Anti-DLL3 antibody and its use

Abstract

To provide an anti-DLL3 antibody and its use. [Solution] Anti-DLL3 antibodies are provided, including mouse antibodies, humanized antibodies, and those having a further optimized CDR sequence. These antibodies have superior binding specificity and improved activity compared to other anti-DLL3 antibodies under development. In various embodiments, the disclosure provides antibodies and antigen-binding fragments specific to the human DLL3 protein. Experimental testing demonstrates that these newly identified antibodies can bind strongly and specifically to the human DLL3 protein without interacting with DLL1 and DLL4 variants. These antibodies also cross-react with cynomolgus monkey DLL3 protein, facilitating preclinical testing.

Description

[Background technology]

[0001] background Lung cancer is the leading cause of cancer death worldwide, with small cell lung cancer (SCLC) accounting for approximately 15% of those cases. Unlike other cancer types, SCLC is highly invasive and can metastasize early. Clinical outcomes include frequent SCLC recurrence and drug resistance. In addition, overall survival (OS) for SCLC is not high due to the lack of targeted therapies and challenges in early detection. Immunotherapy, such as chemotherapy combined with a PD-L1 monoclonal antibody (durvalumab), is available for first-line treatment of advanced small cell lung cancer (ES-SCLC), but OS has not significantly improved. Therefore, there is an urgent need for improved treatments for SCLC.

[0002] Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand in the Notch ligand family. In contrast to other members of the family such as DLL1 and DLL4, the human DLL3 protein has a distinct structure, consisting of one delta / serate / LAG-2 (DSL) domain, six epidermal growth factor (EGF)-like repeats, a 21-amino acid (aa) transmembrane domain (TM), and a 105aa intracellular domain (ICD).

[0003] DLL3 is highly expressed on the cell surface in SCLC tumors and other tumor types of neuroendocrine origin, including glioblastoma multiforme (GBM), large cell neuroendocrine lung tumor (LCNEC), metastatic melanoma, small cell bladder cancer (SCBC), and neuroendocrine prostate cancer (NEPC). The DLL3 protein is expressed by over 80% of all SCLC tumors with a high degree of homogeneity across neonatal cells. In contrast, minimal expression is observed in normal tissues (e.g., neurons, islet cells, and pituitary cells), and this is exclusively cytoplasmic. In preclinical models, DLL3 expression promotes SCLC cell migration and invasion through mechanisms involved in the regulation of the epithelial-mesenchymal transition protein Snail.

[0004] The distinct expression patterns of DLL3 in SCLC and other neuroendocrine tumors have enabled the development of therapies using DLL3 to specifically target these tumor types. Currently, multiple ongoing clinical trials in SCLC and other neuroendocrine tumors are evaluating these DLL3-specific drugs. Several types of DLL3-targeting modalities, including bispecific benzodiazepines (BsAbs), chimeric antigen receptor T-cell therapy (CAR-T), and antibody-drug conjugations (ADCs), are currently under clinical investigation.

[0005] Tallatamab (AMG757), a bispecific T-cell engager (BiTE) molecule with extended half-lives that targets DLL3 and CD3 and simultaneously induces T-cell-mediated oncolysis, demonstrated a durable response and manageable safety in patients with relapsed / refractory SCLC in the Phase I trial DeLLphi-300 (NCT03319940). The objective response rate (ORR) was 23.4% (95% CI, 15.7–32.5), including 2 complete responses and 23 partial responses. The median duration of response (DoR) was 12.3 months (95% CI, 6.6–14.9). The disease control rate (DCR) was 51.4% (95% CI, 41.5–61.2). The median progression-free survival (PFS) and overall survival (OS) were 3.7 months (95% CI, 2.1–5.4) and 13.2 months (95% CI, 10.5–not reached), respectively.

[0006] Other DLL3-based T-cell engagers, including BI764532 from Boehringer-Ingelheim and HPN328 from Harpoon Therapeutics, have also shown promising antitumor efficacy in their individual Phase I clinical trials. Robalpituzumab tesillin (Rova-T) is an ADC containing the DLL3-targeting antibody lovalpituzumab, linked to the DNA crosslinking agent pyrrolobenzodiazepine (PDB) by a protease-cleavable linker. In the Phase 3 TAHOE trial comparing Rova-T to topotecan as a second-line therapy in DLL3-positive advanced or metastatic SCLC, Rova-T exhibited inferior OS and higher toxicity rates. AMG 119, the first CAR-T cell therapy for SCLC, is currently suspended from enrollment, but in its Phase I trial, it was associated with a manageable safety profile and promising antitumor activity in five adult subjects. LB2102, an autologous CAR-T cell therapy, was recently approved by the FDA for Phase I clinical development for the treatment of adult patients with ES-SCLC. Overall, the exploration of different types of drugs that target DLL3 would hopefully lead to more options for SCLC treatment. [Overview of the project] [Means for solving the problem]

[0007] overview In various embodiments, this disclosure provides antibodies and antigen-binding fragments specific to the human DLL3 protein. Experimental testing demonstrates that these newly identified antibodies can bind strongly and specifically to the human DLL3 protein without interacting with DLL1 and DLL4 variants. These antibodies also cross-react with cynomolgus monkey DLL3 protein, facilitating preclinical testing.

[0008] One embodiment of the present disclosure is an antibody or antigen-binding fragment having specificity for human delta-like ligand 3 (DLL3) protein and comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3, wherein (a) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 111-117; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 40; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 42; or (b) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL (c) The CDR3 contains the amino acid sequences of SEQ ID NOs. 13-18; (d) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 19-24; (e) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 25-30; (e) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 31-36; or (f) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 43-48

[0009] In some embodiments, (a) VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 111-117; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 40; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 42.

[0010] In some embodiments, VH includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 65-69 and 104-110, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs. 65-69 and 104-110. In some embodiments, VL includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 70-73, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs. 70-73. In some embodiments, VH includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 104-110, and VL includes the amino acid sequence of SEQ ID NO. 73.

[0011] In some embodiments, (b)VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 13 to 18.

[0012] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% sequence identity to SEQ ID NO: 1, and VL comprises the amino acid sequence of 2, or a peptide having at least 90% sequence identity to SEQ ID NO: 2.

[0013] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 49 and a light chain containing the amino acid sequence of SEQ ID NO: 50.

[0014] In some embodiments, (c)VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 19 to 24.

[0015] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% sequence identity to SEQ ID NO: 3, and VL comprises the amino acid sequence of 4, or a peptide having at least 90% sequence identity to SEQ ID NO: 4.

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 51 and a light chain containing the amino acid sequence of SEQ ID NO: 52.

[0017] In some embodiments, (d)VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 25-30.

[0018] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 5, or a peptide having at least 90% sequence identity to SEQ ID NO: 5, and VL comprises the amino acid sequence of 6, or a peptide having at least 90% sequence identity to SEQ ID NO: 6.

[0019] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 53 and a light chain containing the amino acid sequence of SEQ ID NO: 54.

[0020] In some embodiments, (e)VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 31 to 36.

[0021] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 7, or a peptide having at least 90% sequence identity to SEQ ID NO: 7, and VL comprises the amino acid sequence of SEQ ID NO: 8, or a peptide having at least 90% sequence identity to SEQ ID NO: 8.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 55, and a light chain comprising the amino acid sequence of SEQ ID NO: 56.

[0023] In some embodiments, (f) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each comprise the amino acid sequences of SEQ ID NOs: 43 to 48, respectively.

[0024] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 11, or a peptide having at least 90% sequence identity to SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 12, or a peptide having at least 90% sequence identity to SEQ ID NO: 12.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a light chain comprising the amino acid sequence of SEQ ID NO: 60.

[0026] In one embodiment, also provided is an antibody or antigen-binding fragment thereof that has specificity for human delta-like ligand 3 (DLL3) protein and competes with the antibody or fragment thereof of the present disclosure for binding to DLL3 protein.

[0027] In one embodiment, also provided is an antibody or antigen-binding fragment thereof that has specificity for human delta-like ligand 3 (DLL3) protein and binds to the EGF3-4 domain or the EGF6 domain.

[0028] Multispecific antibodies comprising the antigen-binding fragment of the present disclosure and one or more antibodies or antigen-binding fragments having binding specificity for a target antigen other than DLL3 are also provided.

[0029] Yet another embodiment provides a chimeric antigen receptor (CAR) comprising the antigen-binding fragment of the present disclosure, a transmembrane domain, a co-stimulatory domain, and a CD3ζ intracellular domain.

[0030] Methods and uses for treating cancer are also provided. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, prostate cancer, urinary tract cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer. In some embodiments, the cancer is small cell lung cancer (SCLC). [Brief explanation of the drawing]

[0031] [Figure 1] Figures 1A-1C show ELISA binding of anti-DLL3 antibodies to human / cynomolgus monkey / mouse DLL3 proteins with his tag.

[0032] [Figure 2] Figures 2A and 2B show ELISA conjugation of anti-DLL3 antibodies to human paralogs DLL1 and DLL4.

[0033] [Figure 3] Figure 3 shows the epitope domain mapping results for the anti-DLL3 antibody.

[0034] [Figure 4] Figures 4A and 4B show cell-based binding of anti-DLL3 antibodies to human or cynomolgus monkey HEK293 cell lines expressing DLL3.

[0035] [Figure 5] Figures 5A and 5B show cell-based binding of anti-DLL3 antibodies to tumor cell lines, including SHP77 and NCI-H82.

[0036] [Figure 6] Figures 6A and 6B show cell-based binding of anti-DLL3 antibodies to human paralogs DLL1 and DLL4 expressed in CHO-K1 cells.

