diphenylbut-l-enyl)phenoxy]-N,N-dimethylethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®). Further commercially or clinically available anti-cancer drugs include bortezomib (VELCADE®, Millennium Pharm.), Sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SARAS AR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (Cremophor-free), albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXAN®, NEOSAR®); vinorelbine (NAVELBINE®); capecitabine (XELODA®, Roche), tamoxifen (NOLVADEX®); tamoxifen citrate, FARESTON® (toremifme citrate), MEGASE® (megestrol acetate), AROMASIN® (exemestane;Pfizer), formestany, fadrozole, RIVISOR® (vorozole), FEMARA®, and ARIMIDEX® (anastrozole, AstraZeneca); dabrafumib (TAFINLAR®, GlaxoSmithKline); dasatinib (SPRYCEL®, Bristol-Myers Squibb) Squibb); trametinib (MEKINIST®, GlaxoSmithKline); nilotinib (TASIGNA®, Novartis), troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; vinorelbine and esperamycin, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0166] The compositions of the present technology may be administered to a subject in need thereof as a single bolus, or the dosing regimen may include multiple doses administered at various times after the appearance of the tumor. Administration can be by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intracranially, intratumorally, intrathecally, or topically. Administration includes self-administration and administration by another. It should also be understood that the various modes of treatment of medical conditions as described include complete treatment as well as less-than-complete treatment, and are intended to mean "substantial" in that some biologically or medically relevant result is achieved.

[0167] In some embodiments, the antibodies of the present technology comprise pharmaceutical formulations that can be administered to a subject in need thereof in one or more doses. Dosage regimens can be adjusted to provide the desired response (e.g., a therapeutic response). Typically, an effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect ranges from about 0.000001 mg / kilogram of body weight / day to about 10,000 mg / kilogram of body weight / day. Typically, the dosage range is from about 0.0001 mg / kilogram of body weight / day to about 100 mg / kilogram of body weight / day. For administration of an anti-DLL3 antibody, the dosage ranges from about 0.0001 to 100 mg / kg of the subject's body weight, more usually 0.01 to 5 mg / kg weekly, every two weeks, or every three weeks. For example, the dosage can be within the range of 1 mg / kg or 10 mg / kg weekly, every two weeks, or every three weeks, or 1 to 10 mg / kg weekly, every two weeks, or every three weeks. In one embodiment, a single dose of the antibody ranges from 0.1 to 10,000 micrograms / kg of body weight. In one embodiment, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. The anti-DLL3 antibody may be administered multiple times. The interval between single doses may be hourly, daily, weekly, monthly, or yearly. The interval may be irregular, as indicated by measuring the blood level of the antibody in the subject. In some methods, the dosage is adjusted to achieve a serum antibody concentration in the subject of about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, about 125 μg / mL to about 175 μg / mL, or about 150 μg / mL to about 200 μg / mL. Alternatively, the anti-DLL3 antibody may be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the subject. The dosage and frequency of administration vary depending on whether treatment is preventive or therapeutic.In preventive application, a relatively low dosage is administered at relatively infrequent intervals for a long period of time.In therapeutic application, a relatively high dosage is sometimes required at relatively short intervals until the progression of disease is reduced or terminated, or until the patient shows partial or complete alleviation of the symptoms of disease.Then, patient can be administered a preventive dosage regimen.

