Anti-DLL3 antibodies and uses thereof

Abstract

Anti-DLL3 antibodies are provided, including murine antibodies, humanized antibodies, and antibodies having further optimized CDR sequences. The antibodies have excellent binding specificity and improved activity compared to other anti-DLL3 antibodies that are being developed.

Description

Background Technology

[0001] Lung cancer is the leading cause of cancer death worldwide, with small cell lung cancer (SCLC) accounting for approximately 15%. Unlike other types of cancer, SCLC is highly aggressive and can metastasize at an early stage. Clinical outcomes include frequent SCLC recurrence and drug resistance. Furthermore, due to the lack of targeted therapies and challenges in early detection, the overall survival (OS) rate for SCLC is low. Although immunotherapy can be used as first-line treatment for extensive-stage small cell lung cancer (ES-SCLC), such as using a PD-L1 monoclonal antibody (durvalumab) in combination with chemotherapy, OS has not been significantly improved. Therefore, there is an urgent need for improved SCLC therapies.

[0002] Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand in the Notch ligand family. The human DLL3 protein has a unique structure compared to other members of this family, such as DLL1 and DLL4, consisting of a Delta / Serrate / LAG-2 (DSL) domain, six epidermal growth factor (EGF)-like repeat sequences, a 21-acid (aa) transmembrane domain (TM), and a 105 aa intracellular domain (ICD).

[0003] DLL3 is highly expressed on the cell surface of SCLC tumors and other neuroendocrine-originating tumor types, including glioblastoma multiforme (GBM), large cell neuroendocrine lung cancer (LCNEC), metastatic melanoma, small cell bladder cancer (SCBC), and neuroendocrine prostate cancer (NEPC). Overall, over 80% of SCLC tumors express DLL3 protein, and this expression is highly consistent across tumor cells. In contrast, very slight expression is observed in normal tissues such as neurons, pancreatic islet cells, and pituitary cells, and is limited to the cytoplasm. In preclinical models, DLL3 expression promotes SCLC cell migration and invasion through a mechanism involving the control of the epithelial-mesenchymal transition protein Snail.

[0004] The unique expression pattern of DLL3 in SCLC and other neuroendocrine tumors makes it possible to develop therapeutics that specifically target these tumor types using DLL3. Currently, multiple clinical studies in SCLC and other neuroendocrine tumors are evaluating these DLL3-specific drugs. Several types of approaches targeting DLL3, including bispecific antibodies (BsAbs), chimeric antigen receptor T-cell therapy (CAR-T), and antibody-drug conjugates (ADCs), are currently under clinical investigation.

[0005] Talatamab (AMG757) is a bispecific T-cell connective (BiTE) molecule with an extended half-life (targeting both DLL3 and CD3 to induce T-cell-mediated tumor lysis). In the Phase I DeLLphi-300 study (NCT03319940), it demonstrated manageable safety and encouraging durability of response in patients with relapsed / refractory SCLC. The objective response rate (ORR) was 23.4% (95% CI, 15.7 to 32.5), including two complete responses and 23 partial responses. The median duration of response (DoR) was 12.3 months (95% CI, 6.6 to 14.9). The disease control rate (DCR) was 51.4% (95% CI, 41.5 to 61.2). The median progression-free survival (PFS) and overall survival (OS) were 3.7 months (95% CI, 2.1 to 5.4) and 13.2 months (95% CI, 10.5 to not reached), respectively.

[0006] Other DLL3-based T-cell adaptors, including BI764532 from Boehringer Ingelheim and HPN328 from Harpoon Therapeutics, have also shown promising anti-tumor efficacy in their respective Phase I clinical trials. Rova-T is an ADC containing the DLL3-targeting antibody lova-tuzumab, linked to the DNA cross-linking agent pyrrolobenzodiazepine (PDB) via a protease-cleavable linker. In the Phase 3 TAHOE study comparing Rova-T with topotecan as a second-line therapy for advanced or metastatic SCLC with high DLL3 expression, Rova-T exhibited poor overall survival (OS) and a higher toxicity rate. AMG 119, the first CAR-T cell therapy for SCLC, showed manageable safety profiles and promising anti-tumor activity in five adult subjects in its Phase I study, but enrollment is currently paused. LB2102 is an autologous CAR-T cell therapy that was recently approved by the FDA for Phase I clinical development in the treatment of adult patients with ES-SCLC. Overall, exploring different types of drugs targeting DLL3 holds promise for providing more options for SCLC treatment. Summary of the Invention

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

[0008] One embodiment of this disclosure provides an antibody or antigen-binding fragment thereof that is specific for human Delta-like ligand 3 (DLL3) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3, and the VL comprises VL CDR1, VL CDR2, and VL CDR3, wherein: (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 NO: 38 and 111-117; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39; the 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 comprises the amino acid sequence of SEQ ID NO: 42; (b) the VH CDR1, VH CDR2, VH CDR3, and VL CDR3... CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 13-18; (c) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 19-24; (d) VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 25-30; (e) VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 31-36; or (f) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: The amino acid sequence of 43-48.

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

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

[0011] In some embodiments, (b) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 13-18.

[0012] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 1 or a peptide having at least 90% sequence identity with SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 2 or a peptide having at least 90% sequence identity with 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) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 19-24.

[0015] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 90% sequence identity with SEQ ID NO: 3, and the VL comprises the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 90% sequence identity with 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, wherein (d) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 each contain the amino acid sequences of SEQ ID NO: 25-30.

[0018] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 5 or a peptide having at least 90% sequence identity with SEQ ID NO: 5, and the VL comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 90% sequence identity with 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) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 31-36.

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

[0022] In some embodiments, the antibody or its antigen-binding fragment comprises 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.

[0023] In some embodiments, (f) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 43-48.

[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 with 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 with SEQ ID NO: 12.

[0025] In some embodiments, the antibody or its antigen-binding fragment comprises 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.

[0026] In one embodiment, an antibody or antigen-binding fragment thereof is also provided that is specific for human Delta-like ligand 3 (DLL3) protein, which competes with the antibody or fragment thereof disclosed herein for binding to the DLL3 protein.

[0027] In one embodiment, an antibody or an antigen-binding fragment thereof is also provided, which is specific for human Delta-like ligand 3 (DLL3) protein, which binds to an EGF3-4 domain or an EGF6 domain.

[0028] A multispecific antibody is also provided, comprising the antigen-binding fragment disclosed herein and one or more antibodies or antigen-binding fragments having binding specificity to non-DLL3 target antigens.

[0029] Another embodiment provides a chimeric antigen receptor (CAR) comprising the antigen-binding fragment disclosed herein, a transmembrane domain, a co-stimulatory domain, and a CD3ξ 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). Attached Figure Description

[0031] Figures 1A-1C The ELISA shows the binding of the anti-DLL3 antibody to the his-tagged human / cynomolgus / mouse DLL3 protein.

[0032] Figure 2A and Figure 2B The ELISA results show the binding of anti-DLL3 antibody to human paralogs DLL1 and DLL4.

[0033] Figure 3 The epitope domain localization results of the anti-DLL3 antibody are shown.

[0034] Figure 4A and Figure 4B The cell-based binding of the anti-DLL3 antibody to HEK293 cell lines expressing human or cynomolgus monkey DLL3 is shown.

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

[0036] Figure 6A and Figure 6B The cell-based binding of the anti-DLL3 antibody to human paralogs DLL1 and DLL4 expressed on CHO-K1 cells is shown.

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

[0038] Figure 8A and Figure 8B The cell-based binding of the anti-DLL3 humanized antibody to HEK293 cell lines expressing human or cynomolgus monkey DLL3 is shown.

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

[0040] Figure 10A and Figure 10B The cell-based binding of the anti-DLL3 humanized antibody to human paralogs DLL1 and DLL4 expressed on CHO-K1 cells is shown.

[0041] Figure 11A and Figure 11B The study demonstrated cell-based binding of anti-DLL3 humanized and PTM-deactivated antibodies to HEK293 cells and SHP77 cells expressing human DLL3. Detailed Implementation

[0042] definition

[0043] It should be noted that the terms “a” or “an” refer to one or more of the entities described; for example, “an antibody” should be understood to represent one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein.

[0044] 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 can be a complete antibody as well as any antigen-binding fragment or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule having the biological activity of binding an antigen. Examples of such molecules include, but are not limited to, the complementarity-determining region (CDR) of the heavy or light chain or its ligand-binding portion, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region, or any portion thereof, or at least a portion of the binding protein.

