DLL3 binding molecule and use thereof

Abstract

Provided are an anti-DLL3 antibody or an antigen-binding fragment, multispecific antigen-binding construct, immune effector cell, antibody conjugate or pharmaceutical composition thereof, and a use thereof. Also provided are a polynucleotide, an expression vector, and a method for producing the anti-DLL3 antibody or the antigen-binding fragment thereof.

Description

DLL3 binding molecules and their uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202410381377.X filed on March 29, 2024, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to DLL3 binding molecules and uses thereof. Background Art

[0004] Delta-Like Ligand 3 (DLL3) is a single-pass transmembrane protein attached to the cell surface and belongs to the Notch ligand family. Unlike other Notch ligands, DLL3 is normally expressed in the Golgi apparatus and appears on the cell surface when overexpressed. Studies have found that DLL3 is highly expressed in small cell lung cancer (SCLC) and other neuroendocrine tumors, such as large cell neuroendocrine carcinoma (LCNEC). Furthermore, studies have found that DLL3 is also expressed in other tumor types of neuroendocrine origin, such as melanoma, glioblastoma multiforme, small cell bladder cancer, metastatic castration-resistant prostate cancer, and neuroendocrine lung tumors. In tumor cells, DLL3 is specifically expressed on the tumor cell surface. In contrast, only a few normal cell types (such as neurons, pancreatic islet cells, and pituitary cells) express DLL3, and in normal cells, DLL3 expression is confined to the cytoplasm.

[0005] The DLL3 expression profile (high, homogeneous cell surface expression in tumors versus low, cytoplasmic expression in subsets of normal tissues) enables the development of therapies that specifically target tumor cells (e.g., SCLC cells) with DLL3. Several ongoing clinical studies are evaluating the efficacy of these DLL3-specific drugs against tumors (e.g., SCLC and other neuroendocrine tumors).

[0006] A variety of methods targeting DLL3 are being explored in preclinical and clinical settings, including antibody-drug conjugates (ADCs), T cell engager (TCE) molecules, and chimeric antigen receptor (CAR) therapies. TMAn antibody-drug conjugate developed by AbbVie targets DLL3 in small cell lung cancer (SCLC) tumors. It is internalized and releases the toxin pyrrolobenzodiazepine, which induces cell death. TCE molecules such as Amgen's tarlatamab, Harpoon Therapeutics' HPN328, Boehringer Ingelheim's BI 764532, Qilu Pharmaceutical's QLS31904, Roche's RO7616789, and Phanes Therapeutics' PT217 are all in Phase I clinical trials. Tarlatamab (AMG 757), developed by Amgen, has been optimized to extend the half-life of the BiTE molecule, activating T cells to target DLL3-expressing tumors. A Phase I clinical study of AMG 757 in SCLC patients (NCT03319940) is ongoing. Chimeric antigen receptor T-cell therapy genetically modifies the patient's autologous T cells to express chimeric receptors for tumor antigens, and then reinjects these cells into the patient's body to attack and kill target cells. AMG119 is an adoptive cell therapy that modifies the patient's autologous T cells to express a transmembrane chimeric receptor (CAR) targeting DLL3 and attack DLL3-positive cells. Currently, a Phase I clinical study (NCT03392064) evaluating the safety, tolerability, and efficacy of AMG 119 for the treatment of SCLC is underway.

[0007] As an inhibitory ligand for Notch signaling, DLL3 is closely associated with various tumors. In SCLC, in particular, extensive basic and clinical research evidence indicates that DLL3 is a promising therapeutic target. Therefore, exploring antibody drugs targeting DLL3 for cancer treatment is expected to provide more options for cancer treatment. Summary of the Invention

[0008] In a first aspect, the invention provides a DLL3 binding molecule comprising a heavy chain variable region, wherein the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3, and wherein the HCDR1 comprises the sequence of SEQ ID NO: 18 or 32, or an amino acid sequence that differs from the sequence of SEQ ID NO: 18 or 32 by no more than 2 amino acid additions, deletions or substitutions; and / or the HCDR2 comprises the sequence of SEQ ID NO: 69, 74, 79, 80, 87 or 90, or an amino acid sequence that differs from the sequence of SEQ ID NO: 69, 74, 79, 80, 87 or 90 by no more than 2 amino acid additions, deletions or substitutions; and / or the HCDR3 comprises the sequence of SEQ ID NO: NO:126, 127, 128, 129, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151 or 152, or an amino acid sequence that differs from the sequence of SEQ ID NO:126, 127, 128, 129, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151 or 152 by amino acid addition, deletion or substitution of no more than 2 amino acids.

[0009] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 60, and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 19, 60, and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 61, and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 20, 62, and 127, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 63, and 128, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 21, 64, and 129, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 21, 64, and 129, respectively. NO: 22, 65, 126; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 23, 65, 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 66, 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 64, 131, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 19, 68, 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 68, 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 20, 69, 132, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 21, 69, 133, respectively. NO: 24, 66, 126; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 70, 133, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 70, 134, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 71, 134, respectively; or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 72, 135, respectively;or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 73 and 134, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 25, 74 and 135, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 26, 75 and 136, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 27, 76 and 137, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 25, 77 and 138, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 25, 78 and 139, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 79 and 140, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 27, 76 and 137, respectively. NO: 26, 80, 141; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 81, 142, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 82, 143, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 28, 83, 144, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 29, 84, 143, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 85, 145, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 30, 75, 136, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 31, 75, 137, respectively. NO: 31, 85, 146; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 32, 86, 147, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 85, 148, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 33, 87, 149, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 88, 150, respectively;Or HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 18, 89 and 151; or HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 34, 90 and 152.

[0010] In one embodiment, the heavy chain variable region comprises 1) an amino acid sequence of SEQ ID NO: 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, or 217; NO:174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 187, 188, 189, 190, 1 91, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 2 08, 209, 210, 211, 212, 213, 214, 215, 216, or 217; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 208, 209, 210, 211, 212, 213, 214, 215, 216, or 217; or 17, 180, 181, 182, 183, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216 or 217, wherein the amino acid sequence has one or more substitutions, additions and / or deletions compared to the amino acid sequence of

[0011] In one embodiment, the DLL3 binding molecule is a heavy chain antibody and / or is a fully humanized antibody.

[0012] In one embodiment, the DLL3 binding molecule comprises 1) an amino acid sequence of SEQ ID NO: 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, or 265; NO:222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 233, 234, 235, 236, 237, 238, 2 39, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 2 56, 257, 258, 259, 260, 261, 262, 263, 264 or 265 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity; or 3) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 56, 257, 258, 259, 260, 261, 262, 263, 264 or 265; , 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 330, 331, 332, 333, 334, 335

[0013] In a second aspect, the invention provides a multispecific antigen-binding construct comprising a first antigen-binding moiety that binds DLL3, wherein the first antigen-binding moiety comprises the DLL3-binding molecule of the first aspect of the invention. In a preferred embodiment, the multispecific antigen-binding construct comprises a first antigen-binding moiety that binds DLL3 and a second antigen-binding moiety that binds a second antigen, wherein the first antigen-binding moiety comprises the DLL3-binding molecule of the first aspect of the invention.

[0014] In a third aspect, the present invention provides a chimeric antigen receptor comprising the DLL3 binding molecule of the first aspect of the invention.

[0015] In a fourth aspect, the present invention provides an immune effector cell expressing the chimeric antigen receptor of the third aspect of the present invention on its surface.

[0016] In a fifth aspect, the invention provides an antibody conjugate comprising the DLL3 binding molecule of the first aspect of the invention or the multispecific antigen-binding construct of the second aspect of the invention conjugated to at least one therapeutic agent.

[0017] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the DLL3 binding molecule of the first aspect of the invention, the multispecific antigen-binding construct of the second aspect of the invention, the immune effector cell of the fourth aspect of the invention or the antibody conjugate of the fifth aspect of the invention, and a pharmaceutically acceptable carrier.

[0018] In a seventh aspect, the present invention provides use of the DLL3 binding molecule of the first aspect of the invention, the multispecific antigen-binding construct of the second aspect of the invention, the immune effector cell of the fourth aspect of the invention, the antibody conjugate of the fifth aspect of the invention, or the pharmaceutical composition of the sixth aspect of the invention in the preparation of a medicament for treating cancer.