[0168] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology, wherein the antibody is configured to localize to a tumor expressing DLL3 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactivity level emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (%ID / g). The reference value can be calculated by measuring the radioactivity level present in non-tumor (normal) tissue and calculating the average radioactivity level present in the non-tumor (normal) tissue ± standard deviation by computer. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0169] In some embodiments, the subject has been diagnosed with or is suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0170] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle-emitting isotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu and 67Examples of alpha particle radioactive isotopes include Cu. 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At and 255 Examples of Auger emitters include Fm. 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl and 203 Pb. In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., by the N297A mutation in the Fc region, which results in aglycosylation). The therapeutic efficacy of such immunoconjugates can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio by computer. In some embodiments, the immunoconjugate has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0171] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-DLL3 antibody described herein provides a therapeutic benefit without causing substantial toxicity to the subject. Toxicity of the anti-DLL3 antibodies described herein can be assessed by standard pharmaceutical procedures in cell culture or experimental animals, e.g., LD 50 (lethal dose for 50% of the population) or LD100 The therapeutic index can be determined by determining the lethal dose (the dose that is lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a non-toxic dosage range for use in humans. The dosage of the anti-DLL3 antibody described herein lies within a range of circulating concentrations that includes the effective dose with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the subject's condition. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

[0172] Formulation of Pharmaceutical Compositions. According to the methods of the present technology, the anti-DLL3 antibody can be incorporated into a pharmaceutical composition suitable for administration. The pharmaceutical composition generally comprises a recombinant or substantially purified antibody and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. The pharmaceutically acceptable carrier is determined in part by the particular composition to be administered, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of pharmaceutical compositions for administering antibody compositions (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18 th (See, e.g., U.S. Pat. No. 6,1990.) Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0173] The terms "pharmaceutically acceptable," "physiologically acceptable," and their grammatical variations are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that the material can be administered to a subject without causing any undesirable physiological effects to the extent that they interfere with the administration of the composition. For example, a "pharmaceutically acceptable excipient" generally refers to a safe, non-toxic excipient that is useful in preparing the desired pharmaceutical composition, including excipients that are acceptable for veterinary use and for human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. "Pharmaceutically acceptable salts and esters" refer to salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the composition can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane- and arene-sulfonic acids, e.g., methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the anti-DLL3 antibody, e.g., C 1-6Examples include alkyl esters. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a mono-acid-mono-salt or ester or a di-salt or ester; similarly, when two or more acidic groups are present, some or all of such groups may be salified or esterified. The anti-DLL3 antibodies named in this technology may exist in unsalted or unesterified form, or in salified and / or esterified form, and the naming of such anti-DLL3 antibodies is intended to include both the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain embodiments of the present technology may exist in two or more stereoisomeric forms, and the naming of such anti-DLL3 antibodies is intended to include all single stereoisomers and all mixtures (whether racemic or not) of such stereoisomers. Those skilled in the art will have no difficulty in determining the appropriate timing, sequence, and dosage of administration of particular drugs and compositions of the present technology.

[0174] Examples of such carrier or diluent include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution and 5% human serum albumin.Non-aqueous vehicles such as liposomes and hardened oils can also be used.The use of such media and compounds for pharmaceutically active substances is well known in the art.Unless any conventional media or compound is incompatible with anti-DLL3 antibody, its use in composition is considered.Additional active compounds can also be incorporated into composition. The pharmaceutical compositions of the present technology are formulated to be compatible with their intended administration route. The anti-DLL3 antibody compositions of the present technology can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally, or intramuscularly, or as an inhalant. The anti-DLL3 antibody can also be administered in combination with other substances that are at least partially effective in treating various DLL3-related cancers.

[0175] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: sterile diluents such as water for injection, saline solution, hydrogenated oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetic acid, citric acid, or phosphate; and compounds for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0176] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is desirable to include isotonic compounds in the composition, for example, sugars, polyalcohols such as mannitol and sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be brought about by including in the composition a compound that delays absorption, for example, aluminum monostearate and gelatin.

[0177] Sterile injectable solutions can be prepared by incorporating the anti-DLL3 antibody of the present technology in the required amount in a suitable solvent with one or a combination of the above-listed components as needed, followed by filtration sterilization.Generally, dispersions are prepared by incorporating the anti-DLL3 antibody into a sterile vehicle containing a basic dispersion medium and other components required from the above-listed components.For sterile powders for preparing sterile injectable solutions, the method of preparation is vacuum drying and lyophilization, which obtains a powder of the active ingredient and any additional desired ingredients from the solution that has been previously sterile-filtered.The antibody of the present technology can be administered in the form of depot injection or indwelling preparation, which can be formulated in a way that allows the active ingredient to be released in a sustained or pulsatile manner.