[0045] Full-length antibodies consist of two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains may be referred to as “VH” and “VL”, respectively. The variable regions in both chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (including the light chain (LC) CDRs of LC-CDR1, LC-CDR2, and LC-CDR3, and the heavy chain (HC) CDRs of HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody-antigen binding fragments disclosed herein can be defined or identified using the following conventions: Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three cored red reticulum (CDRs) of either the heavy or light chain are inserted between flanking segments called 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 classified based on the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further subdivided into subclasses, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0046] As used herein, the term "half-antibody" refers to an immunoglobulin heavy chain associated with an immunoglobulin light chain. Those skilled in the art will readily understand that a half-antibody may encompass a fragment thereof and may also have an antigen-binding domain composed of a single variable domain, such as those derived from camelids.

[0047] As used herein, the term "single-chain half antibody" refers to a single-chain polypeptide comprising a VL domain, optionally a CL domain, a linker, a VH domain, optionally a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the domains are positioned relative to each other in the N-terminal to C-terminal direction as follows: VL-linker-VH-hinge-CH2-CH3, VL-linker-VH-partial hinge-CH2-CH3, VL-linker-VH-hinge variant-CH2-CH3, or VL-CL-linker-VH-CH1-hinge-CH2-CH3.

[0048] An antibody expressing a "single-domain antibody" (sdAb) or "single-variable-domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) is capable of conferring antigen binding ability. In other words, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single-domain antibodies include those derived from camelids (lambs and camels) and cartilaginous fish (e.g., nurse sharks), as well as those derived from human and mouse antibodies through recombinant methods (Nature [Nature] (1989) 341:544-546; Dev Comp Immunol [Developmental and Comparative Immunology] (2006) 30:43-56; Trend Biochem Sci [Trends in Biochemistry] (2001) 26:230-235; Trends Biotechnol [Trends in Biotechnology] (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; Febs Lett [Circular of the Federation of European Biochemical Societies] (1994) 339:285-290; WO 00 / 29004; WO 02 / 051870). When sdAb contains only heavy chains, it is interchangeable with “VHH” or “single heavy chain variable domain antibody” or “nanobody”.

[0049] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spikelers, and dimeric antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that exerts its antibody effect by binding to a specific antigen to form a complex.

[0050] In the context of antibodies, "Fab" refers to a monovalent antigen-binding fragment of the antibody, which consists of a light chain (including both variable and constant regions) linked by disulfide bonds to the variable region and a first constant region of a heavy chain. Fab can be obtained by digesting the antibody with papain at residues proximal to the N-terminus of the disulfide bonds between the heavy chains in the hinge region.

[0051] “Fab” refers to a Fab fragment that contains a portion of the hinge region. It can be obtained by digesting an antibody with pepsin at the C-terminus of the disulfide bond between the heavy chains in the hinge region. Therefore, a small number of residues in the hinge region (including one or more cysteine ​​residues) are different from Fab.

[0052] "F(ab)2" refers to the dimer of Fab', which contains two light chains and a portion of two heavy chains.

[0053] "Single-chain variable fragment" or "scFv" refers to the immunoglobulin heavy chain (V). H ) and light chains (V L ) Fusion proteins with variable regions. In some respects, these regions are linked by short linker peptides of ten to approximately 25 amino acids in length. The linker peptides can be enriched with glycine for flexibility, and serine or threonine for improved solubility, and can be linked to V H N-terminus and V L The C-terminus is removed, and vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0054] The term antibody encompasses a wide range of polypeptide classes that can be distinguished by biochemical methods. Those skilled in the art will understand that heavy chains can be classified as γ, μ, α, δ, or ε (gamma, mu, alpha, delta, epsilon), including several subclasses (e.g., γ1-γ4). It is the properties of this chain that determine the "class" of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes) For exampleIgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to confer functional specificity. In view of this disclosure, those skilled in the art can readily identify these classes and modified versions of isotypes, which are accordingly covered within the scope of this disclosure. All immunoglobulin classes are obviously covered within the scope of this disclosure, and the following discussion will generally refer to immunoglobulin molecules of the IgG class. With regard to IgG, a standard immunoglobulin molecule comprises 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 Daltons. These four chains are typically linked by disulfide bonds in a “Y” configuration, wherein the light chain begins at the opening of the “Y”, wraps around the heavy chain, and extends into the variable region.

[0055] The antibodies, antigen-binding moieties, variants, or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate-derived, or chimeric antibodies, single-chain antibodies, and epitope-binding fragments. For example Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including...) For example Anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0056] Light chains can be classified as κ or λ (kappa, lambda). Each heavy chain class can bind to either a κ or λ light chain. Generally, the light and heavy chains are covalently bonded to each other, and when the immunoglobulin is produced by hybridoma cells, B cells, or genetically engineered host cells, the "tail" portions of the two heavy chains are bonded to each other via covalent disulfide bonds or non-covalent bonds. In this heavy chain, the amino acid sequence extends from the N-terminus at the Y-configuration bifurcation to the C-terminus at the base of each chain.

[0057] Both the light and heavy chains are divided into regions with structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains (VK and VH) of both the light and heavy chains determine antigen recognition and specificity. Conversely, the constant domains (CK and CH1, CH2, or CH3) of the light and heavy chains confer important biological properties, such as secretion, transplacental activity, Fc receptor binding, and complement binding. By convention, the farther the constant domain is from the antibody's antigen-binding site or N-terminus, the higher its number. 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.

[0058] As described above, the variable region enables the antibody to selectively recognize and specifically bind to epitopes on antigens. That is, a subset of the antibody's VK and VH domains, or complementarity-determining regions (CDRs), binds to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site located at the end of each arm of this Y-configuration. More specifically, this antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each VH and VK chain. In some cases, such as certain immunoglobulin molecules derived from camel species or engineered based on camel immunoglobulins, the immunoglobulin molecules are complete and may consist only of heavy chains, lacking light chains. See example like Hamers-Casterman et al. , Nature [Nature] 363:446-448 (1993).

[0059] In naturally occurring antibodies, each antigen-binding domain contains six "complementarity-determining regions" or "CDRs," which are short, discontinuous sequences of amino acids precisely positioned to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids within these domains, called "framework" regions, exhibit low intermolecular variability. Framework regions primarily adopt a β-sheet conformation, and CDRs form loops that connect β-sheet structures and, in some cases, form part of the β-sheet structure. Thus, the framework regions act as a scaffold, positioning the CDRs in the correct orientation through interchain, non-covalent interactions. The antigen-binding domain formed by these positioned CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its homologous epitope. For any given heavy or light chain variable region, those skilled in the art can readily identify the amino acids containing the CDR and framework regions, respectively, as they have been precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI.Biol [Journal of Molecular Biology], 196:901-917 (1987)).

[0060] Where a term used and / or accepted in the art has two or more definitions, the definition of the term as used herein is intended to include all such meanings unless expressly stated otherwise. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe discontinuous antigen-binding sites found in the variable regions of both heavy-chain and light-chain polypeptides. This particular region has been described in the following literature: Kabat et al. US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al. , J. MoI. Biol[Journal of Molecular Biology]. 196:901-917 (1987), these references are incorporated herein by reference in their entirety. According to the two definitions of CDR by Kabat and Chothia, overlap or subsets of amino acid residues are included when comparing them. However, the application of any definition used to refer to a CDR of an antibody or its variants is intended to be within the scope of the terminology defined and used herein. For comparison, the corresponding amino acid residues covering these CDRs as defined in the above-cited references are shown in the table below. The exact number of residues covering a particular CDR varies depending on the sequence and size of the CDR. Those skilled in the art can generally determine which residues contain a particular CDR based on the amino acid sequence of the variable region of the antibody.

[0061]

[0062] Kabat et al. also defined a variable domain sequence numbering system applicable to any antibody. Those skilled in the art can explicitly assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat number" refers to the numbering system described in the following reference: Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0063] In addition to the table above, the Kabat numbering system describes these CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (approximately 9 residues after the first cysteine ​​residue), consists of approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, consists of approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty-third amino acid residue after the end of CDR-H2; consists of 3-25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (approximately after one cysteine ​​residue); consists of approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1, consists of approximately 7 residues. CDR-L3 begins at approximately the thirtieth residue following the end of CDR-L2 (i.e., after a cysteine ​​residue); it comprises approximately 7-11 residues and ends at sequence F or WGXG, where X is any amino acid.

[0064] The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, these antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be derived from condricthoids (e.g., from sharks).