[0037] [Figure 7]Figure 7 shows the ELISA binding of an anti-DLL3 humanized antibody to the human DLL3 protein with the his tag.

[0038] [Figure 8] Figures 8A and 8B show cell-based binding of anti-DLL3 humanized antibodies to human or cynomolgus monkey DLL3-expressing HEK293 cell lines.

[0039] [Figure 9] Figures 9A and 9B show cell-based binding of anti-DLL3 humanized antibodies to tumor cell lines, including SHP77 and NCI-H82.

[0040] [Figure 10] Figures 10A and 10B show cell-based binding of anti-DLL3 humanized antibodies to human paralogs DLL1 and DLL4 expressed in CHO-K1 cells.

[0041] [Figure 11] Figures 11A and 11B show cell-based binding of humanized, PTM-removed anti-DLL3 antibodies to HEK293 and SHP77 cells expressing human DLL3. [Modes for carrying out the invention]

[0042] Detailed explanation definition It should be noted that the term "a" or "an" entity refers to one or more such entities; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0043] As used herein, “antibody” or “antigen-binding moiety” refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be a whole antibody and any antigen-binding fragment or single chain thereof. Thus, the term “antibody” includes any protein or peptide containing a molecule that includes at least a portion of an immunoglobulin molecule having biological activity for binding to an antigen. Such examples include, but are not limited to, a complementarity-determining region (CDR) of a heavy or light chain, or its ligand-binding moiety, a variable region of a heavy or light chain, a constant region of a heavy or light chain, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0044] Full-length antibodies consist of two heavy chains and two light chains. Variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains are sometimes referred to as "VH" and "VL," respectively. The variable regions in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain (LC)CDRs including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC)CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or specified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). Three CDRs of the heavy or light chain are inserted between adjacent stretches known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgGA1 (α1 heavy chain), or IgGA2 (α2 heavy chain).

[0045] As used herein, the term "half-antibody" refers to one immunoglobulin heavy chain associated with one immunoglobulin light chain. Those skilled in the art will readily recognize that a half-antibody may contain its fragments and may also have an antigen-binding domain consisting of a single variable domain, for example, originating from camelids.

[0046] The term "single-chain half-antibody," as used herein, refers to a single-chain polypeptide comprising a VL domain, optionally a CL domain, a tether, a VH domain, optionally a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the domains are located relative to each other in the direction from the N-terminus to the C-terminus, as follows: VL-tether-VH-hinge-CH2-CH3, VL-tether-VH-partial-hinge-CH2-CH3, VL-tether-VH-hinge variant-CH2-CH3, or VL-CL-tether-VH-CH1-hinge-CH2-CH3.

[0047] The terms "single-domain antibody" (sdAb) or "single-variable-domain (SVD) antibody" generally refer to antibodies in which a single variable domain (VH or VL) can confer antigen binding. In other words, a single variable domain does not require interaction with another variable domain to recognize the target antigen. Examples of single-domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks), as well as those derived from recombinant methods using human and mouse antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO2005 / 035572; WO03 / 035694; Febs Lett (1994) 339:285-290; WO00 / 29004; WO02 / 051870). If the sdAb contains only the heavy chain, it can be used interchangeably with "VHH" or "single heavy chain variable domain antibody" or "nanobody".

[0048] The terms “antibody fragment” or “antigen-binding fragment,” as used herein, refer to portions of an antibody, such as F(ab')2, F(ab)2,Fab', Fab, Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen recognized by an intact antibody. The term “antibody fragment” includes aptamers, Spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen and forming a complex.

[0049] In relation to antibodies, "Fab" refers to a monovalent antigen-binding fragment of an antibody consisting of a single light chain (both variable and constant regions) attached to the variable and first constant regions of a single heavy chain by disulfide bonds. Fab can be obtained by papain digestion of the antibody at a residue proximal to the N-terminus of the disulfide bond between the heavy chains in the hinge region.

[0050] "Fab'" refers to a Fab fragment that includes a portion of the hinge region, which can be obtained by pepsin digestion of the antibody at a residue proximal to the C-terminus of the disulfide bond between the heavy chains of the hinge region, thus distinguishing it from Fab by a few residues (including one or more cysteines) in the hinge region.

[0051] "F(ab)2" refers to a dimer of Fab' that contains two light chains and two heavy chain segments.

[0052] "Single-stranded variable fragment" or "scFv" refers to the heavy chain (V) of immunoglobulins. H ) and light chain (V L This refers to a fusion protein of the variable region of ). In some embodiments, the region is connected by a short linker peptide of 10 to about 25 amino acids. The linker may be rich in glycine for flexibility, and serine or threonine for solubility, and V H The N-terminus of V LIt can be attached to the C-terminus of or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. The scFv molecule is known in the art and is described, for example, in U.S. Patent No. 5,892,019.

[0053] The term antibody encompasses a wide range of polypeptide classes that can be biochemically distinguished. Those skilled in the art will recognize that the heavy chain is classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) and several subclasses within them (e.g., γ1-γ4). This is a characteristic of the chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and known to confer functional identification. Modified versions of each of these classes and isotypes are readily recognizable to those skilled in the art from the perspective of this disclosure and are therefore within the scope of this disclosure. All immunoglobulin classes are explicitly within the scope of this disclosure, and the following discussion generally focuses on the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule contains two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons, and two identical heavy-chain polypeptides with a molecular weight of 53,000–70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, where the light chains begin at the mouth of the "Y" and flank the heavy chains that continue through a variable region.

[0054] The antibodies, their antigen-binding moieties, variants, or derivatives of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primate-like antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv(scFv), single-chain antibodies, disulfide-linked Fv(sdFv), fragments containing any of the VK or VH domains, fragments produced by Fab expression libraries, and anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulins or antibody molecules of the present disclosure may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass of immunoglobulin molecules.

[0055] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can bind to either a kappa or lambda light chain. Generally, light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide linkage or non-covalent linkage when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell. In heavy chains, the amino acid sequence extends from the N-terminus of the Y-configuration junction to the C-terminus at the bottom of each chain.

[0056] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is recognized that the variable domains of both the light (VK) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CK) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, transplacental transport, Fc receptor binding, and complement binding. In the conventional numbering of constant region domains, they increase as they move away from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0057] As shown above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VK and VH domains of the antibody, or subsets of the complementarity-determining regions (CDRs), combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on the VH and VK chains, respectively (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some examples, for instance, certain immunoglobulin molecules may originate from or be engineered based on camelid immunoglobulins, and a complete immunoglobulin molecule may consist only of a heavy chain, without a light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0058] In naturally occurring antibodies, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, discontinuous sequences of amino acids specifically positioned to form the antigen-binding domain, assuming the antibody's three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, referred to as the "framework" region, exhibit low intermolecular variability. The framework region primarily employs a β-sheet conformation, while the CDRs connect the β-sheet structure, forming loops that, in some cases, form parts of it. Thus, the framework region acts to form a scaffold that provides the correct orientation of the CDRs through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines surface complementarity to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its homologous epitopes. The amino acids constituting the CDRs and framework regions, respectively, can be readily identified for any given heavy or light chain variable region by those skilled in the art, as they are precisely defined. (See "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987).)

[0059] Where there are two or more definitions of a term used and / or permitted in the art, the definition of the term, when used herein, is intended to include all such meanings unless the opposite is expressly stated. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe discontinuous antigen combination sites found within the variable regions of both heavy-chain and light-chain polypeptides. This particular region is described by Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J. MoI. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entirety. The definitions of CDR by Kabat and Chothia include duplication or subsets of amino acid residues when compared to one another. Nevertheless, the application of either definition to refer to the CDR of an antibody or its variant is intended to be within the scope of the terms defined and used herein. For comparison, the appropriate amino acid residues that comprise the CDR as defined by each of the references cited above are shown in the table below. The exact residue numbers that comprise a particular CDR vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR that gives rise to the variable region amino acid sequence of an antibody. [Table 11]

[0060] Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. Those skilled in the art can uniquely assign this “Kabat numbering” system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system presented by Kabat et al., US Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0061] In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), contains approximately 5-7 amino acids, and ends with the following tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, contains approximately 16-19 amino acids, and ends with the following arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2, contains 3-25 amino acids, and ends with the sequence WGXG (where X is any amino acid). CDR-L1 begins at approximately residue 24 (i.e., after the cysteine ​​residue), contains approximately 10-17 residues, and ends with the following tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1, contains approximately 7 residues. CDR-L3 begins approximately 33 residues after the terminal end of CDR-L2 (i.e., after the cysteine ​​residue), contains approximately 7-11 residues, and ends with the sequence F or WGXG (where X is any amino acid).

[0062] The antibodies disclosed herein may be of any animal origin, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be of chondricthoid origin (e.g., shark origin).

[0063] As used herein, the term “heavy chain constant region” includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge domain (e.g., upper, middle, and / or lower hinge regions), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, antigen-binding polypeptides for use in this disclosure may comprise: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this disclosure comprises a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in this disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As shown above, those skilled in the art will understand that the heavy chain constant regions may be modified so that they differ in amino acid sequences from those derived from naturally occurring immunoglobulin molecules.

[0064] The heavy chain constant region of antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may include a hinge region that is partly derived from an IgG1 molecule and partly from an IgG3 molecule. In yet another example, the heavy chain portion may include a chimeric hinge that is partly derived from an IgG1 molecule and partly from an IgG4 molecule.