[0019] In an eighth aspect, the present invention provides a polynucleotide encoding the DLL3 binding molecule of the first aspect of the invention or the multispecific antigen-binding construct of the second aspect of the invention.

[0020] In a ninth aspect, the present invention provides an expression vector comprising the polynucleotide of the eighth aspect of the present invention.

[0021] In a tenth aspect, the present invention provides a host cell comprising the polynucleotide of the eighth aspect of the present invention or the expression vector of the ninth aspect of the present invention.

[0022] In an eleventh aspect, the present invention provides a method of producing the DLL3-binding molecule of the first aspect of the invention or the multispecific antigen-binding construct of the second aspect of the invention, the method comprising:

[0023] a) culturing the host cell of the tenth aspect of the invention under suitable conditions to express the DLL3 binding molecule of the first aspect of the invention or the multispecific antigen-binding construct of the second aspect of the invention; and

[0024] b) isolating the DLL3 binding molecule or multispecific antigen-binding construct from the host cell or culture thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 1A-1D show the binding activity of candidate antibodies to human DLL3 protein.

[0026] 2A-2D show the binding activity of candidate antibodies to cynomolgus monkey DLL3 protein.

[0027] 3A-3D show the binding activity of candidate antibodies to mouse DLL3 protein.

[0028] Figures 4A-4D show the binding activity of candidate antibodies to CHO-K1 cells overexpressing human DLL3.

[0029] Figures 5A-5D show the binding activity of candidate antibodies to CHO-K1 cells overexpressing cynomolgus monkey DLL3.

[0030] Figures 6A-6D show the binding activity of candidate antibodies to CHO-K1 parent cells.

[0031] 7A-7D show the binding activity of candidate antibodies to tumor cells SHP-77.

[0032] 8A-8D show the binding activity of candidate antibodies to tumor cells DMS79.

[0033] 9A-9B show the internalization of candidate antibodies in CHO-K1 cells overexpressing human DLL3.

[0034] 10A-10B show the internalization of candidate antibodies in SHP-77 tumor cells.

[0035] Figure 11A shows the plasmid construction information of the extracellular domain or subdomain of human DLL3 protein. Figure 11B shows the transfection efficiency of transiently transfected HEK293T cells.

[0036] 12A-12J show the epitope regions to which candidate and reference antibodies bind to human DLL3 protein. DETAILED DESCRIPTION

[0037] definition

[0038] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0039] As used herein, "at least one" or "one or more" may mean 1, 2, 3, 4, 5, 6, 7, 8 or more.

[0040] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0041] As used herein, the term "and / or" connecting multiple elements should be understood to include both individual and combined options. In other words, "and / or" includes "and" and "or." For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. More elements qualified with "and / or" are understood in a similar manner and include any one thereof and any combination thereof.

[0042] Unless otherwise indicated, any numerical value or numerical range, such as concentration or concentration range, is understood to be modified by the term "about" in any case. Thus, numerical values ​​generally include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions within the range, unless the context clearly indicates otherwise.

[0043] As used herein, the term "Delta-Like Ligand 3 (DLL3)" refers to a single-pass transmembrane protein attached to the cell surface and a member of the Notch ligand family. Human DLL3 protein consists of 619 amino acids, with the extracellular domain (aa 27-492) containing 466 amino acids, including a Notch-binding DSL domain (aa 176-215) followed by six epidermal growth factor-like domains (EGF-like repeats) (aa 216-465). The extracellular domain of human DLL3 shares 84% ​​amino acid identity with the extracellular segment of mouse DLL3 (aa 27-488).

[0044] As used herein, "antibody" refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen binding site. Antibodies encompass antibody fragments. As used herein, the term "antibody" includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, fully humanized antibodies, heavy chain antibodies, nanobodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b). In preferred embodiments, the antibodies of the present invention are heavy chain antibodies, and / or are fully humanized antibodies.

[0045] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region (e.g., one or more CDRs and / or one or more antibody binding sites) of the antibody that binds to an antigen and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments can include multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding regions), results in an antibody that immunospecifically binds to an antigen.

[0046] As used herein, the terms "heavy-chain-only antibody" and "heavy-chain antibody" are used interchangeably and in their broadest sense to refer to an antibody that lacks conventional antibody light chains and comprises only a heavy chain variable region and a heavy chain constant region that does not comprise CH1 (e.g., an Fc fragment).

[0047] "Fc fragment" generally refers to a crystallizable fragment of a conventional antibody or heavy chain antibody after papain digestion. Generally speaking, the Fc fragment of IgG and heavy chain antibodies can include part of the hinge region, CH2 and CH3. Herein, the Fc fragment can include at least part of the hinge region (e.g., all or part of the hinge region), CH2 and CH3.

[0048] The variable region (i.e., "binding domain") allows the binding molecule to selectively recognize an epitope on an antigen and specifically bind to the epitope. That is, for example, the light chain variable region (VL) domain and the heavy chain variable region (VH) domain of the binding molecule of an antibody, or these complementary determining regions (CDR) subgroups combine to form a variable region that determines a three-dimensional antigen binding site. More specifically, the antigen binding site is determined by three CDRs on each VH and VL chain. For example, the heavy chain antibody of the present invention has an antigen binding site determined by three CDRs on the VH chain. These "complementary determining regions" or "CDRs" are short, non-continuous sequences of amino acids that are specifically positioned to form a binding domain as the antibody adopts its three-dimensional configuration in an aqueous environment. The remaining amino acids in the binding domain are called "framework (FR)" regions, which show smaller intermolecular differences. The binding domain formed by the positioned CDRs determines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its complementary epitope. The amino acids that make up the CDRs and framework regions, respectively, in any given heavy or light chain variable region can be identified by conventional methods (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. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties). Herein, the CDRs (CDRL or LCDR) of the light chain variable region may be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region may be referred to as HCDR1, HCDR2, and HCDR3.

[0049] In the present invention, the amino acid sequences of CDRs are all shown according to the Chothia definition rules (the sequences in the claims of the present invention are also shown according to the Chothia definition rules). However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on antibody sequence variability (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), AbM (Martin, ACR and J. Allen (2007) "Bioinformatics tools for antibody engineering," in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp.95–118), Contact (MacCallum, RM et al., (1996) J. Mol. Biol. 262:732-745), IMGT (Lefranc, M.-P., 2011(6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. In this article, multiple CDR numbering systems may be used for the same variable region, such as Chothia, Abm, Kabat, Contact, and IMGT. It should be understood by those skilled in the art that although the CDRs defined by different numbering systems may be different, the CDRs corresponding to the same numbering system represent effective antigen binding sites that can bind to antigen epitopes.Unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined by any of the above-mentioned known schemes described herein. Although the scope of protection claimed in the claims of the present invention is based on the sequences shown in the Chothia definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0050] Thus, when referring to antibodies defined by specific CDR sequences defined herein, the scope of said antibodies also encompasses antibodies whose variable region sequences comprise said specific CDR sequences, but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0051] As used herein, the terms "framework region" and "framework region" are used interchangeably. As used herein, the terms "framework region," "framework region," or "FR" residues refer to those amino acid residues in the antibody variable region excluding the CDR sequences as defined above.

[0052] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a sulfhydryl group that can form a disulfide bond or bridge a second sulfhydryl group.

[0053] As used herein, the term "humanized antibody" refers to an antibody in which a non-human antibody is modified to increase sequence homology with a human antibody. Humanized antibodies generally retain the antigen-binding ability of the non-human antibody from which they are derived and have lower immunogenicity to the human body. Humanized antibodies can be obtained by engineering any non-human species antibody or an antibody (e.g., a chimeric antibody) comprising a sequence of a non-human species. Non-human species may, for example, include mice, rats, rabbits, alpacas, sharks, or non-human primates. The technology for obtaining humanized antibodies from non-human antibodies is well known to those skilled in the art. For example, the CDR sequences of non-human antibodies (e.g., mouse antibodies) are transplanted into human antibody framework regions. In some cases, in order to maintain the antigen-binding ability and / or stability of the humanized antibody, key amino acid residues of the non-human antibody (e.g., murine antibody) framework sequence can be retained in the human antibody framework region, i.e., "back mutations" are performed (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21): 6851-6855; Neuberger et al. (1984) Nature 312: 604-608).