[0065] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide used in this disclosure may comprise a polypeptide chain containing a CH1 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide disclosed herein comprises a polypeptide chain containing a CH3 domain. Additionally, antibodies used in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant regions can be modified so that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0066] The heavy chain constant regions of antibodies disclosed herein may originate from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may contain 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 contain a hinge region partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In yet another example, the heavy chain portion may contain a chimeric hinge partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0067] 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 comprises at least one of a constant κ domain or a constant λ domain.

[0068] A "light chain-heavy chain pair" refers to a combination of a light chain and a heavy chain that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0069] As previously mentioned, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the N-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" includes the first (closest to the N-terminus) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the N-terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0070] The “CH1 domain” (also known as the “C1” of the “H1” domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0071] As used herein, the term "hinge region" refers to the portion of a heavy-chain molecule that links the CH1 domain to the CH2 domain; in IgG, this region corresponds to Glu216 to Pro230 of human IgG1, according to the Burton EU numbering system. Molec. Immunol. [Molecular Immunology], 22:161-206 (1985)). The hinge region of other IgG isotypes can be compared with the IgG1 sequence by placing the first and last cysteine ​​residues forming the inter-heavy chain SS 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: the upper, middle, and lower hinge domains (Roux). et al. , J. Immunol [Journal of Immunology] 161:4083 (1998)).

[0072] As used herein, the term "CH2 domain" includes the extended portion of the heavy chain molecule, for example, using conventional numbering schemes, from approximately residue 244 to residue 360 ​​of the antibody (Kabat numbering system, residues 244 to 360; EU numbering system, residues 231 to 340). See Kabat et al. US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique because it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

[0073] The “CH3 domain” (also known as the “C3 domain”) contains a string of residues in the Fc region from the C-terminus to the CH2 domain (i.e. from about amino acid residue 341 to the C-terminus of the antibody sequence, usually at amino acid residues 446 or 447 of IgG, EU numbering system).

[0074] The terms “Fc region,” “Fc domain,” or “crystallizable region fragment” used herein are used to define the C-terminal region of the immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the Fc region of the human IgG heavy chain is generally defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, compositions of complete antibodies may include antibody populations with all K447 residues removed, antibody populations without K447 residues removed, and antibody populations having a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0075] As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group, which can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, with the two heavy chains linked by two disulfide bonds located at positions 239 and 242 (positions 226 or 229, EU numbering system) using the Kabat numbering system.

[0076] As used herein, the term "chimeric antibody" should be understood to mean any antibody in which the immune-reactive region or site is derived from or obtained from a first species, and the constant region (which, according to this disclosure, may be whole, partial, or modified) is derived from a second species. In some embodiments, the target binding region or site will be derived from a non-human source (e.g., mouse or primate), and the constant region will be of human origin.

[0077] In this document, "humanized antibody" is used to describe an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but at least a portion of the VH and / or VL sequences have 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 specifically binds to a target antigen and contains a frame (FR) region and a complementarity-determining region (CDR), wherein the frame region has a substantially identical amino acid sequence to that of a human antibody, and the complementarity-determining region has a substantially identical amino acid sequence to that 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. Humanized antibodies substantially comprise all of at least one, typically two, variable domains (Fab, Fab', F(ab')2, Fv), where all or substantially all CDR regions within the variable domains correspond to the CDR regions of non-human immunoglobulins (i.e., donor antibodies), and all or substantially all frame regions are frame regions of human immunoglobulin common sequences. In one embodiment, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region (Fc) of a human immunoglobulin. In some embodiments, the humanized antibody contains a light chain and at least a variable domain of the heavy chain. The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only one humanized light chain. In some embodiments, the humanized antibody contains only one humanized heavy chain. In specific embodiments, the humanized antibody contains only the humanized variable domain of the light chain and / or the humanized heavy chain.

[0078] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody or antibody moiety binds. If two antibodies or antibody moiety competitively bind to an antigen, they can bind to the same epitope within the antigen.

[0079] "Specific binding" or "specific to" generally refers to an antibody binding to an epitope via its antigen-binding domain, and this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds more easily to a particular epitope via its antigen-binding domain than to a random, unrelated epitope. In this paper, the term "specificity" is used to confirm the relative affinity of a particular antibody for a particular epitope. For example, antibody "A" can be considered more specific to a given epitope than antibody "B," or antibody "A" can be said to have a higher specificity for binding to epitope "C" than to its specificity for binding to related epitope "D."

[0080] As used herein, the term "treat" refers to both therapeutic treatment and preventative or preventative measures aimed at preventing or slowing (alleviating) physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization (i.e., non-deterioration) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and mitigation (whether partial or complete), whether detectable or undetectable. "Treatment" can also refer to extended survival compared to expected survival without treatment. Those in need of treatment include those who already have a condition or disorder, those who are susceptible to a condition or disorder, or those whose condition or disorder requires prevention.

[0081] The terms "subject," "individual," "animal," "patient," or "mammal" refer to any subject who requires diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, domestic animals, farm and zoo animals, racing animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0082] As used herein, phrases such as “to a patient in need of treatment” or “subject in need of treatment” include subjects, such as mammalian subjects, who will benefit from the administration of the antibodies or compositions disclosed herein for purposes such as detection, diagnostic procedures, and / or treatment.

[0083] Anti-DLL3 antibody

[0084] As demonstrated in the accompanying experimental examples, the inventors of this invention were able to generate anti-DLL3 antibodies 9E8D8, 36B7F3, 129H2B9, 148C3A7, 310P3C5, and 362H3D3 (Table 1), all of which exhibit high binding affinity to the human DLL3 protein. This binding is specific because they do not bind to DLL1 or DLL4.

[0085] Domain localization revealed that some of these antibodies bind to certain DLL3 domains (Table A) that had not been previously targeted. For example, 129H2B9 and 148C3A7 bind to the EGF3-4 domain of human DLL3 expressed on cells (S312-E389 of UniProt NO. Q9NYJ7). 9E8D8 and 310P3C5 specifically bind to the EGF6 domain of human DLL3 expressed on cells (R429 to E465 of UniProt NO. Q9NYJ7). In contrast, the benchmark antibody DLL3#3 binds to the proximal extracellular domain (F466 to L492 of UniProt NO. Q9NYJ7), and another benchmark antibody, DLL3-4-001, specifically binds to EGF3.

[0086] Table A. DLL3 Domain Sequence

[0087] According to one embodiment of this disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or the antigen-binding fragment thereof has binding specificity to human DLL3 protein. In some embodiments, the antibody or the antigen-binding fragment thereof includes a heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3 and a light chain variable region (VL) containing VL CDR1, VL CDR2, and VLCDR3.

[0088] 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 the 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 the amino acid sequence selected from the group consisting of SEQ ID NO: 42.

[0089] In some embodiments, VH CDR2 is PTM-derisked. The tested PTM-derisked versions include SEQ ID NO: 111-117 (Table 10B), wherein the N residue is substituted with A, F, H, R, V, W, or Y. 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: 111, 112, 113, 114, 115, 116, or 117; VH CDR3 comprises the 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 the amino acid sequence selected from the group consisting of SEQ ID NO: 42.

[0090] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO: 65-69 and 104-110, while retaining the VH CDR or its PTM re-risked version. In some embodiments, VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO: 70-73, while retaining the VL CDR or its PTM re-risked version.

[0091] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104-110, and VL comprises the amino acid sequence of SEQ ID NO: 73.

[0092] In some embodiments, an antibody that binds to the same epitope on DLL3 as 310P3C5 and its antigen-binding fragment are also provided. In some embodiments, an antibody that binds to the EGF6 domain of the DLL3 protein and its antigen-binding fragment are provided. In some embodiments, an antibody that competes with 310P3C5 for binding to DLL3 and its antigen-binding fragment are also provided.

[0093] 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 comprise the amino acid sequence of SEQ ID NO: 13-18.

[0094] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 1, while retaining the VH CDR or its PTM re-risking version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2, while retaining the VL CDR or its PTM re-risking version.

[0095] 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.

[0096] In some embodiments, an antibody that binds to the same epitope on DLL3 as 9E8D8 and its antigen-binding fragment are also provided. In some embodiments, an antibody that binds to the EGF6 domain of the DLL3 protein and its antigen-binding fragment are provided. In some embodiments, an antibody that competes with 9E8D8 for binding to DLL3 and its antigen-binding fragment are also provided.

[0097] 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 comprise the amino acid sequence of SEQ ID NO: 19-24.

[0098] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 3, while retaining the VH CDR or its PTM re-risked version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 4, while retaining the VL CDR or its PTM re-risked version.

[0099] 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.