[0065] As used herein, the term “light chain constant region” includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0066] A "light-heavy chain pair" refers to a set of light and heavy chains that can form a dimer via a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0067] As previously shown, the subunit structures and three-dimensional arrangements of the constant regions of various immunoglobulin classes are well known. As used herein, the term “VH domain” refers to the amino-terminal variable domain of an immunoglobulin heavy chain, and the term “CH1 domain” refers to the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal with respect to the hinge region of the immunoglobulin heavy chain molecule.

[0068] The "CH1 domain" (also known as the "C1" of the "H1" domain) typically extends from approximately 118 amino acids to approximately 215 amino acids (EU numbering system).

[0069] As used herein, the term “hinge region” refers to the portion of the heavy chain molecule that binds the CH1 domain to the CH2 domain, which corresponds to the region in IgG from Glu216 to Pro230 in human IgG1 in the EU numbering system (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the interhemolytic disulfide bond in the same position. This hinge region is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0070] As used herein, the term “CH2 domain” includes the portion of the heavy chain molecule extending, for example, from approximately residues 244 to 360 of an antibody, using the conventional numbering scheme (residues 244–360, Kabat numbering system; and residues 231–340, EU numbering system; see Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983)). The CH2 domain is unique in that it does not closely pair with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. The CH3 domain, which extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues, has also been well described.

[0071] The "CH3 domain" (also called the "C3" domain) includes a sequence of residues from the C-terminus to the CH2 domain in the Fc region (i.e., approximately amino acid residues 341 to the C-terminus of the antibody sequence, typically amino acid residues 446 or 447 of IgG, according to the EU numbering system).

[0072] The terms “Fc region,” “Fc domain,” or “fragmentary crystalline region” as used herein are used to define the C-terminal region of an immunoglobulin heavy chain and include native sequence Fc regions and variant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is typically defined as the segment from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Therefore, a composition of intact antibodies may include antibody populations with all K447 residues removed, antibody populations with and without K447 residues, and antibody populations having mixtures of antibodies with and without K447 residues. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0073] As used herein, the term “disulfide bond” includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a second thiol group and a thiol group that can form a disulfide bond or bridge with it. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system).

[0074] As used herein, the term “chimeric antibody” applies to any antibody in which an immunoreactive region or site is obtained from or derived from a first species, and a constant region (which may be intact, partially or modified according to this disclosure) is obtained from a second species. In certain embodiments, the target-binding region or site is of non-human origin (e.g., mouse or primate), and the constant region is human.

[0075] The term "humanized antibody" is used herein to describe an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences has been modified to be more "human-like," i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody, or a variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest and contains a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementation-determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR means a CDR having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, typically two, variable domains (Fab, Fab', F(ab')2, Fv), where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin (i.e., a donor antibody), and all or substantially all of the framework region corresponds to that of a human immunoglobulin consensus sequence. In some embodiments, the humanized antibody also comprises at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domains of the light chain and the heavy chain. The antibody may also contain the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only the humanized light chain. In some embodiments, the humanized antibody contains only the humanized heavy chain. In specific embodiments, the humanized antibody contains only the humanized variable domains of the light chain and / or the humanized heavy chain.

[0076] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0077] "Specifically binding" or "having specificity for" generally means that an antibody binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more easily than it would to a random, unrelated epitope. The term "specificity" is used herein to describe the relative affinity of a particular antibody to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B", or antibody "A" may be said to bind to epitope "C" with higher specificity than it has for the related epitope "D".

[0078] As used herein, the terms “to treat” or “treatment” refer to both therapeutic treatment and preventive or protective measures, the purpose of which is to prevent or slow (reduce) the progression of undesirable physiological changes or impairments, such as cancer. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in disease severity, stabilization (i.e., non-worsening) of the disease, delay or slowing of disease progression, recovery or mitigation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” may also mean extending survival compared to the expected survival without treatment. Persons requiring treatment include those who already have a condition or impairment, those who are prone to developing a condition or impairment, or those for whom a condition or impairment will be prevented.

[0079] "Subject," "individual," "animal," "patient," or "mammal" means any subject, especially a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0080] Where used herein, phrases such as “patient requiring treatment” or “subject requiring treatment” include, for example, a subject, e.g., a mammalian subject, who would benefit from the administration of an antibody or composition of this disclosure used for detection, a diagnostic procedure, and / or treatment. Anti-DLL3 antibody

[0081] As demonstrated in the attached experimental examples, the inventors have enabled the generation of anti-DLL3 antibodies 9E8D8, 36B7F3, 129H2B9, 148C3A7, 310P3C5, and 362H3D3 (Table 1), all of which exhibit high binding affinity to the human DLL3 protein. The binding is specific, as they did not bind to DLL1 or DLL4.

[0082] Domain mapping showed that some of these antibodies bind to specific DLL3 domains (Table A) that had not been previously targeted. For example, 129H2B9 and 148C3A7 bound to the EGF3-4 domains of human DLL3 expressed in cells (UniProt number Q9NYJ7, S312-E389). 9E8D8 and 310P3C5 specifically bound to the EGF6 domain of human DLL3 expressed in cells (UniProt number Q9NYJ7, R429-E465). In contrast, benchmark antibody DLL3#3 bound to the membrane-proximal extracellular domain (UniProt number Q9NYJ7, F466-L492), while another benchmark antibody, DLL3-4-001, specifically bound to EGF3. [Table A]

[0083] An antibody or an antigen-binding fragment thereof is provided according to one embodiment of the present disclosure. In some embodiments, the antibody or the antigen-binding fragment has binding specificity to the human DLL3 protein. In some embodiments, the antibody or the antigen-binding fragment comprises a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3.

[0084] In some embodiments, an antibody or antigen-binding fragment derived from antibody 310P3C5 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 38; VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 40; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 42.

[0085] In some embodiments, VH CDR2 is free from the risk of PTM. The PTM-free versions to be tested include SEQ ID NOs. 111-117 (Table 10B), where the N residue is substituted with A, F, H, R, V, W, or Y. In some embodiments, VH CDR1 includes the amino acid sequence of SEQ ID NO 37; VH CDR2 includes the amino acid sequence of SEQ ID NOs. 111, 112, 113, 114, 115, 116, or 117; VH CDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NO 39; VL CDR1 includes the amino acid sequence of SEQ ID NO 40; VL CDR2 includes the amino acid sequence of SEQ ID NO 41; and VL CDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NO 42.

[0086] In some embodiments, VH includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 65-69 and 104-110, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs. 65-69 and 104-110, while retaining a re-risked version of the VH CDR or its PTM. In some embodiments, VL includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 70-73, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs. 70-73, while retaining a re-risked version of the VL CDR or its PTM.

[0087] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 104-110, and VL comprises the amino acid sequence of SEQ ID NO: 73.

[0088] In some embodiments, antibodies and antigen-binding fragments that bind to the same epitopes on DLL3 as 310P3C5 are also provided. In some embodiments, antibodies and antigen-binding fragments that bind to the EGF6 domain of the DLL3 protein are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with 310P3C5 for binding to DLL3 are also provided.

[0089] In some embodiments, an antibody or antigen-binding fragment derived from antibody 9E8D8 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 13-18.

[0090] In some embodiments, VH includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1, while retaining the amino acid sequence of SEQ ID NO: 1, or a re-risked version of the VH CDR or its PTM. In some embodiments, VL includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2, while retaining the amino acid sequence of SEQ ID NO: 2, or a re-risked version of the VL CDR or its PTM.

[0091] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 49, and the light chain comprises the amino acid sequence of SEQ ID NO: 50.

[0092] In some embodiments, antibodies and antigen-binding fragments that bind to the same epitopes on DLL3 as 9E8D8 are also provided. In some embodiments, antibodies and antigen-binding fragments that bind to the EGF6 domain of the DLL3 protein are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with 9E8D8 for binding to DLL3 are also provided.

[0093] In some embodiments, an antibody or antigen-binding fragment derived from antibody 36B7F3 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 19-24.

[0094] In some embodiments, VH includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3, while retaining the amino acid sequence of SEQ ID NO: 3, or a re-risked version of the VH CDR or its PTM. In some embodiments, VL includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, while retaining the amino acid sequence of SEQ ID NO: 4, or a re-risked version of the VL CDR or its PTM.

[0095] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 3, and VL comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 51, and the light chain comprises the amino acid sequence of SEQ ID NO: 52.

[0096] In some embodiments, antibodies and antigen-binding fragments that bind to the same epitopes on DLL3 as 36B7F3 are also provided. In some embodiments, antibodies and antigen-binding fragments that bind to the membrane-proximal extracellular domain of the DLL3 protein are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with 36B7F3 for binding to DLL3 are also provided.

[0097] In some embodiments, antibody- or antigen-binding fragments derived from antibody 129H2B9 are provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 25-30.

[0098] In some embodiments, VH includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 5, while retaining the amino acid sequence of SEQ ID NO: 5, or a re-risked version of the VH CDR or its PTM. In some embodiments, VL includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6, while retaining the amino acid sequence of SEQ ID NO: 6, or a re-risked version of the VL CDR or its PTM.

[0099] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 5, and VL comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 53, and the light chain comprises the amino acid sequence of SEQ ID NO: 54.

[0100] In some embodiments, antibodies and antigen-binding fragments that bind to the same epitopes on DLL3 as 129H2B9 are also provided. In some embodiments, antibodies and antigen-binding fragments that bind to the EGF3-4 domains of the DLL3 protein are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with 129H2B9 for binding to DLL3 are also provided.

[0101] In some embodiments, an antibody or antigen-binding fragment derived from antibody 148C3A7 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 31-36.