[0054] In the present invention, the term "fully human antibody" or "fully humanized antibody" generally refers to an antibody expressed by a genetically engineered animal by transferring a human antibody-encoding gene into an antibody gene-deficient animal. All parts of the antibody (including the variable and constant regions) are encoded by genes of human origin. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

[0055] As used herein, "percent (%) sequence identity" or "sequence identity" of amino acid sequences has an art-recognized definition and refers to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). This can be determined using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0056] As used herein, an amino acid sequence that is "derived from" or "derived from" a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence. For example, an amino acid sequence derived from the heavy chain constant region of a human immunoglobulin may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the wild-type sequence of the heavy chain constant region of the human immunoglobulin from which it is derived.

[0057] The non-critical regions in the polypeptide (e.g., CDR regions of antibodies, non-critical amino acids in the framework regions, amino acids in the constant regions) can be modified, for example, by substitution, addition, and / or deletion of one or more amino acids without changing the function of the polypeptide. In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and generally can be made without changing the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in the non-essential regions of a polypeptide do not substantially change biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p.224).

[0058] "Affinity" or "binding affinity" is a measure of the strength of the non-covalent binding between an antibody and an antigen. Affinity can be determined using conventional techniques known in the art, such as biofilm interferometry (e.g., using the Octet Fortebio assay system), radioimmunoassay, surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS).

[0059] "Specific binding" generally refers to a binding molecule, such as an antibody or a fragment, variant, or derivative thereof, that binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. By this definition, a binding molecule is said to "specifically bind" to an epitope when it is easier to bind to an epitope through its antigen binding domain than to a random, unrelated epitope. The term "specificity" is used herein to qualitatively analyze the relative affinity of an antibody for binding to an epitope. For example, it can be considered that binding molecule "A" has a higher specificity for a given epitope than binding molecule "B," or it can be said that binding molecule "A" specifically binds to epitope "C" with a higher specificity than its specificity for a related epitope "D."

[0060] As used herein, the term "EC 50 ”, also known as the “half-maximal effective concentration”, is the concentration of a drug, antibody, or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time.

[0061] As used herein, the term "endocytosis" refers to the process by which a substance to be internalized is enclosed by the cell membrane and then buds into a vesicle containing the internalized substance, bringing the external substance into the cell. This is one way that substances are transported across the cytoplasmic membrane. The term "antibody internalization" refers to the induction of receptor-mediated endocytosis when a cell surface receptor binds to its specific antibody. As described herein, DLL3 antibodies can mediate the internalization of cell-surface-expressed DLL3 protein by binding to the extracellular portion of DLL3. The endocytic effect of antibodies can be measured using conventional techniques known in the art, such as labeling with internalizing antibodies.

[0062] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It will be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, with deoxythymidylic acid in the DNA coding strand sequence corresponding to uridine in the RNA sequence it encodes.

[0063] As used herein, an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules that encode provided DLL3 binding molecules.

[0064] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and / or polypeptides.

[0065] As used herein, "vector" is a medium for introducing exogenous polynucleotides into a host cell, and when the vector is transformed into an appropriate host cell, the exogenous polynucleotides are amplified or expressed. The vector usually remains free, but can be designed to integrate a gene or part thereof into a chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes virus vectors, pox virus vectors, and baculovirus vectors, etc.

[0066] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells, such as HEK 293 cells.

[0067] Terms such as "treating" or "treating" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, alleviate, reduce the symptoms of an existing, diagnosed pathological condition or disorder, and / or arrest or slow the progression of an existing, diagnosed pathological condition or disorder. Terms such as "preventing," "preventing," "avoiding," "containment," and the like refer to preventative or prophylactic measures that prevent the progression of an undiagnosed target pathological condition or disorder. Thus, a "subject in need thereof" can include a subject already suffering from a disease; a subject susceptible to a disease; and a subject in need of prevention of a disease.

[0068] As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

[0069] The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule or other drug that is effective for "treating" a disease or condition in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; block or stop cancer cell division, reduce or block the increase in tumor size; inhibit, for example, suppress, block, prevent, stop, delay or reverse cancer cell infiltration into peripheral organs, including, for example, the spread of cancer to soft tissue and bone; inhibit, for example, suppress, block, prevent, shrink, stop, delay or reverse tumor metastasis; inhibit, for example, suppress, block, prevent, stop, delay or reverse tumor growth; alleviate to some extent one or more symptoms associated with cancer, reduce morbidity and mortality; improve quality of life; or a combination of these effects. To the extent that a drug prevents growth and / or kills existing cancer cells, it can refer to being cytostatic and / or cytotoxic.

[0070] As used herein, the term "subject" refers to a mammal, such as a human.

[0071] The antibody numbers used herein (such as PR013234, PR013235, ..., PR013402, PR013406, etc. or similar numbers) are only used to distinguish or identify antibodies or products, and are not intended to indicate that such identification is a feature of the antibody or product of the present invention. It will be understood by those skilled in the art that, for example, other antibodies or products may also use such identification for the purpose of distinction or identification, but do not refer to the same or equivalent antibodies or products. Similarly, the similar numbers or identifications used in the embodiments are only for illustrative convenience, and the antibodies or products of the present invention are defined by the features described in the appended claims.

[0072] DLL3 binding molecules

[0073] The present invention provides a DLL3 binding molecule. Generally speaking, a DLL3 binding molecule can include any molecule that specifically binds to DLL3.

[0074] In some embodiments, the DLL3 binding molecule is a polypeptide or protein, such as an antibody.

[0075] In a preferred embodiment, the DLL3 binding molecule is an antibody or antigen-binding fragment thereof directed against DLL3.

[0076] DLL3-binding molecules include synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, fully humanized antibodies, heavy chain antibodies, nanobodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the foregoing.

[0077] In one embodiment, the DLL3 binding molecule is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region.

[0078] In one embodiment, the DLL3 binding molecule is a heavy chain antibody or antigen-binding fragment thereof comprising a heavy chain variable region.

[0079] In one embodiment, the DLL3 binding molecule is a heavy chain antibody. In another embodiment, the DLL3 binding molecule is a fully humanized antibody. In a preferred embodiment, the DLL3 binding molecule is a heavy chain antibody and is a fully humanized antibody.

[0080] In one embodiment, the heavy chain variable region comprised by DLL3 can be fused to another molecule; preferably, the other molecule is the Fc domain of an immunoglobulin; most preferably, the other molecule is the Fc domain of immunoglobulin G1 (IgG1); most preferably, the other molecule is the Fc domain of human immunoglobulin G1 (IgG1).

[0081] In one embodiment, the DLL3 binding molecule is a heavy chain antibody comprising a heavy chain variable region and the Fc domain of human IgG1.

[0082] In one embodiment, a DLL3 binding molecule comprises a heavy chain variable region, wherein the heavy chain variable region comprises a HCDR1, a HCDR2, and a HCDR3, and wherein HCDR1 comprises the sequence of SEQ ID NO: 18 or 32, or an amino acid sequence that differs from the sequence of SEQ ID NO: 18 or 32 by no more than 2 amino acid additions, deletions or substitutions; and / or HCDR2 comprises the sequence of SEQ ID NO: 69, 74, 79, 80, 87 or 90, or an amino acid sequence that differs from the sequence of SEQ ID NO: 69, 74, 79, 80, 87 or 90 by no more than 2 amino acid additions, deletions or substitutions; and / or HCDR3 comprises the sequence of SEQ ID NO: NO:126, 127, 128, 129, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151 or 152, or an amino acid sequence that differs from the sequence of SEQ ID NO:126, 127, 128, 129, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151 or 152 by amino acid addition, deletion or substitution of no more than 2 amino acids.

[0083] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with N (SEQ ID NO: 19; GFNFSDY).

[0084] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In one embodiment, the amino acid at position 4 in SEQ ID NO: 18 is substituted with L (SEQ ID NO: 20; GFTLSDY).

[0085] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In one embodiment, the amino acid at position 4 in SEQ ID NO: 18 is substituted with 1 (SEQ ID NO: 21; GFTISDY).