[0100] In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on DLL3 as 36B7F3 are also provided. In some embodiments, an antibody and its antigen-binding fragment that bind to the proximal extracellular domain of the DLL3 protein are provided. In some embodiments, an antibody and its antigen-binding fragment that compete with 36B7F3 for binding to DLL3 are also provided.

[0101] In some embodiments, an antibody or antigen-binding fragment derived from antibody 129H2B9 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 25-30.

[0102] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 5, while retaining the VH CDR or its PTM re-risking version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 6, while retaining the VL CDR or its PTM re-risking version.

[0103] 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.

[0104] In some embodiments, an antibody that binds to the same epitope on DLL3 as 129H2B9 and its antigen-binding fragment are also provided. In some embodiments, an antibody that binds to the EGF3-4 domain of the DLL3 protein and its antigen-binding fragment are provided. In some embodiments, an antibody that competes with 129H2B9 for binding to DLL3 and its antigen-binding fragment are also provided.

[0105] 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 comprise the amino acid sequences of SEQ ID NO: 31-36.

[0106] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 7 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 7, while retaining the VH CDR or its PTM re-risking version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 8, while retaining the VL CDR or its PTM re-risking version.

[0107] 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.

[0108] In some embodiments, an antibody that binds to the same epitope on DLL3 as 148C3A7 and its antigen-binding fragment are also provided. In some embodiments, an antibody that binds to the EGF3-4 domain of the DLL3 protein and its antigen-binding fragment are provided. In some embodiments, an antibody that competes with 148C3A7 for binding to DLL3 and its antigen-binding fragment are also provided.

[0109] In some embodiments, an antibody or antigen-binding fragment derived from antibody 362H3D3 is provided. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 43-48.

[0110] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 11, while retaining the VH CDR or its PTM re-risking version. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 12 or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 12, while retaining the VL CDR or its PTM re-risking version.

[0111] 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.

[0112] In some embodiments, an antibody and its antigen-binding fragment that bind to the same epitope on DLL3 as 362H3D3 are also provided. In some embodiments, an antibody and its antigen-binding fragment that bind to the proximal extracellular domain of the DLL3 protein are provided. In some embodiments, an antibody and its antigen-binding fragment that compete with 362H3D3 for binding to DLL3 are also provided.

[0113] In some embodiments, an antibody-antigen binding fragment is also provided, comprising a CDR sequence derived from the CDR sequence disclosed herein, having one, two, or three amino acid substitutions, deletions, and / or additions.

[0114] Multifunctional group molecules

[0115] Multifunctional molecules include antibody or antigen-binding fragments specific to DLL3 (such as those disclosed herein) and one or more antibody or antigen-binding fragments specific to a second antigen or a different epitope on DLL3.

[0116] In some embodiments, the second antigen is a protein expressed on immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells.

[0117] 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.

[0118] Different forms of bispecific antibodies are also provided. In some embodiments, each of the anti-DLL3 fragment and the second fragment is independently selected from Fab fragments, single-chain variable fragments (scFv), or single-domain antibodies. In some embodiments, the bispecific antibody further includes an Fc fragment.

[0119] Bifunctional molecules that include not only antibodies or antigen-binding fragments are also provided. As molecules targeting tumor antigens, antibodies or antigen-binding fragments specific to DLL3 (such as those described herein) can optionally be combined with immune cytokines or ligands via peptide linkers. The linked immune cytokines or ligands 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 blockade effects with local immunomodulation at the tumor site.

[0120] Chimeric antigen receptor

[0121] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising an antibody or fragment thereof disclosed herein as a targeting unit. In some embodiments, the CAR comprises an antibody or fragment thereof disclosed herein, a transmembrane domain, a co-stimulatory domain, and a CD3ε intracellular domain.

[0122] The transmembrane domain can be designed to fuse with an extracellular domain, including an antibody or fragment, optionally via a hinge domain. It can also fuse with an intracellular domain, such as a co-stimulatory domain. In some embodiments, the transmembrane domain may include a native transmembrane region of a co-stimulatory domain (e.g., a TM region of CD28 or 4-1BB used as a co-stimulatory domain) or a native transmembrane domain of a hinge region (e.g., a TM region of CD8α or CD28 used as a hinge domain).

[0123] In some embodiments, a transmembrane domain may include a sequence that crosses the cell membrane but extends into the cytoplasm and / or extracellular space of the cell. For example, a transmembrane domain may include a transmembrane sequence that may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids extending into the cytoplasm and / or extracellular space of the cell. Thus, a transmembrane domain includes a transmembrane region and may further include one or more amino acids extending into the inner or outer surface of the membrane itself; such sequences may still be considered "transmembrane domains".

[0124] In some embodiments, the transmembrane domain fuses with the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine duplexes (GS), glycine-serine-glycine triplets (GSG), or alanine-alanine-alanine triplets (AAA) provide suitable linkers.

[0125] 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 activation domain. Examples of co-stimulatory domains include, but are not limited to, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), and CD27 (T 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 α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (tactile), CD100 (SEMA4D), CD 103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 ( KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A) , CD158F2 (KIR2DL5B), CD158K (KTR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), 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) Inducible T cell costimulatory molecules (ICOS), LFA-1 (CD11a / CD18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2R β, IL-2R γ, IL-7R α, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc γ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activating NK cell receptor, Toll ligand receptor and fragments or combinations thereof.

[0126] In some embodiments, the cytoplasmic portion of the CAR further includes a signal transduction / activation domain. In one embodiment, the signal transduction / activation domain is a CD3ε domain, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the CD3ε domain.

[0127] Polynucleotides, mRNA, and methods for expressing or preparing antibodies

[0128] This disclosure also provides polynucleotide or nucleic acid molecules encoding antibodies, variants or derivatives thereof, or CARs disclosed herein. The polynucleotides disclosed herein may encode the entire heavy and light chain variable regions of antigen-binding peptides, variants or derivatives thereof, on the same or different polynucleotide molecules. Furthermore, the polynucleotides disclosed herein may encode portions of the heavy and light chain variable regions of antigen-binding peptides, variants or derivatives thereof, on the same or separate polynucleotide molecules.

[0129] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA may be introduced into target cells to express an antibody or a fragment thereof.

[0130] mRNA can be synthesized using any of a variety of known methods. For example, mRNA can be synthesized via in vitro transcription (IVT). In short, IVT typically uses a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions will vary depending on the specific application.

[0131] In some embodiments, to prepare mRNA encoding an antibody, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter (e.g., a T3, T7, or SP6 promoter) for in vitro transcription, followed by the desired nucleotide sequence for encoding the desired antibody (e.g., encoding a heavy or light chain) and a termination signal.

[0132] The mRNA sequence encoding the desired antibody (e.g., encoding the heavy or light chain) can be determined using standard methods and incorporated into a DNA template. For example, virtual reverse translation based on a degenerate genetic code can be performed, starting with the desired amino acid sequence (e.g., the desired heavy or light chain sequence). Optimization algorithms can then be used to select appropriate codons. Typically, the G / C ratio can be optimized to achieve the highest possible G / C ratio, while the frequency of the tRNA can be considered as much as possible based on codon usage. The optimized RNA sequence can be constructed and displayed, for example, using a suitable display device, and compared with the original (wild-type) sequence. Secondary structures can also be analyzed to calculate the stability and instability properties of the RNA, or its regions can be calculated separately.

[0133] mRNA can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to enhance its stability. Modifications to mRNA can include, for example, modifications to the nucleotides of the RNA. Therefore, modified mRNA can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleoside analogs or derivatives that are purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, and 1-methyl-inosine. 5-Uracil, 5-dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyluracil, 5 -Methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, huaistin, and amide phosphates, thiophosphates, peptide nucleotides, methylphosphonates, 7-denitroguanine, 5-methylcytosine, and inosine. The preparation of such analogues is known to those skilled in the art, for example, from U.S. Patent Nos. 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 disclosures of which are incorporated herein by reference in their entirety.

[0134] In some embodiments, mRNA (e.g., mRNA encoding both heavy and light chains) may contain RNA backbone modifications. Typically, backbone modifications are modifications that chemically modify the phosphate groups of the backbone of the nucleotides contained in the RNA. Exemplary backbone modifications generally include, but are not limited to, modifications from the group consisting of methylphosphonates, methylaminophosphates, aminophosphates, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphate)), borophosphates, positively charged guanidine groups, etc., which means replacing the phosphodiester bonds with other anionic, cationic, or neutral groups.