[0102] In some embodiments, VH includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7, while retaining the amino acid sequence of SEQ ID NO: 7, or a re-risked version of the VH CDR or its PTM. In some embodiments, VL includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 8, while retaining the amino acid sequence of SEQ ID NO: 8, or a re-risked version of the VL CDR or its PTM.

[0103] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 7, and VL comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 55, and the light chain comprises the amino acid sequence of SEQ ID NO: 56.

[0104] In some embodiments, antibodies and antigen-binding fragments that bind to the same epitopes on DLL3 as 148C3A7 are also provided. In some embodiments, antibodies and antigen-binding fragments that bind to the EGF3-4 domains of the DLL3 protein are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with 148C3A7 for binding to DLL3 are also provided.

[0105] In some embodiments, antibody- or antigen-binding fragments derived from antibody 362H3D3 are provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 43-48.

[0106] In some embodiments, VH includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11, while retaining the amino acid sequence of SEQ ID NO: 11, or a re-risked version of the VH CDR or its PTM. In some embodiments, VL includes a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12, while retaining the amino acid sequence of SEQ ID NO: 12, or a re-risked version of the VL CDR or its PTM.

[0107] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 11, and VL comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 59, and the light chain comprises the amino acid sequence of SEQ ID NO: 60.

[0108] In some embodiments, antibodies and antigen-binding fragments that bind to the same epitopes on DLL3 as 362H3D3 are also provided. In some embodiments, antibodies and antigen-binding fragments that bind to the membrane-proximal extracellular domain of the DLL3 protein are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with 362H3D3 for binding to DLL3 are also provided.

[0109] In some embodiments, antibody and antigen-binding fragments are also provided that include CDR sequences derived from the disclosed CDR sequences, with one, two, or three amino acid substitutions, deletions, and / or additions. multifunctional molecules

[0110] The multifunctional molecule comprises an antibody or antigen-binding fragment specific to DLL3, for example, those disclosed herein, and one or more antibodies or antigen-binding fragments having specificity to a second antigen or different epitopes on DLL3.

[0111] In some embodiments, the second antigen is a protein expressed in immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells.

[0112] In some embodiments, the second antigen is CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

[0113] Bispecific antibodies in different formats are also provided. In some embodiments, the anti-DLL3 fragment and the second fragment are each independently selected from a Fab fragment, a single-stranded variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0114] Bifunctional molecules that do not consist solely of antibodies or antigen-binding fragments are also provided. As molecules targeting tumor antigens, DLL3-specific antibodies or antigen-binding fragments, such as those described herein, can be combined with immune cytokines or ligands via a peptide linker as needed. Examples of immune cytokines or ligands that can be linked include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. Such bifunctional molecules can combine immune checkpoint blocking effects with local immune modulation at the tumor site. Chimeric antigen receptor

[0115] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising the antibody or a fragment thereof as a targeting unit. In some embodiments, the CAR comprises the antibody or a fragment thereof, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

[0116] The transmembrane domain may be designed to fuse to an extracellular domain containing an antibody or fragment, optionally via a hinge domain. It may also fuse to an intracellular domain, such as a costimulatory domain. In some embodiments, the transmembrane domain may include the native transmembrane region of a costimulatory domain (e.g., the TM region of CD28 or 4-1BB used as the costimulatory domain) or the native transmembrane domain of a hinge domain (e.g., the TM region of CD8 alpha or CD28 used as the hinge domain).

[0117] In some embodiments, a transmembrane domain may include sequences that extend into the cell membrane but also into the cell's cytoplasm and / or extracellular space. For example, a transmembrane may include a membrane-extending sequence that itself may further contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cell's cytoplasm and / or extracellular space. Thus, a transmembrane domain may include a membrane-extending region and further contain amino acids(s) that extend beyond the internal or external surface of the membrane itself, and such sequences may still be considered a “transmembrane domain”.

[0118] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably 2 to 10 amino acids in length, can form a linkage between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine doublets (GS), glycine-serine-glycine triplets (GSG), or alanine-alanine-alanine triplets (AAA) provide suitable linkers.

[0119] In some embodiments, the CAR further includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between the transmembrane domain and the activating domain. Examples of co-stimulatory domains include, but are not limited to, CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (T CD28 (FRSF7), CD28 (CD28T), CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KI R2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), C D158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD 229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(T FSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(KG2D), CD319(SLAMF7), CD335(K-p46 ), CD336(K-p44), CD337(K-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRTAM), CD357(TNFRSF18) Examples include inducible T cell costimulators (ICOS), LFA-1 (CD11a / CD18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activated NK cell receptors, Toll ligand receptors, and fragments or combinations thereof.

[0120] In some embodiments, the cytoplasmic portion of the CAR also includes a signaling / activation domain. In one embodiment, the signaling / activation domain is either a CD3ζ domain or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the CD3ζ domain. Methods for expressing or preparing polynucleotides, mRNA, and antibodies.

[0121] This disclosure also provides polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof, or CARs. The polynucleotides of this disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of this disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules.

[0122] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into target cells to express an antibody or a fragment thereof.

[0123] mRNA may be synthesized according to any of the various known methods. For example, mRNA may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system which may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions vary depending on the specific application.

[0124] In some embodiments, the DNA template is transcribed in vitro for the preparation of antibody-encoding mRNA. A suitable DNA template typically has a promoter for in vitro transcription, e.g., a T3, T7, or SP6 promoter, followed by a desired nucleotide sequence and a stop signal for the mRNA encoding the desired antibody (e.g., encoding the heavy or light chain).

[0125] The mRNA sequence encoding the desired antibody (e.g., the heavy or light chain) may be determined and incorporated into the DNA template using standard methods. For example, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence), virtual backtranslation is performed based on the denatured genetic code. An optimization algorithm may then be used for selecting suitable codons. Typically, the G / C content can be optimized to achieve the highest possible G / C content, while giving the most consideration to the frequency of tRNA, on the other hand, according to codon usage frequency. The optimized RNA sequence can be established and displayed, for example, with the help of a suitable display device, and compared to the original (wild-type) sequence. The secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties, or regions of the RNA, respectively.

[0126] mRNA may be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to enhance stability. Modifications to mRNA may include, for example, nucleotide modifications of the RNA. Modified mRNA may therefore include, for example, skeletal modifications, sugar modifications or base modifications. In some embodiments, the mRNA encoding an antibody (e.g., heavy chain and light chain encoding mRNA) may be, but is not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs, or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl- Adenine, N6-isopentenyl-adenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, inosine, 1-methylinosine, pseudouracil (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil They may also be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxyuracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate(v), 1-methyl-pseuduracil, queosine, 13-D-mannosyl-queosine, weybutoxosine, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.Preparations of such analogs are known to those skilled in the art, for example, from U.S. Patents No. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the full extent of which their disclosures are included herein by reference.

[0127] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphate groups of the nucleotide backbone contained in the RNA are chemically altered. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoamides, phosphoramides, phosphorothioates (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphates, and positively charged guanidinium groups, meaning that the phosphodiester linkage is replaced by other anionic, cationic, or neutral groups.

[0128] In some embodiments, mRNA (e.g., mRNA encoding the heavy and light chains) may contain sugar modifications. Typical sugar modifications are chemical modifications of sugars in nucleotides, which are not limited to, 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribo It contains sugar modifications selected from the group consisting of nucleotides, 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracithidine 5'-triphosphate, 2'-arauridine 5'-triphosphate), or azido triphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0129] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain modifications to the bases of nucleotides (base modification). Modified nucleotides containing base modifications are also called base-modified nucleotides. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, 6 - Examples include azacitidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate, or xanthosine 5'-triphosphate.

[0130] Typically, mRNA synthesis involves the addition of a "cap" at the N-terminus (5') and a "tail" at the C-terminus (3'). The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" plays a role in protecting mRNA from exonuclease degradation.

[0131] Therefore, in some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) contains a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphate via guanylyltransferase to form a 5'5' triphosphate linkage; and the 7-nitrogen of guanine is then methylated by methyltransferase. Examples of cap structures, though not limited to, include m7G(5')ppp(5'(A,G(5')ppp(5)A and G(5)ppp(5')G.

[0132] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) includes a 3' poly(A) tail structure. The poly(A) tail on the 3' end of the mRNA typically contains about 10–300 adenosine nucleotides (e.g., about 10–200 adenosine nucleotides, about 10–175 adenosine nucleotides, about 10–150 adenosine nucleotides, about 10–125 adenosine nucleotides, 10–100 adenosine nucleotides, about 10–75 adenosine nucleotides, about 20–70 adenosine nucleotides, or about 20–60 adenosine nucleotides). In some embodiments, mRNA encoding antibodies (e.g., mRNA encoding heavy and light chains) includes a 3' poly(C) tail structure. A suitable polyC tail on the 3' end of mRNA typically contains about 10–200 cytosine nucleotides (e.g., about 10–150 cytosine nucleotides, about 10–100 cytosine nucleotides, about 20–70 cytosine nucleotides, about 20–60 cytosine nucleotides, or about 10–40 cytosine nucleotides). The polyC tail may be attached to or substituted for the polyA tail.

[0133] In some embodiments, the mRNA (e.g., mRNA encoding heavy and light chains) includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as iron-responsive elements. In some embodiments, the 5' untranslated region may be about 50 to 500 nucleotides long (e.g., about 50 to 400 nucleotides, about 50 to 300 nucleotides, about 50 to 200 nucleotides, or about 50 to 100 nucleotides).

[0134] In some embodiments, the 5' region of mRNA (e.g., mRNA encoding heavy and light chains) includes a sequence encoding a signal peptide, such as those described herein. In certain embodiments, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the sequence encoding the signal peptide is directly or indirectly ligated at the N-terminus to the sequence encoding the heavy or light chain.