[0086] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 4 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with N, and the amino acid at position 4 in SEQ ID NO: 18 is substituted with I (SEQ ID NO: 22; GFNISDY).

[0087] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 4 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with N, and the amino acid at position 4 in SEQ ID NO: 18 is substituted with L (SEQ ID NO: 23; GFNLSDY).

[0088] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 5 are substituted. In one embodiment, the amino acid at position 3 of SEQ ID NO: 18 is substituted with N, and the amino acid at position 5 of SEQ ID NO: 18 is substituted with N (SEQ ID NO: 24; GFNFNDY).

[0089] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 18 is substituted with S (SEQ ID NO: 25; GFTFSSY).

[0090] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 18 is substituted with N (SEQ ID NO: 26; GFTFSNY).

[0091] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 7 is substituted. In one embodiment, the amino acid at position 7 in SEQ ID NO: 18 is substituted with H (SEQ ID NO: 27; GFTFSDH).

[0092] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 18 is substituted with T (SEQ ID NO: 28; GFTFSTY).

[0093] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with A (SEQ ID NO: 29; GFAFSDY).

[0094] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with I, and the amino acid at position 6 in SEQ ID NO: 18 is substituted with N (SEQ ID NO: 30; GFIFSNY).

[0095] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 3 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 18 is substituted with I, and the amino acid at position 6 in SEQ ID NO: 18 is substituted with S (SEQ ID NO: 31; GFIFSSY).

[0096] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 4 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 4 in SEQ ID NO: 18 is substituted with I, and the amino acid at position 6 in SEQ ID NO: 18 is substituted with N (SEQ ID NO: 33; GFTISNY).

[0097] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; GFTFSDY), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 18 is substituted with H (SEQ ID NO: 34; GFTFSHY).

[0098] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 2 is substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 69 is substituted with G (SEQ ID NO: 60; SGSGST).

[0099] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 3 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO:69 is substituted with an A, and the amino acid at position 3 in SEQ ID NO:69 is substituted with an N (SEQ ID NO:61; SANGST).

[0100] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 1 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 1 and the amino acid at position 2 are substituted. In one embodiment, the amino acid at position 1 in SEQ ID NO:69 is substituted with T, and the amino acid at position 2 in SEQ ID NO:69 is substituted with G (SEQ ID NO:62; TGSGST).

[0101] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 5 is substituted. In one embodiment, the amino acid at position 5 in SEQ ID NO: 69 is substituted with N (SEQ ID NO: 63; SSSGNT).

[0102] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 69 is substituted with N (SEQ ID NO: 64; SSNGST).

[0103] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 5 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO:69 is substituted with G, and the amino acid at position 5 in SEQ ID NO:69 is substituted with R (SEQ ID NO:65; SGSGRT).

[0104] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 3 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO:69 is substituted with G, and the amino acid at position 3 in SEQ ID NO:69 is substituted with N (SEQ ID NO:66; SGNGST).

[0105] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 69 (SEQ ID NO: 69; SSSGST), wherein the amino acid at position 5 is substituted. In one embodiment, the amino acid at position 5 in SEQ ID NO: 69 is substituted with G (SEQ ID NO: 67; SSSGGT).

[0106] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO:69 is substituted with G, and the amino acid at position 6 in SEQ ID NO:69 is substituted with I (SEQ ID NO:68; SGSGSI).

[0107] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 5 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 5 of SEQ ID NO:69 is substituted with D, and the amino acid at position 6 of SEQ ID NO:69 is substituted with S (SEQ ID NO:70; SSSGDS).

[0108] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 5 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 5 of SEQ ID NO:69 is substituted with E, and the amino acid at position 6 of SEQ ID NO:69 is substituted with S (SEQ ID NO:71; SSSGES).

[0109] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 5 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 5 of SEQ ID NO:69 is substituted with N, and the amino acid at position 6 of SEQ ID NO:69 is substituted with S (SEQ ID NO:73; SSSGNS).

[0110] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 3 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO:69 is substituted with D, and the amino acid at position 6 in SEQ ID NO:69 is substituted with S (SEQ ID NO:75; SSDGSS).

[0111] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO:69 is substituted with G, and the amino acid at position 6 in SEQ ID NO:69 is substituted with D (SEQ ID NO:76; SGSGSD).

[0112] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 3 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO:69 is substituted with D, and the amino acid at position 6 in SEQ ID NO:69 is substituted with D (SEQ ID NO:82; SSDGSD).

[0113] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 3 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO:69 is substituted with D, and the amino acid at position 6 in SEQ ID NO:69 is substituted with N (SEQ ID NO:83; SSDGSN).

[0114] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO:69 (SEQ ID NO:69; SSSGST), wherein the amino acid at position 2 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO:69 is substituted with T, and the amino acid at position 6 in SEQ ID NO:69 is substituted with P (SEQ ID NO:89; STSGSP).

[0115] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 74 (SEQ ID NO: 74; SGSDNN), wherein the amino acid at position 1 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 74 (SEQ ID NO: 74; SGSDNN), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 74 (SEQ ID NO: 74; SGSDNN), wherein the amino acid at position 1 and the amino acid at position 3 are substituted. In one embodiment, the amino acid at position 1 of SEQ ID NO: 74 is substituted with N, and the amino acid at position 3 of SEQ ID NO: 74 is substituted with N (SEQ ID NO: 72; NGNDNN).

[0116] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 74 (SEQ ID NO: 74; SGSDNN), wherein the amino acid at position 5 is substituted. In one embodiment, the amino acid at position 5 in SEQ ID NO: 74 is substituted with D (SEQ ID NO: 86; SGSDDN).

[0117] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 2 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 79 is substituted with a G, and the amino acid at position 6 in SEQ ID NO: 79 is substituted with an N (SEQ ID NO: 77; NGDGSN).

[0118] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 79 is substituted with Y (SEQ ID NO: 78; NSDGSY).

[0119] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 3 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 5 is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 3 and the amino acid at position 5 are substituted. In one embodiment, the amino acid at position 3 of SEQ ID NO: 79 is substituted with N, and the amino acid at position 5 of SEQ ID NO: 79 is substituted with N (SEQ ID NO: 81; NSNGNT).

[0120] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 79 is substituted with D (SEQ ID NO: 84; NSDGSD).

[0121] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 79 is substituted with N (SEQ ID NO: 85; NSDGSN).

[0122] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 79 (SEQ ID NO: 79; NSDGST), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 79 is substituted with S (SEQ ID NO: 88; NSDGSS).

[0123] In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 126 (SEQ ID NO: 126; ENGNSDY), wherein the amino acid at position 2 is substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 126 is substituted with S (SEQ ID NO: 131; ESGNSDY).

[0124] In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 127 (SEQ ID NO: 127; GLGEFSLGI), wherein the amino acid at position 9 is substituted. In one embodiment, the amino acid at position 9 in SEQ ID NO: 127 is substituted with S (SEQ ID NO: 130; GLGEFSLGS).

[0125] In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 127 (SEQ ID NO: 127; GLGEFSLGI), wherein the amino acid at position 5 is substituted. In one embodiment, the amino acid at position 5 in SEQ ID NO: 127 is substituted with L (SEQ ID NO: 132; GLGELSLGI).

[0126] In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 133 (SEQ ID NO: 133; ERGSTGGWFDP), wherein the amino acid at position 2 is substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 133 is substituted with K (SEQ ID NO: 134; EKGSTGGWFDP).

[0127] In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 139 (SEQ ID NO: 139; GGDTVVVPEFDY), wherein the amino acid at position 2 is substituted. In some embodiments, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 139 (SEQ ID NO: 139; GGDTVVVPEFDY), wherein the amino acid at position 7 is substituted. In some embodiments, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 139 (SEQ ID NO: 139; GGDTVVVPEFDY), wherein the amino acid at position 2 and the amino acid at position 7 are substituted. In one embodiment, the amino acid at position 2 in SEQ ID NO: 139 is substituted with D, and the amino acid at position 7 in SEQ ID NO: 139 is substituted with L (SEQ ID NO: 145; GDDTVVLPEFDY).

[0128] In one embodiment, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34.

[0129] In one embodiment, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90.

[0130] In one embodiment, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 or 152.