[0178] Oral compositions generally contain an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, anti-DLL3 antibodies may be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash; the compound in the fluid carrier is applied orally, swished, and expectorated or swallowed. Pharmaceutically compatible binding compounds and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating compound such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin, or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring.

[0179] For administration by inhalation, the anti-DLL3 antibodies are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer. Systemic administration can be via transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, anti-DLL3 antibodies are formulated into ointments, salves, gels or creams as are generally known in the art. Anti-DLL3 antibodies can also be prepared as pharmaceutical compositions in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0180] In one embodiment, anti-DLL3 antibodies are formulated with carriers that protect the anti-DLL3 antibodies from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0181] C. Kit The present technology provides a kit for detecting and / or treating DLL3-associated cancer, comprising at least one immunoglobulin-related composition (e.g., any antibody or antigen-binding fragment described herein) or a functional variant (e.g., a substitution variant) thereof. The above-mentioned components of the kit of the present technology may be packaged in a suitable container and labeled for the diagnosis and / or treatment of DLL3-associated cancer. The above-mentioned components may be stored in unit or multi-dose containers, such as sealed ampoules, vials, bottles, syringes, and test tubes, as aqueous, preferably sterile solutions or as lyophilized, preferably sterile, formulations for reconstitution. The kit may further comprise a second container holding a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients of the pharmaceutical composition and saline solution. Furthermore, the kit may include instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The kits may further include more containers containing pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. The kits may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and culture media for one or more suitable hosts. The kits may also include instructions customarily included in commercially available packaging of therapeutic or diagnostic agents, containing information regarding, e.g., the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings regarding the use of such therapeutic or diagnostic agent.

[0182] The kit is useful for detecting the presence of immunoreactive DLL3 in any biological sample, including, but not limited to, serum, plasma, lymph, cyst fluid, urine, stool, cerebrospinal fluid, ascites, or blood, and in any bodily fluid, including a biopsy sample of bodily tissue. For example, the kit may include one or more humanized, chimeric, or bispecific anti-DLL3 antibodies (or antigen-binding fragments thereof) of the present technology capable of binding to DLL3 in a biological sample, a means for determining the amount of DLL3 in the sample, and a means for comparing the amount of immunoreactive DLL3 in the sample with a standard. One or more of the anti-DLL3 antibodies may be labeled. The kit components (e.g., reagents) may be packaged in suitable containers. The kit may further include instructions for using the kit to detect immunoreactive DLL3.

[0183] For antibody-based kits, the kits can include, for example, 1) a first antibody, e.g., a humanized, chimeric, or bispecific anti-DLL3 antibody (or antigen-binding fragment thereof) of the present technology attached to a solid support that binds to a DLL3 polypeptide, and, optionally, 2) a second, different antibody that binds to either the DLL3 polypeptide or the first antibody and is conjugated to a detectable label.

[0184] The kit may also include, for example, a buffer, a preservative, or a protein stabilizer. The kit may further include components necessary for detecting the detectable label, such as an enzyme or a substrate. The kit may also include a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit may be enclosed in an individual container, and all of the various containers may be in a single package along with instructions for interpreting the results of the assay performed using the kit. The kit of the present technology may include a written product on or in the kit container. The written product describes how to use the reagents contained in the kit for treating DLL3-associated cancer in a subject in need thereof, for example, for detecting DLL3 polypeptide in vitro or in vivo. In certain embodiments, the use of the reagents may follow the methods of the present technology. [Example]

[0185] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way. The following examples demonstrate the preparation, characterization, and use of exemplary anti-DLL3 antibodies of the present technology.