[0135] This technology may be used to deliver antibodies that can be produced against any antibody known in the art and any desired antigen using standard methods. The present invention may be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0136] Methods for producing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of this disclosure are entirely human. Fully human antibodies can be produced using techniques described in the art and techniques described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous locus has been deactivated. Exemplary techniques that can be used to produce such antibodies are described in U.S. Patents 6,150,584; 6,458,592; and 6,420,140, ​​which are incorporated herein by reference in their entirety. Treatment and Use

[0137] As described herein, the antibodies, variants, derivatives, or antibody-drug conjugates of this disclosure may be used in certain treatment and diagnostic methods.

[0138] This disclosure further covers antibody-based therapies, which include administering the antibodies, fragments, or antibody-drug conjugates of this disclosure to patients, e.g., animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Examples of therapeutic compounds of this disclosure include, but are not limited to, the antibodies of this disclosure (including their variants and derivatives described herein) and nucleic acids or polynucleotides encoding the antibodies of this disclosure (including their variants and derivatives described herein).

[0139] The antibodies of this disclosure can also be used to treat or inhibit cancer. As provided above, DLL3 can be overexpressed in tumor cells, particularly in tumors of the liver, stomach, pancreas, esophagus, ovaries, and lungs. Inhibition of DLL3 has been shown to be useful for treating tumors.

[0140] Accordingly, in some embodiments, a method is provided for treating cancer in patients who require treatment of cancer. In one embodiment, the method involves administering an effective amount of the antibody, fragment, or antibody-drug conjugate of the present disclosure to the patient. In some embodiments, at least one of the cancer cells in the patient (e.g., stromal cells) overexpresses DLL3.

[0141] Cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy, are also provided in this disclosure. Suitable cells (or alternatively, cells engineered to express the anti-DLL3 antibody of this disclosure) can be used, which are transduced by a vector encoding or in contact with a CAR containing the anti-DLL3 antibody of this disclosure. Upon such contact or manipulation, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient may have any of the types of cancer disclosed herein. The cells (e.g., T cells) may be, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

[0142] In some embodiments, the cells were isolated from the cancer patient themselves. In some embodiments, the cells were provided by a donor or from a cell bank. When cells are isolated from the cancer patient, undesirable immune responses can be minimized.

[0143] Non-exclusive examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of the following: gastric, pancreatic, esophageal, ovarian, and lung cancers, and cutaneous T-cell lymphoma. In some embodiments, the cancer is small cell lung cancer (SCLC).

[0144] Additional diseases or conditions associated with increased cell viability that may be treated, prevented, diagnosed and / or prognosticated by the antibodies or variants thereof of this disclosure include, but are not limited to, malignant tumors and related disorders, e.g., leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphoblastic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström hypergammaglobulinemia, heavy chain disease, and solid tumors, e.g., sarcomas and carcinomas, e.g., fibrosarcoma, mucosal sarcoma. Ligosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic This includes the progression and / or metastasis of cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminomas, embryonic carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0145] The specific dosage and treatment regimen for any particular patient depends on various factors, including the specific antibody used, its variant or derivative, the patient's age, weight, overall health, sex, and diet, as well as the timing of administration, rate of excretion, drug combination, and the severity of the specific disease being treated. Determining such factors by healthcare professionals is within the normal skill of the art. The dosage also depends on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0146] Methods of administering antibodies, fragments, or antibody-drug conjugates include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding polypeptides or compositions may be administered by any convenient route, for example, by injection or bolus injection, by absorption via the epithelium or inner layers of the skin mucosa (e.g., oral mucosa, rectal and intestinal mucosa), or in combination with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of this disclosure may be administered orally, rectally, parenterally, intracisional, vaginal, intraperitoneal, topically (such as by powder, ointment, drop, or transdermal patch), buccal, or as oral or nasal sprays.

[0147] As used herein, the term "parenteral" refers to methods of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0148] Administration may be systemic or local. In addition, it may be desirable to introduce the antibodies of this disclosure into the central nervous system by any preferred route, including intracerebroventricular and intrathecal injection, and intracerebroventricular injection may be facilitated by an intracerebroventricular catheter attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be used, for example, by the use of an inhaler or nebulizer and a formulation having an aerosolizing agent.

[0149] It may be desirable to administer the antigen-binding polypeptide or composition of this disclosure topically to an area requiring treatment, which may be achieved, for example, by local injection during surgery, local application, for example, local application in conjunction with wound dressing after surgery, by injection, by means of catheter, by means of suppository, or by means of implant, the implant being made of porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. Preferably, when administering a protein, including an antibody, of this disclosure, care should be taken to use a material that does not absorb the protein.

[0150] The amount of the antibodies, fragments, or antibody-drug conjugates of this disclosure that are effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. In addition, in vitro assays may be used, if necessary, to help identify the optimal dose range. The exact dose used in the formulation should also depend on the route of administration and the severity of the disease, disorder, or condition, and should be determined according to the practitioner's judgment and the circumstances of each patient. The effective dose may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0151] As a general suggestion, the dosage of the antibodies, fragments, or antibody-drug conjugates of this disclosure administered to patients is typically 0.001 mg / kg of patient body weight to 100 mg / kg of patient body weight, 0.01 mg / kg of patient body weight to 20 mg / kg of patient body weight, or 0.5 mg / kg of patient body weight to 10 mg / kg of patient body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other species, due to the immune response to exogenous polypeptides. Therefore, lower doses and lower frequencies of administration of human antibodies are often possible. Furthermore, the dosage and frequency of administration of the antibodies of this disclosure can be reduced by enhancing antibody uptake and tissue penetration (e.g., into the brain) through modifications such as lipidization.

[0152] In additional embodiments, the compositions of the Disclosure are administered in combination with cytokines. Cytokines that may be administered with the compositions of the Disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0153] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as radiotherapy. composition

[0154] This disclosure also provides pharmaceutical compositions, which comprise an effective amount of an antibody, fragment, or antibody-drug conjugate and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0155] In specific embodiments, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory agency, or is listed in the United States Pharmacopeia or any other commonly recognized pharmacopoeia for use in animals, or more specifically, in humans. Furthermore, “pharmaceutically acceptable carriers” are generally non-toxic solid, semi-solid, or liquid fillers, diluents, mounting materials, or any type of formulation adjuvants.

[0156] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids, such as water, and oils, such as those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, white powder, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. The composition may also contain, if desired, small amounts of wetting or emulsifying agents, or pH buffers, such as acetates, citrates, or phosphates. Antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic pressure, such as sodium chloride or dextrose, are also conceivable. These compositions can take the form of liquids, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated as suppositories using traditional binders and carriers such as triglycerides. Oral formulations may include standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, incorporated herein by reference. Such compositions contain a therapeutically effective amount of antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier, in order to provide a form for appropriate administration to the patient. The formulation must be suitable for the mode of administration.Parental preparations may be enclosed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0157] In some embodiments, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizer and a local anesthetic such as lignocaine to relieve pain at the injection site. Generally, the components are supplied in unit dosage forms, either separately or mixed together, as a dry lyophilized powder or a concentrated substance without water, in a sealed container such as an ampoule or sachet indicating the amount of the active drug. If the composition is to be administered by infusion, it may be administered using an infusion bottle containing sterile pharmaceutical-grade water or saline. If the composition is administered by injection, ampoules of sterile water or saline for injection may be provided so that the components can be mixed before administration. [Examples]

[0158] (Example 1) Production of mouse anti-human DLL3 antibody This example describes the preparation of a mouse anti-human DLL3 monoclonal antibody using hybridoma technology.

[0159] Immunogens: Two immunogens were used during the mouse immunization process. One consisted of the extracellular domain (ECD, UniProt number Q9NYJ7 A27~L492) of the human DLL3 protein fused with a human Fc fragment (hDLL3-hFc, Acro Bio, catalog number DL3-H5255). The other was the ECD of the human DLL3 protein fused with a his tag (hDLL3-his, Acro Bio, catalog number DL3-H52H4).

[0160] Mouse immunization scheme: To produce mouse monoclonal antibodies against human DLL3, BALB / c and C57BL / 6 mice were immunized intraperitoneally or subcutaneously with hDLL3-hFc or hDLL3-his protein at bi-weekly intervals. Serum titers of immunized mice were monitored by ELISA against human hDLL3-his protein. After several rounds of immunization, mice with sufficient titers were boosted with hDLL3-his protein and selected for fusion.

[0161] Cell fusion and hybridoma screening: Splenocytes from selected mice were fused with mouse myeloma cell line Sp2 / 0 by electrofusion. These hybridoma cells were then seeded in 96 flat-bottom microplates and allowed to secrete mouse antibodies into the supernatant. During the primary screening, positive clones were screened in a high-throughput manner using ELISA for protein binding to hDLL3-his and FACS for cell binding of human DLL3 overexpressed in HEK293 cells (HEK293-hDLL3, customized by Genomeditech) or SCLC cell line SHP77 (ATCC, catalog no. CRL-2195). The following confirmation screening was continued, and clones with nonspecific binding to human DLL1 his-tagged fusion protein (hDLL1-his, Sino Biological, catalog number 11635-H08H) or human DLL4 his-tagged fusion protein (hDLL4-his, Sino Biologial, catalog number 10171-H08H) were removed by ELISA. Clonal binding to cynomolgus monkey DLL3 his-tagged fusion protein (cynoDLL3-his, Acro Bio, catalog number DL3-C52H3) was identified by ELISA.