[0131] In one embodiment, HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34; HCDR2 comprises the amino acid sequence of SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90; and HCDR3 comprises the amino acid sequence of SEQ ID NO: 1 The amino acid sequence shown in NO:126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 or 152.

[0132] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 60 and 126, respectively.

[0133] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 19, 60 and 126, respectively.

[0134] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 61 and 126, respectively.

[0135] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 20, 62 and 127, respectively.

[0136] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 63, and 128, respectively.

[0137] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 21, 64, and 129, respectively.

[0138] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 22, 65 and 126, respectively.

[0139] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 23, 65 and 126, respectively.

[0140] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 66, and 126, respectively.

[0141] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 67, and 130, respectively.

[0142] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 64, and 131, respectively.

[0143] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 19, 68 and 126, respectively.

[0144] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 68, and 126, respectively.

[0145] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 20, 69 and 132, respectively.

[0146] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 24, 66, and 126, respectively.

[0147] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 70 and 133, respectively.

[0148] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 70, and 134, respectively.

[0149] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 71 and 134, respectively.

[0150] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 72, and 135, respectively.

[0151] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 73, and 134, respectively.

[0152] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 74, and 135, respectively.

[0153] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 75, and 136, respectively.

[0154] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 27, 76, and 137, respectively.

[0155] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 77, and 138, respectively.

[0156] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 78 and 139, respectively.

[0157] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 79 and 140, respectively.

[0158] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 80, and 141, respectively.

[0159] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 81 and 142, respectively.

[0160] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 82 and 143, respectively.

[0161] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 28, 83, and 144, respectively.

[0162] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 29, 84, and 143, respectively.

[0163] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 85, and 145, respectively.

[0164] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 30, 75 and 136, respectively.

[0165] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 31, 85 and 146, respectively.

[0166] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 32, 86, and 147, respectively.

[0167] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 85, and 148, respectively.

[0168] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 33, 87 and 149, respectively.

[0169] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 88, and 150, respectively.

[0170] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 89 and 151, respectively.

[0171] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 34, 90 and 152, respectively.

[0172] In one embodiment, the heavy chain variable region comprises 1) an amino acid sequence of SEQ ID NO: 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, or 217; NO:174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 19 0, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 , 208, 209, 210, 211, 212, 213, 214, 215, 216, or 217; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1; 17, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216 or 217, wherein the amino acid sequence has one or more substitutions, additions and / or deletions compared to the amino acid sequence of

[0173] In one embodiment, the heavy chain variable region is fused to another molecule. In a preferred embodiment, the other molecule is the Fc domain of an immunoglobulin. In a more preferred embodiment, the other molecule is the Fc domain of human immunoglobulin G1 (IgG1).

[0174] In one embodiment, a DLL3 binding molecule comprises 1) an amino acid sequence of SEQ ID NO: 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, or 265; NO:222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 23 8, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255 , 256, 257, 258, 259, 260, 261, 262, 263, 264, or 265 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ , 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 330, 331, 332, 333, 334,

[0175] In one embodiment, the DLL3 binding molecule has at least one of the following characteristics:

[0176] 1) Has affinity activity for DLL3 protein;

[0177] 2) has affinity activity for DLL3-positive cells;

[0178] 3) Ability to be internalized into DLL3-positive cells.

[0179] In one embodiment, the DLL3 binding molecules include antibodies to DLL3, such as PR013234, PR013235, PR013236, PR013239, PR013240, PR013241, PR013242, PR013244, PR013246, PR013249, PR013250, PR013252, PR013253, PR013254, PR013256, PR013257, PR013258, PR013259, PR013260, PR013261, PR013262, PR013263, PR013264, PR013363, PR013364, 13240, PR013254, PR013371, PR013377, PR013393, and PR013397.

[0180] Antibody PR013234

[0181] In one aspect, the present invention provides an antibody PR013234 or an antigen-binding fragment thereof against DLL3,

[0182] wherein the antibody comprises a heavy chain variable region, wherein

[0183] The heavy chain variable region comprises:

[0184] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0185] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 60, and

[0186] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0187] In one embodiment, the DLL3 antibody PR013234 comprises a heavy chain variable region (VH),

[0188] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 174.

[0189] In one embodiment, the DLL3 antibody PR013234 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013234 comprises the amino acid sequence of SEQ ID NO: 222, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 222.

[0190] Antibody PR013235

[0191] In another aspect, the present invention provides an antibody PR013235 or an antigen-binding fragment thereof against DLL3,

[0192] wherein the antibody comprises a heavy chain variable region, wherein

[0193] The heavy chain variable region comprises:

[0194] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19,

[0195] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 60, and

[0196] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0197] In one embodiment, the DLL3 antibody PR013235 comprises a heavy chain variable region (VH),

[0198] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 175.

[0199] In one embodiment, the DLL3 antibody PR013235 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013235 comprises the amino acid sequence of SEQ ID NO: 223, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 223.

[0200] Antibody PR013236

[0201] In another aspect, the present invention provides an antibody PR013236 or an antigen-binding fragment thereof against DLL3,

[0202] wherein the antibody comprises a heavy chain variable region, wherein

[0203] The heavy chain variable region comprises:

[0204] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0205] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 61, and

[0206] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0207] In one embodiment, the DLL3 antibody PR013236 comprises a heavy chain variable region (VH),

[0208] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 176.

[0209] In one embodiment, the DLL3 antibody PR013236 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013236 comprises the amino acid sequence of SEQ ID NO: 224, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 224.

[0210] Antibody PR013239

[0211] In another aspect, the present invention provides an antibody PR013239 or an antigen-binding fragment thereof against DLL3,

[0212] wherein the antibody comprises a heavy chain variable region, wherein

[0213] The heavy chain variable region comprises:

[0214] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0215] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 60, and

[0216] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0217] In one embodiment, the DLL3 antibody PR013239 comprises a heavy chain variable region (VH),

[0218] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 177.

[0219] In one embodiment, the DLL3 antibody PR013239 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013239 comprises the amino acid sequence of SEQ ID NO: 225, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 225.

[0220] Antibody PR013240

[0221] In another aspect, the present invention provides an antibody PR013240 or an antigen-binding fragment thereof against DLL3,

[0222] wherein the antibody comprises a heavy chain variable region, wherein

[0223] The heavy chain variable region comprises:

[0224] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20,

[0225] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 62, and

[0226] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 127.

[0227] In one embodiment, the DLL3 antibody PR013240 comprises a heavy chain variable region (VH),

[0228] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 178.

[0229] In one embodiment, the DLL3 antibody PR013240 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013240 comprises the amino acid sequence of SEQ ID NO: 226, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 226.

[0230] Antibody PR013241

[0231] In another aspect, the present invention provides an antibody PR013241 or an antigen-binding fragment thereof against DLL3,

[0232] wherein the antibody comprises a heavy chain variable region, wherein

[0233] The heavy chain variable region comprises:

[0234] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0235] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 63, and

[0236] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 128.

[0237] In one embodiment, the DLL3 antibody PR013241 comprises a heavy chain variable region (VH),

[0238] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 179.

[0239] In one embodiment, the DLL3 antibody PR013241 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013241 comprises the amino acid sequence of SEQ ID NO: 227, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 227.

[0240] Antibody PR013242

[0241] In another aspect, the present invention provides an antibody PR013242 or an antigen-binding fragment thereof against DLL3,

[0242] wherein the antibody comprises a heavy chain variable region, wherein

[0243] The heavy chain variable region comprises:

[0244] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21,

[0245] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 64, and

[0246] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 129.

[0247] In one embodiment, the DLL3 antibody PR013242 comprises a heavy chain variable region (VH),

[0248] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 180 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 180.

[0249] In one embodiment, the DLL3 antibody PR013242 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013242 comprises the amino acid sequence of SEQ ID NO: 228, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 228.

[0250] Antibody PR013244

[0251] In another aspect, the present invention provides an antibody PR013244 or an antigen-binding fragment thereof against DLL3,

[0252] wherein the antibody comprises a heavy chain variable region, wherein

[0253] The heavy chain variable region comprises:

[0254] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22,

[0255] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 65, and

[0256] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0257] In one embodiment, the DLL3 antibody PR013244 comprises a heavy chain variable region (VH),

[0258] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 181.