[0186] Example 1 Monoclonal antibody production The extracellular domain (ECD) of DLL3 corresponding to amino acids Ala27-Ala479, with a C-terminal 6xHis tag (GenBank accession number Q9NYJ7-1), produced in HEK293T cells stably expressing full-length DLL3, was used as the immunogen. Ablexis AlivaMAb Kappa mice (Ablexis, San Diego, CA) carrying a human immunoglobulin repertoire were immunized for three weeks with either soluble DLL3-ECD or stable cells according to standard immunization techniques. Spleen cells and draining lymph node cells from mice with high serum titers specific for DLL3 were harvested and fused with mouse myeloma cells to generate hybridomas using electrofusion. These hybridomas were then screened for the presence of antibodies specifically binding to soluble DLL3-ECD by ELISA, and full-length DLL3 protein on stably expressing 293T cells was confirmed by flow cytometry compared to parental 293 cells. Hybridomas were selected for further investigation by flow cytometric ranking of staining intensity on 293 DLL3 transfectants along with 4°C / 37°C staining as described below.

[0187] Example 2 4 / 37 internalization assay using monoclonal antibodies 6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A Four monoclonal antibodies (6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A) were compared for their ability to internalize DLL3 by staining at 4°C versus 37°C. The reference monoclonal antibody SC16, known to internalize via DLL3 and possessing ADC activity and previously reported in the literature, was used as a positive control for internalization. Exponentially growing NCI-H82 cells were harvested with trypsin / EDTA, washed once with RPMI containing 10% fetal calf serum (FCS), and then cultured at 2 × 10 in DMEM supplemented with 10% FCS. 7 Resuspended at 100 μl (2 × 10 6Cells) were added to a U-bottom 96-well plate. Test monoclonal antibodies (6-G23-F, 2-C8-A, 7-I1-B, or 10-O18-A) or reference monoclonal antibodies were added to separate wells of two plates at a final concentration of 10 μg / ml. Both plates were incubated at 4°C for 30 minutes, then washed twice with chilled RPMI supplemented with 10% FCS and resuspended in RPMI supplemented with 10% FCS. One plate was kept at 4°C (control plate), while the other plate was incubated at 37°C in a CO2 incubator (experimental plate). After 4 hours of incubation in a CO2 incubator at 37°C and the control plate at 4°C, the cells were washed three times with chilled wash buffer (PBS containing 0.5% BSA) at 4°C. Samples were then resuspended in cold wash buffer plus R-Phycoerythrin-AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG (Jackson 115-116-071) at a final concentration of 7 μg / ml in wash buffer. After a 30-minute incubation at 4°C, cells were washed three times with cold wash buffer, either fixed with 0.5% paraformaldehyde in PBS, and analyzed by flow cytometry within 48 hours. The mean fluorescence intensity (MFI) ratio was calculated by dividing the MFI obtained from the control plate (incubated with antibody at 4°C) by the corresponding MFI obtained from the experimental plate (incubated with antibody at 37°C), providing a relative measure of internalization. Higher values ​​indicate greater internalization. As shown in the table below, all monoclonal antibodies (6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A) were able to internalize DLL3 upon binding, although not to the same extent as the reference monoclonal antibodies.

[0188] [Table 3]

[0189] These results demonstrate that the immunoglobulin-related compositions of the present technology undergo internalization via binding to DLL3. Thus, the immunoglobulin-related compositions disclosed herein are useful for delivering therapeutic agents to DLL3-positive cancer cells. Example 3 Quenching Internalization Assay for Monoclonal Antibodies 6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A