[0162] Subcloning screening and sequencing: Positive primary clones from each fusion that met the above criteria were subcloned by limiting dilution to ensure that the hybridoma subclones originated from a single parent cell. The subclones were screened using the same criteria as the primary clones described above. Subclones that exhibited specific binding efficacy to hDLL3 and cynoDLL3 but lacked binding to hDLL1 and hDLL4 were selected for subsequent sequencing.

[0163] The variable region sequence obtained from a mouse antibody was fused with the constant region of human IgG1 to create a chimeric DLL3 mAb. The DNA sequence of the chimeric antibody was cloned into a pcDNA3.4 plasmid and expressed in CHO-K1 cells. Subsequently, the antibody was purified using a protein A affinity chromatography column or beads. The purified chimeric antibody was subjected to serial in vitro screening to determine its affinity, binding ability, specificity, and species cross-reactivity.

[0164] Six chimeric mAbs, including 9E8D8, 36B7F3, 129H2B9, 148C3A7, 310P3C5, and 362H3D3, were selected for further analysis based on their performance in screening assays. The amino acid sequences of the variable regions of the selected chimeric DLL3 antibodies are provided in Table 1 below, and the CDR sequences are summarized in Table 2. The heavy and light chain sequences of all antibodies are listed in Table 3. Two benchmark DLL3 mAbs, DLL3-4-001(CC) (sequence derived from the IMGT database) and DLL3#3 (see WO2019234220), are their individual DLL3 binding elements in two T-cell engagers (TCEs) tarulatamab and BI765432, which are currently undergoing clinical evaluation. [Table 1-1] [Table 1-2] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] (Example 2) Protein binding activity of chimeric monoclonal antibodies targeting DLL3 2.1. ELISA conjugation to human DLL3

[0165] To determine the binding ability of chimeric mAbs to human DLL3 protein, an ELISA-based binding assay was performed as follows. Briefly, hDLL3-his protein was diluted with DPBS buffer at 1 μg / mL and adsorbed to wells of a 96-well microplate at 4°C overnight. After blocking the wells with 1% bovine serum albumin (BSA) to prevent nonspecific binding, a titer determination series of DLL3 chimeric mAbs, benchmark antibodies DLL3-4-001(CC) and DLL3#3, or isotype controls were prepared starting from 100 nM and added to the wells pre-adsorbed with hDLL3-his protein at 3-fold dilutions. The mixtures were incubated at room temperature (RT) for 1 hour. The bound DLL3 mAbs were recognized by a detection antibody against human IgG Fc conjugated with horseradish peroxidase (HRP) (Jackson Immuno, catalog no. 109-035-008). Tetramethylbenzidine (TMB), a substrate for HRP, was added to the wells, and the binding signal was visualized. After sufficient color development, a stop solution was added to the wells. The absorbance of the signal was detected at 450 nm using an Envision multi-label plate reader (PerkinElemer). Graphs and statistical analyses were created using 4-parameter nonlinear regression curve fitting in Graphpad Prism 9 software.

[0166] As shown in Figure 1A, all DLL3 chimeric antibodies efficiently bound to the human DLL3 protein. The EC50 values ​​of the binding curves for each antibody are summarized in Table 4. 2.2 ELISA conjugation to cynomolgus monkey and mouse DLL3 protein

[0167] To determine the cross-reactivity of chimeric Abs to cynomolgus monkey and mouse DLL3, ELISA binding assays were performed as described above. Recombinant cynoDLL3-his (KACTUS, catalog number DLL-CM103) and mouse DLL3 his-tagged protein (mDLL3-his, KACTUS, catalog number DLL-MM103) were used as coating antigens at a concentration of 1 μg / mL.

[0168] As shown in Figures 1B and 1C, all DLL3 chimeric Abs efficiently bound to cynomolgus monkey DLL3 and mouse DLL3 proteins.

[0169] The EC50 values ​​of the binding curves for each antibody are summarized in Table 4. 2.3. ELISA conjugation to human DLL1 and DLL4

[0170] Due to the high sequence similarity between DLL3 and its family member proteins DLL1 and DLL4, it is necessary to confirm the specificity of DLL3 chimeric antibodies.

[0171] To determine the binding ability of chimeric abs to human DLL1 and DLL4 proteins, an ELISA binding assay was performed as previously described. hDLL1-his protein (Sino Biological, Inc., catalog no. 11635-H08H) and hDLL4-his protein (Sino Biological, Inc., catalog no. 10171-H08H) were used as coating antigens at a concentration of 1 μg / mL. DLL1 mAb pidilizumab (CAS no. 1036730-42-3) and DLL4 mAb (Fab)MLCK-2 (Patent No. WO2015005632, ABL Bio) were used as positive controls.

[0172] As shown in Figures 2A and 2B, most DLL3 chimeric abs showed negligible binding to either the hDLL1 or hDLL4 protein, similar to the benchmark antibody. However, 362H3D3 showed detectable binding to both human DLL1 and DLL4 proteins, which may represent the cross-reactivity of this DLL3 ab to the other two family members. [Table 4] 2.4. Affinity measurement

[0173] The binding affinity of chimeric antibodies to human DLL3 protein was determined using Biacore® 8K. Briefly, the antibody (1 or 2 ug / ml) was captured using a Pro-A tip. A single dose (50 or 100 nM) of human DLL3-his protein was injected onto the captured antibody at a flow rate of 30 μL / min. The antigen was associated for 120 seconds and dissociated for 150–400 seconds. Data analysis was performed using Biacore® 8K evaluation software. The results showed that all chimeric DLL3 antibodies exhibited high affinity for human DLL3, which was similar to that of the benchmark antibody (Table 5). [Table 5] 2.5. Domain Mapping of Chimeric DLL3 Antibodies

[0174] To evaluate the precise binding domain of chimeric mAbs to human DLL3, cell-based binding assays were used as follows. Briefly, HEK293 cells stably expressing a series of duplicated ECDs of human DLL3 were used in these assays. The human DLL3 protein (UniProt number Q9NYJ7, A27-K618) has a distinct extracellular structure (A27-L492) consisting of one DSL domain, six EGF-like repeats, and an unstructured membrane proximal extracellular region (MPER) adjacent to the transmembrane domain (TM). As a result, cell lines expressing EGF1 and EGF2 (EGF1+2, A216~E310), EGF2 and EGF3 (EGF2+3, G274~E351), EGF3 and EGF4 (EGF3+4, S312~E389), EGF4 and EGF5 (EGF4+5, R353~R427), EGF5 and EGF6 (EGF5+6, D393~E465), and EGF6 and MPER (EGF6-MPER, R429~L492), conjugated with DLL3 TM and ICD, were constructed (customized by Genomeditech). The indicated DLL3 chimeric Ab, benchmark antibody, or isotype control was diluted to 100 nM in staining buffer (PBS buffer containing 2% FBS) and sterilized in a 96-well microplate at 4°C for 30 minutes, 5 × 10⁶ 4 The cells were incubated with the indicated cells. Antibody binding to the antigen on the cell surface was detected using a goat anti-human IgG(H+L) cross-adsorbed secondary antibody, Alexa Fluor® 488 (ThermoFisher Scientific, catalog number A-11013), at a dilution ratio of 1:2000. The cells were analyzed using a MACSQuant® analyzer 16 flow cytometer (Miltenyi Biotec BV&Co.KG). The data were analyzed using Flowjo 10.0 software. Graphs and statistical analyses were created using 4-parameter nonlinear regression curve fitting in Graphpad Prism 9 software.

[0175] As shown in Figure 3 and Table 6, these DLL3 chimeric antibodies bound to different ECD domains of human DLL3 expressed in HEK293 cells. More specifically, 129H2B9 and 148C3A7 bound to the EGF3-4 domains of human DLL3 expressed in cells. 9E8D8 and 310P3C5 specifically bound to the EGF6 domain of human DLL3 expressed in cells. 36B7 and 362H3D3 bound to the membrane-proximal extracellular domain similar to that of the benchmark antibody DLL3#3. Another benchmark antibody, DLL3-4-001(CC), specifically bound to EGF3, which is consistent with previously reported data. [Table 6] (Example 3) Cell-binding activity of chimeric monoclonal antibodies targeting DLL3 3.1 Binding ability to human DLL3 overexpressed in HEK-293 cells

[0176] To evaluate the binding activity of chimeric mAbs to human DLL3 expressed in cells, cell-based binding assays were used as follows. Briefly, HEK293 cells (HEK293-hDLL3) that stably express high levels of human DLL3 ECD were used in these assays. The indicated DLL3 chimeric Ab, benchmark antibody, or isotype control was diluted 3-fold in staining buffer (PBS buffer containing 2% FBS), starting at a concentration of 50 nM. The antibody dilution was stored in a 96-well microplate at 4°C for 30 minutes, yielding 5 × 10⁶ samples. 4The cells were incubated with the indicated cells. Antibody binding to the antigen on the cell surface was detected using a goat anti-human IgG(H+L) cross-adsorbed secondary antibody, Alexa Fluor® 488 (ThermoFisher Scientific, catalog number A-11013), at a dilution ratio of 1:2000. The cells were analyzed using a MACSQuant® analyzer 16 flow cytometer (Miltenyi Biotec BV&Co.KG). The data were analyzed using Flowjo 10.0 software. Graphs and statistical analyses were created using 4-parameter nonlinear regression curve fitting in Graphpad Prism 9 software.