[0259] In one embodiment, the DLL3 antibody PR013244 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013244 comprises the amino acid sequence of SEQ ID NO: 229, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 229.

[0260] Antibody PR013246

[0261] In another aspect, the present invention provides an antibody PR013246 or an antigen-binding fragment thereof against DLL3,

[0262] wherein the antibody comprises a heavy chain variable region, wherein

[0263] The heavy chain variable region comprises:

[0264] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 23,

[0265] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 65, and

[0266] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0267] In one embodiment, the DLL3 antibody PR013246 comprises a heavy chain variable region (VH),

[0268] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 182.

[0269] In one embodiment, the DLL3 antibody PR013246 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013246 comprises the amino acid sequence of SEQ ID NO: 230, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 230.

[0270] Antibody PR013249

[0271] In another aspect, the present invention provides an antibody PR013249 or an antigen-binding fragment thereof against DLL3,

[0272] wherein the antibody comprises a heavy chain variable region, wherein

[0273] The heavy chain variable region comprises:

[0274] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0275] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 66, and

[0276] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0277] In one embodiment, the DLL3 antibody PR013249 comprises a heavy chain variable region (VH),

[0278] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 183.

[0279] In one embodiment, the DLL3 antibody PR013249 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013249 comprises the amino acid sequence of SEQ ID NO: 231, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 231.

[0280] Antibody PR013250

[0281] In another aspect, the present invention provides an antibody PR013250 or an antigen-binding fragment thereof against DLL3,

[0282] wherein the antibody comprises a heavy chain variable region, wherein

[0283] The heavy chain variable region comprises:

[0284] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0285] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 67, and

[0286] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 130.

[0287] In one embodiment, the DLL3 antibody PR013250 comprises a heavy chain variable region (VH),

[0288] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 184.

[0289] In one embodiment, the DLL3 antibody PR013250 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013250 comprises the amino acid sequence of SEQ ID NO: 232, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 232.

[0290] Antibody PR013252

[0291] In another aspect, the present invention provides an antibody PR013252 or an antigen-binding fragment thereof against DLL3,

[0292] wherein the antibody comprises a heavy chain variable region, wherein

[0293] The heavy chain variable region comprises:

[0294] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0295] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 64, and

[0296] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 131.

[0297] In one embodiment, the DLL3 antibody PR013252 comprises a heavy chain variable region (VH),

[0298] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 185.

[0299] In one embodiment, the DLL3 antibody PR013252 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013252 comprises the amino acid sequence of SEQ ID NO: 233, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 233.

[0300] Antibody PR013253

[0301] In another aspect, the present invention provides an antibody PR013253 or an antigen-binding fragment thereof against DLL3,

[0302] wherein the antibody comprises a heavy chain variable region, wherein

[0303] The heavy chain variable region comprises:

[0304] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19,

[0305] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 68, and

[0306] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0307] In one embodiment, the DLL3 antibody PR013253 comprises a heavy chain variable region (VH),

[0308] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 186.

[0309] In one embodiment, the DLL3 antibody PR013253 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013253 comprises the amino acid sequence of SEQ ID NO: 234, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 234.

[0310] Antibody PR013254

[0311] In another aspect, the present invention provides an antibody PR013254 or an antigen-binding fragment thereof against DLL3,

[0312] wherein the antibody comprises a heavy chain variable region, wherein

[0313] The heavy chain variable region comprises:

[0314] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0315] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 63, and

[0316] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 128.

[0317] In one embodiment, the DLL3 antibody PR013254 comprises a heavy chain variable region (VH),

[0318] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 187.

[0319] In one embodiment, the DLL3 antibody PR013254 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013254 comprises the amino acid sequence of SEQ ID NO: 235, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 235.

[0320] Antibody PR013256

[0321] In another aspect, the present invention provides an antibody PR013256 or an antigen-binding fragment thereof against DLL3,

[0322] wherein the antibody comprises a heavy chain variable region, wherein

[0323] The heavy chain variable region comprises:

[0324] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0325] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 68, and

[0326] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0327] In one embodiment, the DLL3 antibody PR013256 comprises a heavy chain variable region (VH),

[0328] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 188.

[0329] In one embodiment, the DLL3 antibody PR013256 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013256 comprises the amino acid sequence of SEQ ID NO: 236, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 236.

[0330] Antibody PR013257

[0331] In another aspect, the present invention provides an antibody PR013257 or an antigen-binding fragment thereof against DLL3,

[0332] wherein the antibody comprises a heavy chain variable region, wherein

[0333] The heavy chain variable region comprises:

[0334] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20,

[0335] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 69, and

[0336] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 132.

[0337] In one embodiment, the DLL3 antibody PR013257 comprises a heavy chain variable region (VH),

[0338] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 189 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 189.

[0339] In one embodiment, the DLL3 antibody PR013257 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013257 comprises the amino acid sequence of SEQ ID NO: 237, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 237.

[0340] Antibody PR013258

[0341] In another aspect, the present invention provides an antibody PR013258 or an antigen-binding fragment thereof against DLL3,

[0342] wherein the antibody comprises a heavy chain variable region, wherein

[0343] The heavy chain variable region comprises:

[0344] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 24,

[0345] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 66, and

[0346] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0347] In one embodiment, the DLL3 antibody PR013258 comprises a heavy chain variable region (VH),

[0348] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 190 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 190.

[0349] In one embodiment, the DLL3 antibody PR013258 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013258 comprises the amino acid sequence of SEQ ID NO: 238, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 238.

[0350] Antibody PR013259

[0351] In another aspect, the present invention provides an antibody PR013259 or an antigen-binding fragment thereof against DLL3,

[0352] wherein the antibody comprises a heavy chain variable region, wherein

[0353] The heavy chain variable region comprises:

[0354] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0355] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 70, and

[0356] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 133.

[0357] In one embodiment, the DLL3 antibody PR013259 comprises a heavy chain variable region (VH),

[0358] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 191 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 191.

[0359] In one embodiment, the DLL3 antibody PR013259 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013259 comprises the amino acid sequence of SEQ ID NO: 239, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 239.

[0360] Antibody PR013260

[0361] In another aspect, the present invention provides an antibody PR013260 or an antigen-binding fragment thereof against DLL3,

[0362] wherein the antibody comprises a heavy chain variable region, wherein

[0363] The heavy chain variable region comprises:

[0364] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0365] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 70, and

[0366] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134.

[0367] In one embodiment, the DLL3 antibody PR013260 comprises a heavy chain variable region (VH),

[0368] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 192 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 192.

[0369] In one embodiment, the DLL3 antibody PR013260 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013260 comprises the amino acid sequence of SEQ ID NO: 240, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 240.

[0370] Antibody PR013261

[0371] In another aspect, the present invention provides an antibody PR013261 or an antigen-binding fragment thereof against DLL3,

[0372] wherein the antibody comprises a heavy chain variable region, wherein

[0373] The heavy chain variable region comprises:

[0374] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0375] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 71, and

[0376] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134.

[0377] In one embodiment, the DLL3 antibody PR013261 comprises a heavy chain variable region (VH),

[0378] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 193 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 193.

[0379] In one embodiment, the DLL3 antibody PR013261 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013261 comprises the amino acid sequence of SEQ ID NO: 241, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 241.

[0380] Antibody PR013262

[0381] In another aspect, the present invention provides an antibody PR013262 or an antigen-binding fragment thereof against DLL3,

[0382] wherein the antibody comprises a heavy chain variable region, wherein

[0383] The heavy chain variable region comprises:

[0384] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25,

[0385] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 72, and

[0386] HCDR3 comprises the amino acid sequence shown in SEQ ID NO:135.

[0387] In one embodiment, the DLL3 antibody PR013262 comprises a heavy chain variable region (VH),

[0388] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 194 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 194.

[0389] In one embodiment, the DLL3 antibody PR013262 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013262 comprises the amino acid sequence of SEQ ID NO: 242, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 242.

[0390] Antibody PR013263

[0391] In another aspect, the present invention provides an antibody PR013263 or an antigen-binding fragment thereof against DLL3,

[0392] wherein the antibody comprises a heavy chain variable region, wherein

[0393] The heavy chain variable region comprises:

[0394] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0395] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 73, and

[0396] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134.