[0190] To rank monoclonal antibodies for internalization, we used a quenching internalization assay. This method reflects internalization and entry into the endosomal / lysosomal pathway. Goat anti-mouse IgG1 F(ab) (Jackson Immunoresearch 115-007-185) was double-labeled with Dy light Dy650 NHS ester (Thermofisher 02206) and LICOR IRDye QC1 NHS ester (LICOR 929-7030) (this double-labeled antibody is referred to herein as "F(ab) Dy650-QC1"). The principle of this assay is as follows: F(ab) Dy650-QC1 does not fluoresce because the fluorescence of Dy light Dy650 is quenched by IRDye QC1. However, upon internalization, F(ab)Dy650-QC1 is degraded in the endosomal / lysosomal pathway, resulting in the release of IRDye QC1 and the observable fluorescence of Dy light Dy650. Therefore, the fluorescence signal of Dy light Dy650 was used as a measure of internalization via lysosomes. Briefly, exponentially growing NCI-H82 cells were harvested with trypsin / EDTA, washed once with growth medium RPMI supplemented with 10% FCS, resuspended in growth medium, and incubated at 1.25 × 10 6Cells (80 μl) were added per well. A monoclonal DLL3 antibody at a concentration of 200 μg / ml was mixed with 200 μg / ml goat anti-mouse IgG1 Dy650 QC1 at room temperature for 20 minutes, and 20 μl of the mixture was added to the cells. After 30 minutes of incubation at 4°C, the cells were washed twice with growth medium, resuspended in growth medium, and transferred to a 37°C CO2 incubator for 4 hours to allow for internalization. The cells were then washed twice with ice-cold PBS containing 0.5% BSA and analyzed by flow cytometry to determine the mean fluorescence intensity. The mean fluorescence intensity of the control reference monoclonal was set to 100% internalization. As shown in the table below, all four monoclonal antibodies were demonstrated to be internalized and enter the endosomal / lysosomal pathway. [Table 4]

[0191] These results demonstrate that the immunoglobulin-related compositions of the present technology are internalized through binding to DLL3 and enter the phagosome / lysosome compartment of cell.Therefore, the immunoglobulin-related compositions disclosed herein are useful for delivering therapeutic agents to DLL3 positive cancer cells.

[0192] Example 4 Fab ZAP assay against a panel of anti-DLL3 monoclonal antibodies As an alternative method for measuring internalization, we used the Fab ZAP assay. The Fab ZAP assay measures the delivery of toxins into cells via the internalization of anti-DLL3 monoclonal antibodies. The Fab ZAP assay uses F(ab) anti-mouse heavy and light chains conjugated with saporin toxin to tag monoclonal antibodies. A panel of anti-DLL3 monoclonal antibodies was characterized using a kit from Advanced Targeting Systems, following the Fab ZAP assay protocol. Briefly, exponentially growing NCI-H82 cells were harvested with trypsin / EDTA, washed once with RPMI supplemented with 10% FCS, and plated at 5000 cells / well in 96-well white solid plates in 100 μl of RPMI supplemented with 10% FCS. The next day, 25 μl of purified monoclonal antibody (G23-F, 2-C8-A, 7-I1-B, or 10-O18-A) or reference monoclonal antibody was added at a starting concentration of 10 μg / ml and serially diluted 3-fold. Saponin-conjugated F(ab) anti-mouse Ig HL (Fab ZAP) was added to the plate at a final concentration of 4.4 nM. After 3–4 days, an equal volume of Cell Titre Glow (Promega G7571) was added to the plate, followed by 2 minutes of shaking on an orbital shaker. Luminescence was then read using a plate reader after 10 minutes at room temperature. To rule out prozone effects, all monoclonal antibodies were tested at full dose titration. As shown in Figure 1 and the table below, all monoclonal antibodies demonstrated cytotoxic activity comparable to that of the reference monoclonal antibody. Furthermore, in other experiments using these monoclonal antibodies, a mouse IgG1 control monoclonal antibody showed no cytotoxic activity. Thus, these results demonstrate that the cytotoxic activity is mediated through recognition of DLL3 and not through FcR.

[0193] [Table 5] These results demonstrate that the immunoglobulin-related compositions of the present technology can deliver therapeutic agents to tumors that express DLL3 on the cell surface.Therefore, the immunoglobulin-related compositions disclosed herein are useful for delivering therapeutic agents to DLL3-positive cancer cells.