[0177] As shown in Figure 4A, all DLL3 chimeric antibodies efficiently bound to human DLL3 expressed in HEK293 cells in a dose-dependent manner. Notably, the chimeric antibodies 129H2B9, 148C3A7, 310P3C5, and 362H3D3 exhibited superior binding efficacy to human DLL3 expressed in HEK293 cells compared to the benchmark antibodies DLL3-4-001(CC) and DLL3#3, as indicated by either an improved EC50 for DLL3-4-001(CC) or an improved maximal binding signal for DLL3#3. 9E8D8 and 36B7F3 exhibited comparable maximal binding efficacy to human DLL3 expressed in HEK293 cells to that of the EGF3-binding benchmark DLL3-4-001(CC).

[0178] The EC50 values ​​of the binding ability to human DLL3 expressed in cells are summarized in Table 7. 3.2 Binding ability to cynomolgus monkey DLL3 overexpressed in cells

[0179] To evaluate the species cross-reactivity of chimeric mAbs against cynomolgus monkey DLL3 expressed in cells, cell-based binding assays were used according to the previously described protocol. Briefly, HEK293 cells (HEK293-cynoDLL3) stably expressing full-length human cynomolgus monkey DLL3 were constructed (customized by Cusabio). The indicated DLL3 chimeric Ab, benchmark antibody, or isotype control was tested in this assay.

[0180] As shown in Figure 4B, all DLL3 chimeric antibodies efficiently bound to cynomolgus monkey DLL3 expressed in HEK293 cells with equivalent binding efficacy compared to their binding efficacy to human DLL3.

[0181] The EC50 binding affinity of cynomolgus monkey DLL3 expressed in cells is summarized in Table 7. 3.3 Binding to human DLL3 expressed in tumor cells

[0182] To evaluate the binding activity of chimeric mAbs to human DLL3 expressed in tumor cells, human SCLC cell lines SHP77 and NCI-H82, which have low levels of DLL3 expression, were used in cell-based binding assays according to the protocol described in Example 3.1.

[0183] As shown in Figures 5A and 5B, all DLL3 chimeric antibodies efficiently bound to human DLL3 expressed in SCLC tumor cell lines SHP77 or NCI-H82. Interestingly, unlike the binding tendency in HEK293 cells overexpressing DLL3, the binding of DLL3 mAbs 129H2B9 and 148C3A7 to the distal membrane region showed significantly better binding efficacy than membrane-proximal binding mAbs including 9E8D8, 36B7F3, 310P3C5, and 362H3D3 in these two DLL3-expressing tumor cell lines, demonstrating the superiority of the binding ability of the distal membrane binding agents (Figures 5A and 5B). 3.4 Binding ability to human DLL1 and DLL4 overexpressed in cells

[0184] To eliminate nonspecific binding of chimeric mAbs to human DLL1 and DLL4 expressed in cells, cell-based binding assays were used as previously described. Briefly, CHO-K1 cells (CHO-K1-hDLL1 and CHO-K1-hDLL4, Genomeditech) stably expressing full-length human DLL1 and DLL4 were constructed. The human DLL1 and DLL4 sequences used in this example were derived from UniProt (O00548 and Q9NR61). Protein expression was confirmed by FACS analysis using anti-human DLL1 (R&D Systems, MAB1818) and anti-human DLL4 (R&D System, MAB1506) antibodies. The indicated DLL3 chimeric Ab, benchmark antibody, or isotype control was tested in the assay.

[0185] As shown in Figure 6A, in contrast to the significant binding affinity of the positive control DLL1 mAb pidilizumab to DLL1, most of the DLL3 chimeric antibodies, including 9E8D8, 36B7F3, 129H2B9, 148C3A7, and 310P3C5, and two benchmark antibodies, did not show specific binding to human DLL1 expressed in CHO-K1 cells. 362H3D3 showed only slight binding to overexpressed DLL1 in CHO-K1 cells at its highest dose level of 100 nM. Also, similar to the two benchmark antibodies DLL3-4-01(CC) and DLL3#3, all DLL3 chimeric antibodies showed negligible binding to overexpressed DLL4 in CHO-K1 cells, in contrast to the significant binding strength of the DLL4-specific positive control antibody MLCK-2 (Figure 6B). Overall, all chimeric antibodies exhibited high specificity in binding to human DLL3 expressed in cells. [Table 7] (Example 4) Humanization of chimeric DLL3 antibodies

[0186] The variable region of the 310P3C5 chimeric antibody was selected for humanization.

[0187] Briefly, the amino acid sequences of VH and VL were aligned with available databases of human Ig gene sequences to identify the overall best matching human germline Ig gene sequence. Next, the CDRs of the chimeric antibody heavy and light chains were grafted into candidate human germline sequences. A 3D model of the CDR-grafted antibody was generated using Molecular Operating Environment (MOE) to determine whether any critical human amino acids in the framework region required back-mutation to the corresponding mouse amino acid to maintain CDR conformation and antibody function.

[0188] For the heavy chain of 310P3C5, the candidate human germline sequence was IGKV1-18 * 01 or IGKV1-3 * 01 gene. For the light chain of 310P3C5, the candidate human germline sequence was IGKV1-12 * 01 or IGKV1-33 * 01 gene. In the case of the heavy chain, M48I, R67Q, V68A, M70L, T72V and T74K in the framework of IGKV1-18 * 01, and R38K, M48I, V68A, I70L, R72V, T74K and S84R in the framework of IGKV1-3 * 01 were involved in back-mutation. In the case of the light chain, Y49S, Y49S and T69K in the framework regions of IGKV1-33 * 01 and IGKV1-12 * 01 were separately involved in back-mutation.

[0189] Variable regions of humanized antibodies were constructed by selecting different combinations of revertant mutation sites. The sequences of the heavy and light chain variable regions of humanized antibodies for 310P3C5 are listed in Table 8A. The VH and VL pairings for each humanized antibody are listed in Table 8B. The variable regions of the humanized antibodies were then fused to the constant region of human IgG1 for antibody production and functional characterization. The complete sequences of the heavy and light chains of the humanized antibodies are listed in Table 8C. [Table 8A-1] [Table 8A-2] [Table 8B] [Table 8C-1] [Table 8C-2] [Table 8C-3] [Table 8C-4] [Table 8C-5] [Table 8C-6] (Example 5) Antigen-binding properties of humanized antibodies 5.1 Binding to Recombinant Human DLL3

[0190] To evaluate antigen-binding activity, humanized antibodies were subjected to ELISA as previously described. As shown in Figure 7, all humanized 310P3C5 antibodies showed comparable binding efficacy to their parental chimeric antibodies against the human DLL3 protein. 5.2 Affinity Ranking of Humanized Antibodies by Biacore®

[0191] To investigate whether humanized antibodies maintained their binding kinetics, single-dose affinity ranking was performed using Biacore®. Antibodies (2 ug / ml) were captured using a Protein A tip. 100 nM human DLL3-his protein was injected onto the captured antibody at a flow rate of 30 μL / min for 120 seconds. The antigen was dissociated for 400 seconds. The experiment was performed using Biacore® 8K. Data analysis was performed using Biacore® 8K evaluation software.

[0192] The results shown in Table 9 demonstrate that several humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14, and 310P3C5-z15, showed comparable affinity to their chimeric antibodies. [Table 9-1] [Table 9-2] 5.3 Binding to human DLL3 and cynomolgus monkey DLL3 overexpressing HEK293 cells

[0193] To evaluate the binding properties of human DLL3 and cynomolgus monkey DLL3 overexpressed in cells, selected humanized antibodies were analyzed by FACS according to the protocol described in previous examples.

[0194] As shown in Figures 8A and 8B, all selected humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14, and 310P3C5-z15, maintained binding to overexpressed human DLL3 in HEK293 cells compared to their chimeric antibodies, which was significantly superior to the two benchmark antibodies DLL3-4-001(CC) and DLL3#3. Furthermore, all of these humanized mAbs maintained binding to cynomolgus monkey DLL3 expressed in HEK293 cells. 5.4 Binding to human DLL3 expressed in cancer cell lines

[0195] To evaluate the binding properties of selected human DLL3 expressed in cancer cells, the selected humanized antibodies were analyzed by FACS according to the protocol described previously.

[0196] As shown in Figures 9A and 9B, all selected humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14, and 310P3C5-z15, maintained binding to endogenously expressed human DLL3 in tumor cells SHP77 and NCI-H82 compared to their chimeric antibodies, which was better than the benchmark antibody DLL3#3. 5.5 Binding to human DLL1 and DLL4 overexpressed in CHO-K1 cells

[0197] To eliminate nonspecific binding of humanized antibodies to human DLL1 and DLL4 expressed in cells, selected humanized antibodies were analyzed by FACS according to the protocol described in previous examples.

[0198] As shown in Figures 10A and 10B, all humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14, and 310P3C5-z15, showed negligible binding to human DLL1 and DLL4 overexpressed in CHO-K1 cells. (Example 6) Optimization of humanized antibodies

[0199] In certain cases, humanized antibodies are further optimized to improve their developability, including long-term stability, manufacturability, and low heterogeneity. Post-translational modifications (PTMs), such as deamidation, isomerization, glycosylation, and oxidation, which are important factors, can affect developability and further impair the potency, efficacy, and safety of therapeutic antibodies. In this example, computer tools were used to predict PTM-prone sites to facilitate the manipulation of antibodies with better developability. 6.1 Design of PTM site removal

[0200] In this case, MOE was used to predict sites prone to PTM. Amino acid N55, located in the CDR region of the heavy chain of the 310P3C5 antibody, was identified as a potential deamidation site. Therefore, this amino acid was replaced with A, F, H, R, V, W, or Y. The sequence of the variable region of the humanized antibody from which PTM was removed is shown in Table 10A, and the modified VH CDR2 sequence is shown in Table 10B. The variable region of the humanized antibody was then fused to the constant region of human IgG1 for antibody production and functional characterization. [Table 10A] [Table 10B-1] [Table 10B-2] 6.2 Conjugation of PTM-removed humanized antibodies to human DLL3-expressing HEK293 cells

[0201] To evaluate the binding properties to human DLL3 expressed in cells, humanized antibodies with the PTM removed were analyzed by FACS according to the protocol described in previous examples.