[0135] In some embodiments, mRNA (e.g., mRNA encoding both the heavy and light chains) may contain sugar modifications. Typical sugar modifications are chemical modifications to the sugars of the nucleotides it contains, including but not limited to sugar modifications selected from the group consisting of: 2'-deoxy-2'-fluoro-oligonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), and 2'-deoxy-2'-deamine-oligonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate). ), 2'-O-alkyl oligonucleotides, 2'-deoxy-2'-C-alkyl oligonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligonucleotides and their isomers (2'-cytarabine 5'-triphosphate, 2'-cytarabine 5'-triphosphate) or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0136] In some embodiments, mRNA (e.g., mRNA encoding the heavy and light chains) may contain modifications of nucleotide bases (base modifications). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine ribonucleoside 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-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine nucleoside 5'-triphosphate, 7-deadenosine 5'-triphosphate, 7-deadenosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole ribonucleoside 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 xanthine nucleoside 5'-triphosphate.

[0137] Typically, mRNA synthesis involves adding a “cap” at the N-terminus (5’) and a “tail” at the C-terminus (3’). The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the “tail” is to protect the mRNA from degradation by exonucleases.

[0138] Therefore, in some embodiments, the mRNA (e.g., mRNA encoding both the heavy and light chains) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphates via a guanylate transferase, creating a 5'5'5 triphosphate bond; then, the 7-nitrogen of guanine is methylated via a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5)A and G(5)ppp(5')G.

[0139] Therefore, in some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) includes a 3' poly(A) tail. The poly(A) tail at the 3' end of the mRNA typically comprises about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, the mRNA encoding an antibody (e.g., mRNA encoding the heavy and light chains) includes a 3' poly(C) tail. A suitable poly-C tail at the 3' end of mRNA typically comprises about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to or may replace the poly-A tail.

[0140] Therefore, in some embodiments, the mRNA (e.g., mRNA encoding the 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 an iron-responsive element. In some embodiments, the length of the 5' untranslated region can be about 50 to 500 nucleotides (e.g., about 50 to 400 nucleotides, about 50 to 300 nucleotides, about 50 to 200 nucleotides, or about 50 to 100 nucleotides).

[0141] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) includes a sequence encoding a signal peptide, such as those described herein. In a particular embodiment, 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 linked at the N-terminus to a sequence encoding the heavy or light chain.

[0142] The technology of this invention can be used to deliver any antibody known in the art and antibodies that can be generated against a desired antigen using standard methods. This invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or compositions, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0143] Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptide disclosed herein are fully human. Fully human antibodies can be prepared using techniques described in the art and as 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 an antigen challenge, but whose endogenous loci have been deactivated. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents: 6,150,584; 6,458,592; 6,420,140, ​​the contents of which are incorporated herein by reference in their entirety.

[0144] Treatment and Uses

[0145] As described herein, the antibodies, variants, derivatives, or antibody-drug conjugates disclosed herein may be used in certain therapeutic and diagnostic approaches.

[0146] This disclosure further relates to antibody-based therapies involving the administration of the disclosed antibodies, fragments, or antibody-drug conjugates to patients (e.g., animals, mammals, and humans) to treat one or more of the disorders or conditions described herein. The therapeutic compounds disclosed include, but are not limited to, the antibodies disclosed (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the antibodies disclosed (including variants and derivatives thereof as described herein).

[0147] The antibodies disclosed herein can also be used to treat or inhibit cancer. As provided above, DLL3 can be overexpressed in tumor cells, particularly in liver, stomach, pancreas, esophagus, ovary, and lung tumors. Inhibition of DLL3 has been shown to be useful in the treatment of tumors.

[0148] Therefore, in some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method involves administering an effective amount of the antibody, fragment, or antibody-drug conjugate disclosed herein to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient overexpresses DLL3.

[0149] This disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy. Suitable cells can be used, transduced with or contacted with a CAR encoding a CAR comprising the anti-DLL3 antibody disclosed herein (or alternatively engineered to express the anti-DLL3 antibody disclosed herein). After such contact or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer as disclosed herein. Cells (e.g., T cells) may be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, but are not limited thereto.

[0150] In some embodiments, the cells are isolated from the cancer patient's own body. In some embodiments, the cells are provided by a donor or from a cell bank. Isolating cells from a cancer patient can minimize adverse immune responses.

[0151] Non-limiting 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 stomach cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, and cutaneous T-cell lymphoma. In some embodiments, the cancer is small cell lung cancer (SCLC).

[0152] Other diseases or conditions associated with increased cell survival (which can be treated, prevented, diagnosed, and / or predicted by the antibodies disclosed herein or their variants or derivatives) include, but are not limited to, the progression and / or metastasis of malignancies and related disorders, such as 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 lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, and chondrosarcoma). Osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, 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 gland carcinoma, cystadenoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

[0153] The specific dosage and treatment regimen for any given patient will depend on a number of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health condition, sex, diet and timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of healthcare professionals regarding these factors is within the realm of general technical skill in this field. The dosage will also depend 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 using principles of pharmacology and pharmacokinetics well known in the art.

[0154] Methods of administering antibodies, antibody fragments, or antibody-drug conjugates include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or bolus injection, absorption through the epithelial or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides disclosed herein can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0155] As used in this article, the term “parenteral” refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0156] Administration can be systemic or local. Alternatively, it is preferable to introduce the disclosed antibodies into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated by, for example, an intraventricular catheter attached to a reservoir (such as an Ommaya reservoir). Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and formulations containing nebulizing agents.

[0157] Preferably, the antigen-binding peptides or compositions disclosed herein are applied topically to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion during surgery, topical application (e.g., in conjunction with postoperative wound dressings), by injection, via catheter, via suppository, or via implantation of a porous, non-porous, or gelatinous material, including membranes such as sialastic membranes or fibers. Preferably, when administering the proteins (including antibodies) disclosed herein, care must be taken to use materials that do not absorb the proteins.

[0158] The amount of the disclosed antibody, fragment, or antibody-drug conjugate effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined using standard clinical techniques. Additionally, in vitro assays may optionally be used to help determine the ideal dosage range. The precise dosage to be used in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be determined based on the physician's judgment and the individual patient's situation. The effective dosage can be deduced from dose-response curves derived from in vitro or animal model testing systems.

[0159] As a general recommendation, the dosage of the antibodies, fragments, or antibody-drug conjugates disclosed herein administered to patients is typically between 0.001 mg / kg and 100 mg / kg of patient body weight, between 0.01 mg / kg and 20 mg / kg of patient body weight, or between 0.5 mg / kg and 10 mg / kg of patient body weight. Generally, due to the immune response to exogenous peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, the dosage of human antibodies can usually be reduced and the frequency of administration decreased. Furthermore, the dosage and frequency of administration of the antibodies disclosed herein can be reduced by modifications (e.g., lipidation) to enhance antibody uptake and tissue penetration (e.g., into the brain).

[0160] In another embodiment, the compositions disclosed herein are administered in combination with cytokines. Cytokines that can be administered with the compositions disclosed herein 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.

[0161] In another embodiment, the disclosed composition is administered in combination with other therapeutic or preventative regimens, such as radiotherapy.

[0162] Composition

[0163] This disclosure also provides pharmaceutical compositions. Such compositions 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).

[0164] In certain embodiments, the term "pharmaceuticalally acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more particularly in humans. Furthermore, "pharmaceuticalally acceptable carrier" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0165] The term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the drug composition is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for regulating tension, such as sodium chloride or dextrose, are also considered. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated into suppositories with conventional 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 drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide (preferably in purified form) and an appropriate carrier to provide a suitable form of administration to the patient. The formulation should be suitable for the mode of administration. Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0166] In one embodiment, the composition is formulated according to standard procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Generally, the ingredients are provided individually or in combination in unit dosage forms, for example, as lyophilized powders or anhydrous concentrates in sealed containers (such as ampoules or pouches), with the amount of active agent indicated. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, a single ampoule of sterile water for injection or saline can be provided to mix the ingredients prior to administration.

[0167] Example

[0168] Example 1. Production of mouse anti-human DLL3 antibody

[0169] This example describes the use of hybridoma technology to generate mouse anti-human DLL3 monoclonal antibodies.

[0170] Immunogen: Two immunogens are available, one for use in mouse immunization. One consists of the extracellular domain (ECD, UniProt NO. Q9NYJ7, A27-L492) of human DLL3 protein fused with a human Fc fragment (hDLL3-hFc, Acro Bio, catalog number DL3-H5255). The other is an ECD of human DLL3 protein fused with a his tag (hDLL3-his, Acro Bio, catalog number DL3-H52H4).