[0194] Example 5 Epitope binning of a panel of anti-DLL3 monoclonal antibodies The purified anti-DLL3 monoclonal antibody panel and a reference monoclonal antibody were subjected to pairwise epitope binning on a Carterra® array surface plasmon resonance (SPR) assay platform (Carterra® Inc., Salt Lake City, UT). Each monoclonal antibody was tested for capture of histidine-tagged DLL3 antigen (DLL3-His) and for competition with all other antibodies in the panel for binding to DLL3-His. Antibodies were immobilized to an HC200M chip (ligand) using standard amine coupling technology via a printed array method. In each cycle, the entire array was then injected with antigen, followed by a single antibody (analyte). At the end of each cycle, the surface was regenerated to remove the antigen and analyte before a new cycle began. As shown in the table below, three distinct bins were identified in this panel, with 7-I1-B and 2-C8-A mapping to bin 2, 6-G23-F to bin 3, and 10-O18-A to bin 1.

[0195] [Table 6]

[0196] These results demonstrate that the immunoglobulin-related compositions of the present technology bind to three distinct epitopes present in DLL3 protein.Therefore, the immunoglobulin-related compositions disclosed herein can be used in combination with each other to deliver multiple therapeutic agents to tumor cells that express DLL3.

[0197] Example 6 Affinity measurement The binding affinities of four monoclonal antibodies (6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A) were determined by biolayer interferometry (BLI) using an Octet HTX instrument with 0.1% BSA, 0.02% Tween 20 in PBS as the binding buffer and 10 mM glycine, pH 1.7, as the regeneration buffer at 25°C. The four purified monoclonal antibodies (5 μg / mL each) were loaded onto an anti-mouse Fc sensor. The loaded sensor was immersed in a serial dilution of Recombinant Human DLL3 Protein (amino acids Ala27-Ala479, catalog number 9749-DL, R&D Systems) at a starting concentration of 200 nM, with seven 1:3 dilutions. As shown in Figures 2A–2D, binding was concentration-dependent. The dissociation constants (K D ) was calculated using a monovalent (1:1) binding model. As shown in Figure 2E, all monoclonal antibodies had affinities in the subnanomolar range. Figure 7 shows that monoclonal antibodies (mAbs) 6-G23-F, 10-O18-A, and 2-C8-A selectively bind to DLL3 but not to DLL1 or DLL4. 7-I1-B mAb binds to both DLL3 and DLL4 but not to DLL1. These results demonstrate that the immunoglobulin-related compositions of the present technology specifically bind to DLL3 with high affinity, and are therefore useful in methods for detecting DLL3 protein in biological samples.

[0198] Example 7 Binding of monoclonal antibodies to transfected and primary cells Four monoclonal antibodies (6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A) were examined for their ability to bind to mouse and cynomolgus monkey DLL3, as well as endogenous human DLL3, by flow cytometry. To this end, HEK293 cells were transfected with plasmid DNA encoding full-length human, mouse, or cynomolgus monkey DLL3 and used in the experiments. Briefly, 10 6 Transfected HEK293 cells or NCI-H82 primary cells were added to wells of a 96-well U-bottom plate in FACS buffer (0.5% BSA in PBS) and purified monoclonal antibodies were added at 10 μg / ml. After 30 min of incubation at 4°C, the cells were washed three times with FACS buffer and incubated with PE-labeled F(ab)2 anti-mouse IgG H and L second stage antibodies. After a further 30 min of incubation at 4°C, the cells were washed three times with FACS buffer and analyzed by flow cytometry. Data are presented as the ratio of the mean fluorescence intensity of the monoclonal antibody divided by the background staining in the second stage. As shown in the table below, all monoclonal antibodies cross-reacted with cynomolgus monkey DLL3 and detected endogenous DLL3 on NCI-H82 cells, but only 6-G23-F and 7-I1-B bound to mouse DLL3.

[0199] [Table 7] These results demonstrate that the immunoglobulin-related compositions of the present technology are useful in methods for detecting DLL3 protein in biological samples.