[0397] In one embodiment, the DLL3 antibody PR013263 comprises a heavy chain variable region (VH),

[0398] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 195 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 195.

[0399] In one embodiment, the DLL3 antibody PR013263 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013263 comprises the amino acid sequence of SEQ ID NO: 243, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 243.

[0400] Antibody PR013264

[0401] In another aspect, the present invention provides an antibody PR013264 or an antigen-binding fragment thereof against DLL3,

[0402] wherein the antibody comprises a heavy chain variable region, wherein

[0403] The heavy chain variable region comprises:

[0404] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25,

[0405] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 74, and

[0406] HCDR3 comprises the amino acid sequence shown in SEQ ID NO:135.

[0407] In one embodiment, the DLL3 antibody PR013264 comprises a heavy chain variable region (VH),

[0408] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 196 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 196.

[0409] In one embodiment, the DLL3 antibody PR013264 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013264 comprises the amino acid sequence of SEQ ID NO: 244, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 244.

[0410] Antibody PR013363

[0411] In another aspect, the present invention provides an antibody PR013363 or an antigen-binding fragment thereof against DLL3,

[0412] wherein the antibody comprises a heavy chain variable region, wherein

[0413] The heavy chain variable region comprises:

[0414] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26,

[0415] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 75, and

[0416] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 136.

[0417] In one embodiment, the DLL3 antibody PR013363 comprises a heavy chain variable region (VH),

[0418] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 197 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 197.

[0419] In one embodiment, the DLL3 antibody PR013363 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013363 comprises the amino acid sequence of SEQ ID NO: 245, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 245.

[0420] Antibody PR013364

[0421] In another aspect, the present invention provides an antibody PR013364 or an antigen-binding fragment thereof against DLL3,

[0422] wherein the antibody comprises a heavy chain variable region, wherein

[0423] The heavy chain variable region comprises:

[0424] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27,

[0425] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 76, and

[0426] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 137.

[0427] In one embodiment, the DLL3 antibody PR013364 comprises a heavy chain variable region (VH),

[0428] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 198 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 198.

[0429] In one embodiment, the DLL3 antibody PR013364 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013364 comprises the amino acid sequence of SEQ ID NO: 246, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 246.

[0430] Antibody PR013367

[0431] In another aspect, the present invention provides an antibody PR013367 or an antigen-binding fragment thereof against DLL3,

[0432] wherein the antibody comprises a heavy chain variable region, wherein

[0433] The heavy chain variable region comprises:

[0434] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25,

[0435] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 77, and

[0436] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 138.

[0437] In one embodiment, the DLL3 antibody PR013367 comprises a heavy chain variable region (VH),

[0438] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 199 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 199.

[0439] In one embodiment, the DLL3 antibody PR013367 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013367 comprises the amino acid sequence of SEQ ID NO: 247, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 247.

[0440] Antibody PR013368

[0441] In another aspect, the present invention provides an antibody PR013368 or an antigen-binding fragment thereof against DLL3,

[0442] wherein the antibody comprises a heavy chain variable region, wherein

[0443] The heavy chain variable region comprises:

[0444] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25,

[0445] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 78, and

[0446] HCDR3 comprises the amino acid sequence shown in SEQ ID NO:139.

[0447] In one embodiment, the DLL3 antibody PR013368 comprises a heavy chain variable region (VH),

[0448] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 200 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 200.

[0449] In one embodiment, the DLL3 antibody PR013368 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013368 comprises the amino acid sequence of SEQ ID NO: 248, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 248.

[0450] Antibody PR013370

[0451] In another aspect, the present invention provides an antibody PR013370 or an antigen-binding fragment thereof against DLL3,

[0452] wherein the antibody comprises a heavy chain variable region, wherein

[0453] The heavy chain variable region comprises:

[0454] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0455] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 79, and

[0456] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 140.

[0457] In one embodiment, the DLL3 antibody PR013370 comprises a heavy chain variable region (VH),

[0458] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 201 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 201.

[0459] In one embodiment, the DLL3 antibody PR013370 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013370 comprises the amino acid sequence of SEQ ID NO: 249, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 249.

[0460] Antibody PR013371

[0461] In another aspect, the present invention provides an antibody PR013371 or an antigen-binding fragment thereof against DLL3.

[0462] wherein the antibody comprises a heavy chain variable region, wherein

[0463] The heavy chain variable region comprises:

[0464] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26,

[0465] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 80, and

[0466] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 141.

[0467] In one embodiment, the DLL3 antibody PR013371 comprises a heavy chain variable region (VH),

[0468] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 202 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 202.

[0469] In one embodiment, the DLL3 antibody PR013371 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013371 comprises the amino acid sequence of SEQ ID NO: 250, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 250.

[0470] Antibody PR013372

[0471] In another aspect, the present invention provides an antibody PR013372 or an antigen-binding fragment thereof against DLL3,

[0472] wherein the antibody comprises a heavy chain variable region, wherein

[0473] The heavy chain variable region comprises:

[0474] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26,

[0475] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 81, and

[0476] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 142.

[0477] In one embodiment, the DLL3 antibody PR013372 comprises a heavy chain variable region (VH),

[0478] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 203 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 203.

[0479] In one embodiment, the DLL3 antibody PR013372 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013372 comprises the amino acid sequence of SEQ ID NO: 251, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 251.

[0480] Antibody PR013376

[0481] In another aspect, the present invention provides an antibody PR013376 or an antigen-binding fragment thereof against DLL3,

[0482] wherein the antibody comprises a heavy chain variable region, wherein

[0483] The heavy chain variable region comprises:

[0484] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25,

[0485] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 82, and

[0486] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 143.

[0487] In one embodiment, the DLL3 antibody PR013376 comprises a heavy chain variable region (VH),

[0488] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 204 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 204.

[0489] In one embodiment, the DLL3 antibody PR013376 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013376 comprises the amino acid sequence of SEQ ID NO: 252, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 252.

[0490] Antibody PR013377

[0491] In another aspect, the present invention provides an antibody PR013377 or an antigen-binding fragment thereof against DLL3,

[0492] wherein the antibody comprises a heavy chain variable region, wherein

[0493] The heavy chain variable region comprises:

[0494] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0495] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 60, and

[0496] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126.

[0497] In one embodiment, the DLL3 antibody PR013377 comprises a heavy chain variable region (VH),

[0498] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 205 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 205.

[0499] In one embodiment, the DLL3 antibody PR013377 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013377 comprises the amino acid sequence of SEQ ID NO: 253, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 253.

[0500] Antibody PR013378

[0501] In another aspect, the present invention provides an antibody PR013378 or an antigen-binding fragment thereof against DLL3,

[0502] wherein the antibody comprises a heavy chain variable region, wherein

[0503] The heavy chain variable region comprises:

[0504] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 28,

[0505] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 83, and

[0506] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 144.

[0507] In one embodiment, the DLL3 antibody PR013378 comprises a heavy chain variable region (VH),

[0508] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 206 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 206.

[0509] In one embodiment, the DLL3 antibody PR013378 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013378 comprises the amino acid sequence of SEQ ID NO: 254, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 254.

[0510] Antibody PR013381

[0511] In another aspect, the present invention provides an antibody PR013381 or an antigen-binding fragment thereof against DLL3,

[0512] wherein the antibody comprises a heavy chain variable region, wherein

[0513] The heavy chain variable region comprises:

[0514] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 29,

[0515] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 84, and

[0516] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 143.

[0517] In one embodiment, the DLL3 antibody PR013381 comprises a heavy chain variable region (VH),

[0518] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 207 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 207.

[0519] In one embodiment, the DLL3 antibody PR013381 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013381 comprises the amino acid sequence of SEQ ID NO: 255, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 255.

[0520] Antibody PR013382

[0521] In another aspect, the present invention provides an antibody PR013382 or an antigen-binding fragment thereof against DLL3,

[0522] wherein the antibody comprises a heavy chain variable region, wherein

[0523] The heavy chain variable region comprises:

[0524] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26,

[0525] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 85, and

[0526] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 145.

[0527] In one embodiment, the DLL3 antibody PR013382 comprises a heavy chain variable region (VH),

[0528] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 208 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 208.