[0171] Mouse immunization regimen: To generate mouse monoclonal antibodies against human DLL3, BALB / c and C57BL / 6 mice were immunized intraperitoneally or subcutaneously with hDLL3-hFc or hDLL3-his protein every two weeks. Serum titers of immunized mice were monitored using an ELISA targeting human hDLL3-his protein. After several rounds of immunization, mice with sufficient hDLL3-his protein titers were boosted and selected for fusion.

[0172] Cell fusion and hybridoma screening: Selected mouse spleen cells were fused with the mouse myeloma cell line Sp2 / 0 via electrofusion. These hybridoma cells were then plated in 96-well microplates, and mouse antibodies were secreted in the supernatant. In the initial screening process, positive clones were selected using high-throughput screening based on the binding of the hDLL3-his protein as detected by ELISA, and the binding of the ECD of human DLL3 overexpressed on HEK293 cells (HEK293-hDLL3, custom-made by Genomeditech) or the constitutively DLL3-expressing SCLC cell line SHP77 (ATCC, catalog number CRL-2195). Confirmatory screening was then performed, using ELISA to filter out clones that non-specifically bind to human DLL1 his-tag fusion protein (hDLL1-his, Sino Biologial, catalog number 11635-H08H) or human DLL4 his-tag fusion protein (hDLL4-his, Sino Biologial, catalog number 10171-H08H). Clones binding to cynomolgus monkey DLL3 his-tag fusion protein (cynoDLL3-his, Acro Bio, catalog number DL3-C52H3) were identified using ELISA.

[0173] Subcloning screening and sequencing: Subcloning of positive primary clones from each fusion that meet the above criteria was performed using restrictive dilution to ensure that the hybridoma subclones originated from a single parental cell. The selection criteria for subclones were the same as those for the primary clones. Subclones that specifically bind to hDLL3 and cynoDLL3 but not to hDLL1 and hDLL4 were selected for subsequent sequencing.

[0174] The obtained variable region sequence of the mouse antibody was fused with the constant region of human IgG1 to generate a chimeric DLL3 mAb. The DNA sequence of the chimeric antibody was cloned into the pcDNA3.4 plasmid and expressed in CHO-K1 cells, followed by purification of the antibody using a protein A affinity chromatography column or beads. The purified chimeric antibody was subjected to serial in vitro screening to determine affinity, binding capacity, specificity, and species cross-reactivity.

[0175] Based on the performance of the screening assays, six chimeric mAbs (including 9E8D8, 36B7F3, 129H2B9, 148C3A7, 310P3C5, and 362H3D3) were selected for further analysis. 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. Table 3 lists the heavy and light chain sequences of all antibodies. Two benchmark DLL3 mAbs, DLL3-4-001 (CC) (sequence sourced from the IMGT database) and DLL3#3 (see WO 2019234220), are the DLL3 binding elements of two T-cell adaptor (TCE) talatazumab and BI765432, which are currently undergoing clinical evaluation.

[0176] Table 1. Variable region sequence of DLL3 chimeric mAb (underlined CDR)

[0177] Table 2. CDR Sequences (Kabat Numbers)

[0178] Table 3. Sequences of DLL3-chimeric mAbs

[0179] Example 2. Protein binding activity of chimeric monoclonal antibodies targeting DLL3

[0180] 2.1. Combination of ELISA detection with human DLL3

[0181] To determine the binding affinity of the chimeric mAb to human DLL3 protein, an ELISA-based binding assay was performed as follows. Briefly, hDLL3-his protein was diluted to 1 μg / mL with DPBS buffer and adsorbed into the wells of a 96-well microplate and incubated overnight at 4°C. After blocking the wells with 1% bovine serum albumin (BSA) to prevent nonspecific binding, the DLL3 chimeric mAb, the baseline antibody DLL3-4-001 (CC), and DLL3#3 or an isotype control were titrated at 100 nM at 3-fold dilutions and added to the wells pre-adsorbed with hDLL3-his protein. The mixture was incubated at room temperature (RT) for 1 hour. The bound DLL3 mAb was recognized by a detection antibody against human IgG Fc conjugated to horseradish peroxidase (HRP) (Jackson Immuno, catalog number 109-035-008). The substrate of HRP, tetramethylbenzidine (TMB), was added to the wells to visualize the binding signal. After sufficient color development, stop solution was added to the wells. The absorbance of the signal was detected at 450 nm using an Envision multi-label microplate reader (PerkinElmer). A graph was generated using a four-parameter nonlinear regression curve fitted in Graphpad Prism 9 software, and statistical analysis was performed.

[0182] like Figure 1A As shown, all DLL3 chimeric antibodies effectively bound to human DLL3 protein. Table 4 summarizes the EC50 values ​​of the binding curves for each antibody.

[0183] 2.2 ELISA detection of binding to DLL3 protein in cynomolgus monkeys and mice

[0184] To determine the cross-reactivity of the chimeric Ab with 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.

[0185] like Figure 1B and Figure 1C As shown, all DLL3 chimeric Abs effectively bound to cynomolgus monkey DLL3 and mouse DLL3 proteins.

[0186] Table 4 summarizes the EC50 values ​​of the binding curves for each antibody.

[0187] 2.3. ELISA detection combined with human DLL1 and DLL4

[0188] Because family members DLL1 and DLL4 have highly similar sequences to DLL3, it is necessary to determine the specificity of DLL3 chimeric antibodies.

[0189] To determine the binding affinity of the chimeric Ab to human DLL1 and DLL4 proteins, an ELISA binding assay was performed as described above. hDLL1-his protein (Sino Biological, catalog number 11635-H08H) and hDLL4-his protein (Sino Biological, catalog number 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. WO 2015005632, ABL Bio) served as positive controls.

[0190] like Figure 2A and Figure 2B As shown, most DLL3 chimeric antibodies exhibit negligible binding to either hDLL1 or hDLL4 proteins, similar to benchmark antibodies. However, 362H3D3 shows detectable binding to both human DLL1 and DLL4 proteins, which may indicate cross-reactivity of this DLL3 antibody with the other two family members.

[0191] Table 4. Binding activity of DLL3 antibody to antigen protein

[0192] --: No combination

[0193] 2.4. Affinity Measurement

[0194] Use Biacroe TM 8K assays were used to determine the binding affinity of the chimeric antibody to human DLL3 protein. In short, the antibody (1 or 2 μg / ml) was captured using a Pro-A chip. 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. Antigen association was allowed for 120 s, followed by dissociation for 150–400 s. Biacore was used. TM Data analysis was performed using 8K evaluation software. The results showed that all chimeric DLL3 antibodies exhibited high affinity for human DLL3, similar to the baseline antibody (Table 5).

[0195] Table 5. By Biacroe TM Results of chimeric antibody affinity ranking

[0196] 2.5. Domain localization of chimeric DLL3 antibodies

[0197] To evaluate the exact binding domain of the chimeric mAb to human DLL3, the following cell-based binding assays were performed. Briefly, these assays used HEK293 cells with a series of overlapping ECDs stably expressing human DLL3. The human DLL3 protein (UniProt NO. Q9NYJ7, A27-K618) has a unique extracellular structure (A27-L492) consisting of a DSL domain, six EGF-like repeat sequences, and an unstructured proximal extracellular region (MPER) adjacent to the transmembrane domain (TM). Therefore, cell lines expressing TM and ICD conjugates of DLL3 were constructed (custom-made by Genomeditech). These included 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). The specified DLL3 chimeric Ab, benchmark antibody, or allotype control was diluted to 100 nM in staining buffer (PBS buffer containing 2% FBS) and then plated in 96-well microplates with 5 x 10⁻⁶ cells. 4 Selected cells were incubated at 4°C for 30 min. Antibodies binding to cell surface antigens were detected using the goat anti-human IgG (H+L) cross-adsorption secondary antibody Alexa Fluor™ 488 (Thermo Fisher Scientific, catalog number A-11013), at a dilution of 1:2000. Cells were analyzed using a MACSQuant® Analyzer 16 flow cytometer (Miltenyi Biotec BV & Co. KG). Data analysis was performed using Flowjo 10.0 software. Graphs were generated and statistical analysis was performed using a four-parameter nonlinear regression curve fitted in Graphpad Prism 9 software.