[0200] Example 8 Sequencing The variable heavy and light chains of four monoclonal antibodies were isolated by rapid amplification of cDNA ends (RACE) from the corresponding hybridomas, 7-I1-B, 6-G23-F, 2-C8-A, and 10-O18-A. RNA was prepared from lysed hybridomas using the RNAEasy kit (Qiagen). This mRNA was isolated for cDNA synthesis, and PCR products were generated using the RACE kit. The PCR products were then cloned into the TOPO vector, PCR amplified, gel separated, and sequenced. The heavy chain variable domain (V H ) and the light chain variable domain (V L The nucleotide and amino acid sequences of ) are shown in the table below and in Figures 3A-3D (7-I1-B), 4A-4D (2-C8-A), 5A-5D (10-O18-A), and 6A-6D (6-G23-F).

[0201] [Table 8] TIFF2025156278000011.tif229169 TIFF2025156278000012.tif219169 TIFF2025156278000013.tif224169

[0202] Example 9 Treatment of NCI-H209 xenografts Eight-week-old female athymic nude mice were used for this study. NCI-H209 tumor cells were grown to mid-logarithmic phase in RPMI containing 10% fetal calf serum (FCS). Cells were cultured in tissue culture flasks in a humidified incubator at 37°C in a 5% CO2 and 95% air atmosphere. Mice were inoculated with 5x10 cells in 50% Matrigel into the right flank. 6 NCI-H209 tumor cells are implanted subcutaneously in a total volume of 0.2 ml. Eight days after inoculation, tumors reach a mean tumor volume of approximately 150 mm. 3At the time of tumor growth, mice are randomly assigned to groups. On days 8, 12, and 15, mice are intraperitoneally injected with either human IgG1 (1 or 10 mg / kg; n = 5–15 mice) as a negative control, or one or more of the antibody-drug conjugates 6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A (1 or 10 mg / kg; n = 5–15 mice). Tumor growth is measured.

[0203] Antibody-drug conjugates of 6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A were prepared using methods known in the literature. Some methods are disclosed in Examples 3-4 herein. One or more protein toxins (e.g., saporin toxin, Pseudomonas exotoxin, diphtheria toxin), radionuclides, anthracyclines, microtubule inhibitors, mitotic inhibitors, DNA damaging agents, nucleotide analogs, amino acid analogs, and vitamin analogs were conjugated to 6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A to generate 6-G23-F-ADC, 2-C8-A-ADC, 7-I1-B-ADC, and 10-O18-A-ADC, respectively. Eight-week-old female athymic nude mice were used for this study. NCI-H209 tumor cells are grown to mid-logarithmic phase in RPMI containing 10% fetal calf serum (FCS). Cells are cultured in tissue culture flasks in a humidified incubator at 37°C in a 5% CO2 and 95% air atmosphere. Mice are seeded with 5x10 cells in 50% Matrigel on the right flank. 6 NCI-H209 tumor cells are implanted subcutaneously in a total volume of 0.2 ml. Eight days after inoculation, tumors reach a mean tumor volume of approximately 150 mm. 3 At the time of tumor growth, randomly divide the mice into groups. On days 8, 12, and 15, inject the mice intraperitoneally with either human IgG1-ADC (0.11 or 1 mg / kg; n = 5–15 mice) as a negative control, or one or more of 6-G23-F-ADC, 2-C8-A-ADC, 7-I1-B-ADC, or 10-O18-A-ADC (0.1 or 1 mg / kg; n = 5–15 mice). Measure tumor growth.