[0202] As shown in Figures 11A and 11B, humanized antibodies from which all selected PTMs had been removed maintained binding to either human DLL3 overexpressed in HEK-293 cells or endogenously expressed in tumor cell line SHP77 cells, compared to their parent humanized antibody 310P3C5-z15. * * *

[0203] This disclosure is intended to be intended as a single example of each aspect of the disclosure, and its scope should not be limited by the specific embodiments described; any functionally equivalent composition or method is within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to encompass modifications and variations of this disclosure, provided that they are within the scope of the appended claims and their equivalents.

[0204] All publications and patent applications cited herein are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated by reference. In certain embodiments, for example, the following items are provided: (Item 1) An antibody or its antigen-binding fragment having specificity for human delta-like ligand 3 (DLL3) protein, comprising a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, (a) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; The VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 38 and 111-117; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 39; The aforementioned VL CDR1 contains the amino acid sequence of SEQ ID NO: 40; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 41; and The aforementioned VL CDR3 contains the amino acid sequence of SEQ ID NO: 42, (b) Whether VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 13-18; (c) Whether VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 19-24; (d) Whether the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 25-30; (e) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NOs. 31-36; or (f) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NOs. 43-48. An antibody or its antigen-binding fragment. (Item 2) (a) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; The VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 38 and 111-117; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 39; The aforementioned VL CDR1 contains the amino acid sequence of SEQ ID NO: 40; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 41; and The aforementioned VL CDR3 contains the amino acid sequence of SEQ ID NO: 42, The antibody or its antigen-binding fragment as described in item 1. (Item 3) The antibody or antigen-binding fragment thereof according to item 2, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 65-69 and 104-110, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs. 65-69 and 104-110. (Item 4) The antibody or antigen-binding fragment thereof according to item 2 or 3, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 70 to 73, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs. 70 to 73. (Item 5) The antibody or antigen-binding fragment thereof according to item 4, wherein VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 104 to 110, and VL comprises the amino acid sequence of SEQ ID NO: 73. (Item 6) (b) The antibody or antigen-binding fragment described in item 1, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NOs. 13 to 18. (Item 7) The antibody or antigen-binding fragment according to item 6, wherein VH comprises the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% sequence identity to SEQ ID NO: 1, and VL comprises the amino acid sequence of 2, or a peptide having at least 90% sequence identity to SEQ ID NO: 2. (Item 8) An antibody or antigen-binding fragment thereof as described in item 7, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 49 and a light chain containing the amino acid sequence of SEQ ID NO: 50. (Item 9) (c) The antibody or antigen-binding fragment described in item 1, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NOs. 19 to 24. (Item 10) The antibody or antigen-binding fragment according to item 9, wherein VH comprises the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% sequence identity to SEQ ID NO: 3, and VL comprises the amino acid sequence of 4, or a peptide having at least 90% sequence identity to SEQ ID NO: 4. (Item 11) An antibody or antigen-binding fragment thereof as described in item 10, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 51 and a light chain containing the amino acid sequence of SEQ ID NO: 52. (Item 12) (d) The antibody or antigen-binding fragment described in item 1, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NOs. 25 to 30. (Item 13) The antibody or antigen-binding fragment according to item 12, wherein VH comprises the amino acid sequence of SEQ ID NO: 5, or a peptide having at least 90% sequence identity to SEQ ID NO: 5, and VL comprises the amino acid sequence of 6, or a peptide having at least 90% sequence identity to SEQ ID NO: 6. (Item 14) An antibody or antigen-binding fragment thereof as described in item 13, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 53 and a light chain containing the amino acid sequence of SEQ ID NO: 54. (Item 15) (e) The antibody or antigen-binding fragment described in item 1, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NOs. 31 to 36. (Item 16) The antibody or antigen-binding fragment according to item 15, wherein VH comprises the amino acid sequence of SEQ ID NO: 7, or a peptide having at least 90% sequence identity to SEQ ID NO: 7, and VL comprises the amino acid sequence of SEQ ID NO: 8, or a peptide having at least 90% sequence identity to SEQ ID NO: 8. (Item 17) An antibody or antigen-binding fragment thereof as described in item 16, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 55 and a light chain containing the amino acid sequence of SEQ ID NO: 56. (Item 18) (f) The antibody or antigen-binding fragment described in item 1, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequence of SEQ ID NOs. 43 to 48. (Item 19) The antibody or antigen-binding fragment according to item 18, wherein VH comprises the amino acid sequence of SEQ ID NO: 11 or a peptide having at least 90% sequence identity to SEQ ID NO: 11, and VL comprises the amino acid sequence of SEQ ID NO: 12 or a peptide having at least 90% sequence identity to SEQ ID NO: 12. (Item 20) An antibody or antigen-binding fragment thereof as described in item 19, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 59 and a light chain containing the amino acid sequence of SEQ ID NO: 60. (Item 21) An antibody or antigen-binding fragment thereof that has specificity for the human delta-like ligand 3 (DLL3) protein and competes with the antibody or fragment described in any one of items 1 to 20 for binding to the DLL3 protein. (Item 22) An antibody or antigen-binding fragment thereof that has specificity for said human delta-like ligand 3 (DLL3) protein and binds to the EGF3-4 domain or the EGF6 domain. (Item 23) The antibody or fragment thereof according to any one of Items 1 to 22, wherein the antibody or fragment thereof is a bivalent Fab antibody, or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv. (Item 24) The antibody or fragment thereof according to any one of Items 1 to 23, which is humanized. (Item 25) A multispecific antibody comprising the antigen-binding fragment according to any one of Items 1 to 23, and one or more antibodies or antigen-binding fragments having binding specificity for a target antigen other than DLL3. (Item 26) A chimeric antigen receptor (CAR) comprising the antigen-binding fragment according to any one of Items 1 to 25, a transmembrane domain, a co-stimulatory domain, and a CD3ζ intracellular domain. (Item 27) One or more polynucleotides encoding the antibody or antigen-binding fragment thereof according to any one of Items 1 to 25, or the CAR according to Item 26. (Item 28) The polynucleotide according to Item 27, which is one or more mRNAs. (Item 29) The polynucleotide according to Item 28, wherein the mRNA is chemically modified. (Item 30) A cell comprising the polynucleotide according to Item 28 or 29. (Item 31) A composition comprising the antibody or antigen-binding fragment thereof according to any one of Items 1 to 25, or the CAR according to Item 26, and a pharmaceutically acceptable carrier. (Item 32) A method for treating cancer in a patient who requires treatment for cancer, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof as described in any one of items 1 to 25, or a CAR as described in item 26. (Item 33) Use of an antibody or antigen-binding fragment thereof as described in any one of items 1 to 25, or a CAR as described in item 26, for the preparation of a medicine for treating cancer. (Item 34) The method described in item 32 or the use described in item 33, wherein the cancer is selected from the group consisting of ovarian cancer, prostate cancer, urinary tract cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer. (Item 35) The method described in item 32 or the use described in item 33, wherein the cancer is small cell lung cancer (SCLC).

Claims

1. An antibody or its antigen-binding fragment having specificity for human delta-like ligand 3 (DLL3) protein, comprising a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, (a) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 37; The VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 111-117; The VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 40; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 41; and The VL CDR3 contains the amino acid sequence of SEQ ID NO: 42, An antibody or its antigen-binding fragment.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 65-69 and 104-110, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 65-69 and 104-110.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 70-73, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 70-73.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 104 to 110, and the VL comprises the amino acid sequence of SEQ ID NO:

73.

5. An antibody or antigen-binding fragment thereof having specificity for the human delta-like ligand 3 (DLL3) protein and competing with the antibody or fragment thereof described in claim 1 for binding to the DLL3 protein.

6. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof is a bivalent Fab antibody, or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv.

7. A humanized antibody or fragment thereof according to claim 1.

8. A multispecific antibody comprising the antigen-binding fragment described in claim 1, and one or more antibodies or antigen-binding fragments having binding specificity to a target antigen other than DLL3.

9. A chimeric antigen receptor (CAR) comprising the antigen-binding fragment, transmembrane domain, costimulatory domain, and CD3ζ intracellular domain as described in claim 1.

10. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or one or more polynucleotides encoding a CAR according to claim 9.

11. The polynucleotide according to claim 10, which is one or more mRNAs.

12. The polynucleotide according to claim 11, wherein the mRNA is chemically modified.

13. A cell containing polynucleotides as described in claim 11.

14. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a CAR according to claim 9, and a pharmaceutically acceptable carrier.

15. A composition for treating cancer in a patient who requires treatment for cancer, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a CAR according to claim 9.

16. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a CAR according to claim 9, for the preparation of a pharmaceutical for treating cancer.

17. The composition according to claim 15, wherein the cancer is selected from the group consisting of ovarian cancer, prostate cancer, urinary tract cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer.

18. The composition according to claim 15, wherein the cancer is small cell lung cancer (SCLC).

19. The use according to claim 16, wherein the cancer is selected from the group consisting of ovarian cancer, prostate cancer, urinary tract cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer.

20. The use according to claim 16, wherein the cancer is small cell lung cancer (SCLC).

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