[0198] like Figure 3As shown in Table 6, these DLL3 chimeric antibodies bind to different ECD domains of human DLL3 expressed on HEK293 cells. More specifically, 129H2B9 and 148C3A7 bind to the EGF3-4 domain of human DLL3 expressed on cells. 9E8D8 and 310P3C5 specifically bind to the EGF6 domain of human DLL3 expressed on cells. 36B7 and 362H3D3 bind to the proximal extracellular domain, which is similar to the benchmark antibody DLL3#3. Another benchmark antibody, DLL3-4-001 (CC), specifically binds to EGF3, consistent with previously reported data.

[0199] Table 6. Domain-binding properties of chimeric antibodies

[0200] Example 3. Cell-binding activity of chimeric monoclonal antibodies targeting DLL3

[0201] 3.1 Binding ability with human DLL3 overexpressed on HEK-293 cells

[0202] To evaluate the binding activity of chimeric mAbs to human DLL3 expressed on cells, the following cell-based binding assays were performed. Briefly, HEK293 cells (HEK293-hDLL3) stably expressing high levels of human DLL3 ECD were used in these assays. The designated DLL3 chimeric Ab, baseline antibody, or allotype control was diluted 3-fold starting at 50 nM in staining buffer (PBS buffer containing 2% FBS). Antibody dilutions were then plated in 96-well microplates with 5 x 10⁻⁶ microplates. 4 Indicator cells were incubated at 4°C for 30 min. Antibodies binding to cell surface antigens were detected using the goat anti-human IgG (H+L) cross-adsorption secondary antibody Alexa Fluor™ 488 (Thermo Fisher Scientific, catalog number A-11013), at a dilution of 1:2000. Cell analysis was performed using a MACSQuant® Analyzer 16 flow cytometer (Miltenyi Biotec BV & Co. KG). Data analysis was performed using Flowjo 10.0 software. Graphs were generated and statistical analysis was performed using a four-parameter nonlinear regression curve fitted in Graphpad Prism 9 software.

[0203] like Figure 4AAs shown, all DLL3 chimeric antibodies bound efficiently to human DLL3 expressed on HEK293 cells in a dose-dependent manner. Notably, compared to the baseline antibodies DLL3-4-001 (CC) and DLL3#3, the chimeric antibodies 129H2B9, 148C3A7, 310P3C5, and 362H3D3 exhibited higher binding potency to human DLL3 expressed on HEK293 cells, as evidenced by improved EC50 compared to DLL3-4-001 (CC) or improved maximum binding signal compared to DLL3#3. The maximum binding potency of 9E8D8 and 36B7F3 to human DLL3 expressed on HEK293 cells was comparable to that of EGF3 binding to the baseline DLL3-4-001 (CC).

[0204] Table 7 summarizes the EC50 values ​​for binding ability to human DLL3 expressed on cells.

[0205] 3.2 Binding ability with cyno DLL3 overexpressed on cells

[0206] To evaluate the species cross-reactivity of the chimeric mAb with cynomolgus monkey DLL3 expressed on cells, the following cell-based binding assay was performed according to the previously described protocol. Briefly, HEK293 cells stably expressing full-length human cyno DLL3 (HEK293-cynoDLL3) were constructed (custom-made by Cusabio). In this assay, a specified DLL3 chimeric mAb, a baseline antibody, or an isotype control was tested.

[0207] like Figure 4B As shown, all DLL3 chimeric antibodies effectively bound to cyno DLL3 expressed on HEK293 cells, and their binding efficacy was comparable to that of human DLL3.

[0208] Table 7 summarizes the EC50 values ​​for binding ability to cynomolgus monkey DLL3 expressed on cells.

[0209] 3.3 Binding to human DLL3 expressed on tumor cells

[0210] To evaluate the binding activity of the chimeric mAb to human DLL3 expressed on tumor cells, the human SCLC cell lines SHP77 and NCI-H82, which express low levels of DLL3, were used in a cell-based binding assay, following the protocol described in Example 3.1.

[0211] like Figure 5A and Figure 5BAs shown, all DLL3 chimeric antibodies effectively bound to human DLL3 expressed on SCLC tumor cell lines SHP77 or NCI-H82. Interestingly, unlike the binding trend on HEK293 cells overexpressing DLL3, in both of these DLL3-expressing tumor cell lines, DLL3 mAbs 129H2B9 and 148C3A7, which bind to the distal membrane region, showed significantly better binding efficacy than mAbs (including 9E8D8, 36B7F3, 310P3C5, and 362H3D3) that bind to the proximal membrane region, indicating that distal membrane conjugates have stronger binding ability. Figure 5A and Figure 5B ).

[0212] 3.4 Binding ability with overexpressed human DLL1 and DLL4 on cells

[0213] To rule out non-specific binding of the chimeric mAb to human DLL1 and DLL4 expressed on cells, a cell-based binding assay was employed as previously described. Briefly, CHO-K1 cells stably expressing full-length human DLL1 and DLL4 (CHO-K1-hDLL1 and CHO-K1-hDLL4, Genomeditech) were constructed. The sequences of human DLL1 and DLL4 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 Systems, MAB1506) antibodies. Designated DLL3 chimeric Abs, baseline antibodies, or isotype controls were tested in the assay.

[0214] like Figure 6A As shown, in contrast to the significant binding ability of the positive control DLL1 mAb pidiliterizumab to DLL1, no specific binding to human DLL1 expressed on CHO-K1 cells was detected in most DLL3 chimeric antibodies (including 9E8D8, 36B7F3, 129H2B9, 148C3A7, 310P3C5, and two benchmark antibodies). 362H3D3 only showed weak binding to DLL1 overexpressed on CHO-K1 cells at the highest dose level of 100 nM. Furthermore, similar to the two benchmark antibodies DLL3-4-01 (CC) and DLL3#3, the binding of all DLL3 chimeric antibodies to DLL4 overexpressed on CHO-K1 cells was negligible, 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 for binding to human DLL3 expressed on cells.

[0215] Table 7. Binding characteristics of chimeric antibodies to different antigens

[0216] --: No combination

[0217] Example 4. Humanization of chimeric DLL3 antibodies

[0218] The variable region of the 310P3C5 chimeric antibody was selected for humanization.

[0219] In short, the amino acid sequences of VH and VL were compared with existing human Ig gene sequence databases to determine the best-matching human germline Ig gene sequence overall. Then, the chimeric antibody heavy and light chain CDRs were transplanted into candidate germlines. A 3D model of the CDR-transplanted antibody was generated using a Molecular Operating Environment (MOE) to determine if any key human amino acids in the frame region must be reverted to their corresponding mouse amino acids to maintain CDR conformation and antibody function.

[0220] For the heavy chain of 310P3C5, the candidate phylogenetic sequence is IGKV1-18 01 or IGKV1-3 01 Gene. For the light chain 310P3C5, the candidate germline sequence is... IGKV1-12 01 or IGKV1-33 01 Genes. For heavy chains, IGKV1- 18 01 The frame includes M48I, R67Q, V68A, M70L, T72V, and T74K, as well as IGKV1-3 01 R38K, M48I, V68A, I70L, R72V, T74K, and S84R in the framework are involved in reversion mutations. For the light chain, IGKV1-33... 01 and IGKV1-12 Y49S, Y49S, and T69K in the 01 frame region were involved in the reversion mutation.

[0221] Different combinations of reversion mutation sites were selected to generate the variable regions of humanized antibodies. Table 8A lists the sequences of the heavy and light chain variable regions of the 310P3C5 humanized antibody. Table 8B lists the pairings of VH and VL of individual humanized antibodies. The variable regions of the humanized antibodies were then fused with the constant region of human IgG1 for antibody production and functional characterization. Table 8C lists the complete sequences of the heavy and light chains of the humanized antibodies.

[0222] Table 8A. Variable region sequence of humanized antibodies (underline / bold indicates CDR; bold / italic indicates reversion mutation) Change)

[0223] Table 8B. Pairing of humanized antibodies VH and VL

[0224] Table 8C. Sequences of the heavy and light chains of humanized antibodies

[0225] Example 5. Antigen-binding properties of humanized antibodies

[0226] 5.1 Binding with recombinant human DLL3

[0227] To evaluate antigen-binding activity, humanized antibodies were tested using an ELISA assay as described above. Figure 7 As shown, the binding efficacy of all humanized 310P3C5 antibodies to human DLL3 protein is comparable to that of their parental chimeric antibodies.

[0228] 5.2 via Biacore TM Affinity sequencing of humanized antibodies

[0229] To explore whether humanized antibodies can maintain their binding kinetics, Biacore was used. TM Single-dose affinity sequencing was performed. Antibody (2 μg / ml) was captured using a protein A chip. 100 nM human DLL3-his protein was injected onto the captured antibody at a flow rate of 30 μL / min for 120 s. Antigen dissociation was allowed for 400 s. [The data was then processed using Biacore.] TM Experiments were conducted on 8K using Biacore. TM 8K evaluation software is used for data analysis.