[0204] These results would demonstrate that the immunoglobulin-related compositions of the present technology are useful in methods of treating subjects suffering from DLL3-associated cancers (e.g., small cell lung cancer, large cell neuroendocrine carcinoma, pulmonary neuroendocrine carcinoma, extrapulmonary neuroendocrine carcinoma, or melanoma). equivalent The present technology should not be limited by the specific embodiments described in this application, which are intended as single illustrations of individual aspects of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and apparatuses within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the present technology. It is to be understood that the present technology is not limited to particular methods, reagents, compound compositions, or biological systems, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0205] Furthermore, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure also is described in terms of any individual members or subgroups of members of the Markush group. As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of subranges. Recited ranges can be readily recognized as being fully descriptive and valid, with the same range broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. Also, as will be understood by those skilled in the art, all language, such as "up to," "at least," "greater than," and "less than," refers to a range that is inclusive of the recited numbers and can subsequently be broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0206] All patents, patent applications, provisional applications, and publications referenced or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the explicit teachings of this specification.

Claims

1. Heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L and wherein: (a) V H teeth, (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (vi) SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively; V selected from the group consisting of H - CDR1 sequence, V H - CDR2 sequence, and V H - comprising a CDR3 sequence, and / or (b) V L teeth, (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. V selected from the group consisting of L - CDR1 sequence, V L - CDR2 sequence, and V L - an antibody or antigen-binding fragment thereof, comprising a CDR3 sequence.

2. Heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L and wherein: (a) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, and SEQ ID NO:32; and / or (b) V L An antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, and SEQ ID NO:

37.

3. V H Amino acid sequence and V L The amino acid sequence is SEQ ID NO:2 and SEQ ID NO:7 (7-I1-B), respectively; SEQ ID NO:12 and SEQ ID NO:17 (2-C8-A), respectively; SEQ ID NO:22 and SEQ ID NO:27 (10-O18-A), respectively; SEQ ID NO:32 and SEQ ID NO:37 (6-G23-F), respectively; 3. The antibody or antigen-binding fragment of claim 2, selected from the group consisting of:

4. (a) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of any one of SEQ ID NOs: 7, 17, 27, or 37; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of any one of SEQ ID NOs: 2, 12, 22, or 32. An antibody or antigen-binding fragment thereof comprising:

5. 5. The antibody or antigen-binding fragment of any one of claims 1 to 4, further comprising an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE.

6. Antigen-binding fragments include Fab, F(ab') 2 5. The antibody or antigen-binding fragment of any one of claims 1 to 4, which is selected from the group consisting of Fab', scFv and Fv.

7. The antibody according to any one of claims 1 to 5, which is a monoclonal antibody, a chimeric antibody, a humanized antibody or a bispecific antibody.

8. The antibody or antigen-binding fragment of any one of claims 1 to 7, which binds to an epitope present in a mammalian DLL3 polypeptide.

9. 9. The antibody or antigen-binding fragment of claim 8, wherein the epitope is a conformational or non-conformational epitope.

10. 10. The antibody or antigen-binding fragment of claim 8 or 9, wherein the mammalian DLL3 polypeptide has an amino acid sequence comprising amino acid residues 27 to 492 of SEQ ID NO:50 or SEQ ID NO:

51.

11. 11. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 10 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to a substance selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

12. A recombinant nucleic acid sequence encoding the antibody or antigen-binding fragment of any one of claims 1 to 10.

13. A recombinant nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31 and 36.

14. A host cell or vector comprising a recombinant nucleic acid sequence according to claim 12 or claim 13.

15. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 10 and instructions for use.

16. 16. The kit of claim 15, wherein the antibody or antigen-binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label.

17. 11. A method of treating DLL3-associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment is conjugated to at least one additional therapeutic agent.

18. 18. The method of claim 17, wherein the at least one additional therapeutic agent is selected from the group consisting of an isotope, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

19. 19. The method of claim 17 or 18, wherein the DLL3-associated cancer is small cell lung cancer, large cell neuroendocrine carcinoma, pulmonary neuroendocrine carcinoma, extrapulmonary neuroendocrine carcinoma, or melanoma.

20. 1. A method for detecting DLL3 protein levels in a biological sample, comprising: contacting a biological sample with the antibody or antigen-binding fragment of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment is conjugated to a detectable label; detecting a signal produced by the detectable label in the biological sample; A method comprising:

Images