[0230] The results in Table 9 show that several humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14 and 310P3C5-z15, have comparable affinity to their chimeric antibodies.

[0231] Table 9. Affinity ranking results of humanized antibodies

[0232] 5.3 Binding with HEK293 cells overexpressing human DLL3 and cynomolgus monkey DLL3

[0233] To evaluate the binding characteristics with human DLL3 and cynomolgus monkey DLL3 overexpressed on cells, the selected humanized antibody was analyzed by FACS according to the protocol described in the preceding examples.

[0234] like Figure 8A and Figure 8B As shown, compared to chimeric antibodies, all selected humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14, and 310P3C5-z15, showed binding to human DLL3 overexpressed on HEK293 cells, which was significantly superior to the two benchmark antibodies DLL3-4-001 (CC) and DLL3#3. Furthermore, all these humanized mAbs also maintained binding to cyno DLL3 expressed on HEK293 cells.

[0235] 5.4 Binding to human DLL3 expressed on cancer cell lines

[0236] To evaluate the binding properties with human DLL3 expressed on cancer cells, the selected humanized antibody was analyzed by FACS according to the previously described protocol.

[0237] like Figure 9A and Figure 9B As shown, compared with chimeric antibodies, all selected humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14 and 310P3C5-z15, showed that they maintained binding to human DLL3 endogenously expressed on tumor cells SHP77 and NCI-H82, which was superior to the benchmark antibody DLL3#3.

[0238] 5.5 Binding with human DLL1 and DLL4 overexpressed on CHO-K1 cells

[0239] To rule out nonspecific binding of humanized antibodies to human DLL1 and DLL4 expressed on cells, the selected humanized antibodies were analyzed by FACS according to the protocol described in the previous examples.

[0240] like Figure 10A and Figure 10B As shown, all humanized antibodies, including 310P3C5-z4, 310P3C5-z8, 310P3C5-z12, 310P3C5-z14 and 310P3C5-z15, showed negligible binding to human DLL1 and DLL4 overexpressed on CHO-K1 cells.

[0241] Example 6. Optimization of Humanized Antibodies

[0242] In some cases, humanized antibodies are further optimized to improve their developability, including long-term stability, manufacturability, and low heterogeneity. One key factor, post-translational modifications (PTMs), such as deamidation, isomerization, glycosylation, and oxidation, can affect developability and further impair the potency, efficacy, and safety of therapeutic antibodies. In this example, computational tools are used to predict PTM-prone sites to facilitate the engineering of antibodies with better developability.

[0243] 6.1 Design of PTM site removal

[0244] In this case, MOE was used to predict the PTM-prone site. Amino acid N55, located in the CDR region of the 310P3C5 antibody heavy chain, was identified as a potential deamidation site. Therefore, this amino acid was replaced with A, F, H, R, V, W, or Y. Table 10A shows the variable region sequence of the PTM-removed humanized antibody, and Table 10B shows the modified VH CDR2 sequence. The variable region of the humanized antibody was then fused with the constant region of human IgG1 for antibody production and functional characterization.

[0245] Table 10A. Sequences of humanized antibody variable regions with PTM removed (underline indicates CDR; bold / italic indicates predicted) PTM sites and mutations)

[0246] Table 10B. Optimized VH CDR2 sequences

[0247] 6.2 Binding of PTM-removed humanized antibody to HEK293 cells expressing human DLL3

[0248] To evaluate the binding properties with human DLL3 expressed on cells, the PTM-removed humanized antibody was analyzed by FACS according to the protocol described in the preceding examples.

[0249] like Figure 11A and Figure 11B As shown, compared with the parental humanized antibody 310P3C5-z15, all selected PTM-removed humanized antibodies showed binding to human DLL3 overexpressed on HEK-293 cells or endogenously expressed in the tumor cell line SHP77.

[0250]

[0251] The scope of this disclosure is not limited to the particular embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover such modifications and variations, provided they fall within the scope of the appended claims and their equivalents.

[0252] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody or an antigen-binding fragment thereof, which is specific for human Delta-like ligand 3 (DLL3) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3, and the VL comprises VL CDR1, VL CDR2, and VL CDR3, wherein: (a) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 37; The VH CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and 111-117; The VH CDR3 contains the amino acid sequence of SEQ ID NO: 39; The VL CDR1 contains the amino acid sequence of SEQ ID NO: 40; The VL CDR2 contains the amino acid sequence of SEQ ID NO: 41; and The VL CDR3 contains the amino acid sequence of SEQ ID NO: 42; (b) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO:13-18; (c) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO:19-24; (d) The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequences of SEQ ID NO: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 NO: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 NO:43-48.

2. The antibody or its antigen-binding fragment according to claim 1, wherein: (a) The VH CDR1 contains the amino acid sequence of SEQ ID NO: 37; The VH CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 38 and 111-117; The VH CDR3 contains the amino acid sequence of SEQ ID NO: 39; The VL CDR1 contains the amino acid sequence of SEQ ID NO: 40; The VL CDR2 contains the amino acid sequence of SEQ ID NO: 41; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:

42.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110 or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 65-69 and 104-110.

4. The antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73 or a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 70-73.

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

73.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein (b) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 13-18.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 or a peptide having at least 90% sequence identity with SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 2 or a peptide having at least 90% sequence identity with SEQ ID NO:

2.

8. The antibody or antigen-binding fragment thereof according to claim 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.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein (c) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 19-24.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the VH comprises the amino acid sequence of SEQ ID NO: 3 or a peptide having at least 90% sequence identity with SEQ ID NO: 3, and the VL comprises the amino acid sequence of SEQ ID NO: 4 or a peptide having at least 90% sequence identity with SEQ ID NO:

4.

11. The antibody or antigen-binding fragment thereof according to claim 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.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein (d) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 25-30.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5 or a peptide having at least 90% sequence identity with SEQ ID NO: 5, and the VL comprises the amino acid sequence of SEQ ID NO: 6 or a peptide having at least 90% sequence identity with SEQ ID NO:

6.

14. The antibody or antigen-binding fragment thereof according to claim 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.

15. The antibody or antigen-binding fragment thereof according to claim 1, wherein (e) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 31-36.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 or a peptide having at least 90% sequence identity with SEQ ID NO: 7, and the VL comprises the amino acid sequence of SEQ ID NO: 8 or a peptide having at least 90% sequence identity with SEQ ID NO:

8.

17. The antibody or antigen-binding fragment thereof according to claim 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.

18. The antibody or antigen-binding fragment thereof according to claim 1, wherein (f) the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO: 43-48.

19. The antibody or antigen-binding fragment thereof according to claim 18, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 or a peptide having at least 90% sequence identity with 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 with SEQ ID NO:

12.

20. The antibody or antigen-binding fragment thereof according to claim 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.

21. An antibody or an antigen-binding fragment thereof, which is specific for human Delta-like ligand 3 (DLL3) protein and competitively binds to the DLL3 protein with an antibody or a fragment thereof according to any one of claims 1-20.

22. An antibody or an antigen-binding fragment thereof, which is specific for human Delta-like ligand 3 (DLL3) protein and binds to an EGF3-4 domain or an EGF6 domain.

23. The antibody or fragment thereof according to any one of claims 1-22, 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.

24. The antibody or fragment thereof according to any one of claims 1-23, wherein it is humanized.

25. A multispecific antibody comprising an antigen-binding fragment according to any one of claims 1-23 and one or more antibodies or antigen-binding fragments having binding specificity to non-DLL3 target antigens.

26. A chimeric antigen receptor (CAR) comprising an antigen-binding fragment, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain according to any one of claims 1-25.

27. One or more polynucleotides encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-25 or a CAR according to claim 26.

28. The polynucleotide of claim 27, wherein it is one or more mRNAs.

29. The polynucleotide of claim 28, wherein the mRNA is chemically modified.

30. A cell comprising the polynucleotide according to claim 28 or 29.

31. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-25 or a CAR according to claim 26, and a pharmaceutically acceptable carrier.

32. A method of treating cancer in a patient in need, the method comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-25, or a CAR according to claim 26.

33. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-25 or the CAR according to claim 26 for the preparation of a medicament for treating cancer.

34. The method of claim 32 or the use of claim 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.

35. The method of claim 32 or the use of claim 33, wherein the cancer is small cell lung cancer (SCLC).

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