[0529] In one embodiment, the DLL3 antibody PR013382 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013382 comprises the amino acid sequence of SEQ ID NO: 256, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 256.

[0530] Antibody PR013384

[0531] In another aspect, the present invention provides an antibody PR013384 or an antigen-binding fragment thereof against DLL3,

[0532] wherein the antibody comprises a heavy chain variable region, wherein

[0533] The heavy chain variable region comprises:

[0534] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 30,

[0535] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 75, and

[0536] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 136.

[0537] In one embodiment, the DLL3 antibody PR013384 comprises a heavy chain variable region (VH),

[0538] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 209 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 209.

[0539] In one embodiment, the DLL3 antibody PR013384 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013384 comprises the amino acid sequence of SEQ ID NO: 257, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 257.

[0540] Antibody PR013387

[0541] In another aspect, the present invention provides an antibody PR013387 or an antigen-binding fragment thereof against DLL3,

[0542] wherein the antibody comprises a heavy chain variable region, wherein

[0543] The heavy chain variable region comprises:

[0544] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31,

[0545] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 85, and

[0546] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 146.

[0547] In one embodiment, the DLL3 antibody PR013387 comprises a heavy chain variable region (VH),

[0548] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 210 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 210.

[0549] In one embodiment, the DLL3 antibody PR013387 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013387 comprises the amino acid sequence of SEQ ID NO: 258, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 258.

[0550] Antibody PR013392

[0551] In another aspect, the present invention provides an antibody PR013392 or an antigen-binding fragment thereof against DLL3,

[0552] wherein the antibody comprises a heavy chain variable region, wherein

[0553] The heavy chain variable region comprises:

[0554] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0555] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 70, and

[0556] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 134.

[0557] In one embodiment, the DLL3 antibody PR013392 comprises a heavy chain variable region (VH),

[0558] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 211 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 211.

[0559] In one embodiment, the DLL3 antibody PR013392 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013392 comprises the amino acid sequence of SEQ ID NO: 259, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 259.

[0560] Antibody PR013393

[0561] In another aspect, the present invention provides an antibody PR013393 or an antigen-binding fragment thereof against DLL3.

[0562] wherein the antibody comprises a heavy chain variable region, wherein

[0563] The heavy chain variable region comprises:

[0564] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 32,

[0565] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 86, and

[0566] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 147.

[0567] In one embodiment, the DLL3 antibody PR013393 comprises a heavy chain variable region (VH),

[0568] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 212 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 212.

[0569] In one embodiment, the DLL3 antibody PR013393 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013393 comprises the amino acid sequence of SEQ ID NO: 260, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 260.

[0570] Antibody PR013394

[0571] In another aspect, the present invention provides an antibody PR013394 or an antigen-binding fragment thereof against DLL3,

[0572] wherein the antibody comprises a heavy chain variable region, wherein

[0573] The heavy chain variable region comprises:

[0574] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0575] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 85, and

[0576] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 148.

[0577] In one embodiment, the DLL3 antibody PR013394 comprises a heavy chain variable region (VH),

[0578] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 213 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 213.

[0579] In one embodiment, the DLL3 antibody PR013394 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013394 comprises the amino acid sequence of SEQ ID NO: 261, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 261.

[0580] Antibody PR013397

[0581] In another aspect, the present invention provides an antibody PR013397 or an antigen-binding fragment thereof against DLL3,

[0582] wherein the antibody comprises a heavy chain variable region, wherein

[0583] The heavy chain variable region comprises:

[0584] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 33,

[0585] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 87, and

[0586] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 149.

[0587] In one embodiment, the DLL3 antibody PR013397 comprises a heavy chain variable region (VH),

[0588] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 214.

[0589] In one embodiment, the DLL3 antibody PR013397 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013397 comprises the amino acid sequence of SEQ ID NO: 262, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 262.

[0590] Antibody PR013399

[0591] In another aspect, the present invention provides an antibody PR013399 or an antigen-binding fragment thereof against DLL3.

[0592] wherein the antibody comprises a heavy chain variable region, wherein

[0593] The heavy chain variable region comprises:

[0594] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26,

[0595] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 88, and

[0596] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 150.

[0597] In one embodiment, the DLL3 antibody PR013399 comprises a heavy chain variable region (VH),

[0598] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 215 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 215.

[0599] In one embodiment, the DLL3 antibody PR013399 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013399 comprises the amino acid sequence of SEQ ID NO: 263, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 263.

[0600] Antibody PR013402

[0601] In another aspect, the present invention provides an antibody PR013402 or an antigen-binding fragment thereof against DLL3.

[0602] wherein the antibody comprises a heavy chain variable region, wherein

[0603] The heavy chain variable region comprises:

[0604] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18,

[0605] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 89, and

[0606] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 151.

[0607] In one embodiment, the DLL3 antibody PR013402 comprises a heavy chain variable region (VH),

[0608] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 216 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 216.

[0609] In one embodiment, the DLL3 antibody PR013402 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013402 comprises the amino acid sequence of SEQ ID NO: 264, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 264.

[0610] Antibody PR013406

[0611] In another aspect, the present invention provides an antibody PR013406 or an antigen-binding fragment thereof against DLL3,

[0612] wherein the antibody comprises a heavy chain variable region, wherein

[0613] The heavy chain variable region comprises:

[0614] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 34,

[0615] HCDR2 comprising the amino acid sequence shown in SEQ ID NO:90, and

[0616] HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 152.

[0617] In one embodiment, the DLL3 antibody PR013406 comprises a heavy chain variable region (VH),

[0618] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 217 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 217.

[0619] In one embodiment, the DLL3 antibody PR013406 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the DLL3 antibody PR013406 comprises the amino acid sequence of SEQ ID NO: 265, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO: 265.

[0620] As used herein, the term "modification" refers to the presence of changes to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such changes or modifications can be obtained by post-synthetic modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide; or by co-translational or post-translational modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. The form "modified or unmodified" means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide in question is optionally modified, i.e., the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide in question can be modified or unmodified.

[0621] DLL3-binding molecules of the present invention may include modified or unmodified amino acids. For example, during antibody production, transportation, or storage, modifications to the antibody's amino acid sequence may occur, including post-translational modifications (PTMs), particularly in the CDR regions. In the present invention, such PTMs may include modifications known to those skilled in the art, including asparagine deamidation (positions NG, NS, NH, etc.), aspartate isomerization (positions DG, DP, etc.), N-glycosylation (positions NxS / T, where x is any amino acid except proline), and oxidation. Possible PTMs of the DLL3 antibodies of the present invention are shown in Table 1.

[0622] Multispecific antigen-binding constructs

[0623] In a second aspect, the invention provides a multispecific antigen-binding construct comprising a first antigen-binding moiety that binds DLL3, wherein the first antigen-binding moiety comprises the DLL3-binding molecule of the first aspect of the invention. In a preferred embodiment, the multispecific antigen-binding construct comprises a first antigen-binding moiety that binds DLL3 and a second antigen-binding moiety that binds a second antigen, wherein the first antigen-binding moiety comprises the DLL3-binding molecule of the first aspect of the invention.

[0624] As used herein, a "multispecific antigen-binding construct" or "multispecific molecule" refers to a construct that specifically binds to one or more different antigens or one or more different epitopes within the same antigen. A multispecific antigen-binding construct can be a protein, a protein complex, or an antibody. A multispecific antigen-binding construct comprises an antibody or antigen-binding fragment thereof that can be associated or linked to at least one other functional molecule (e.g., another peptide or protein, such as another antibody or ligand for a receptor) to form a molecule that binds to at least two different binding sites or target molecules. A multispecific molecule may have cross-reactivity to other related antigens, for example, to the same antigen from another species (homologous), such as a human or monkey, e.g., a cynomolgus macaque or a chimpanzee, or may bind to an epitope shared between two or more different antigens. Exemplary multispecific molecules include multispecific antibodies, such as bispecific antibodies.

[0625] As used herein, the term "multispecific antibody" refers to an antibody that specifically binds to two or more (e.g., two, three, four, five, or six) different antigenic epitopes. Multispecific antibodies may be, for example, bispecific, trispecific, or tetraspecific antibodies, each capable of specifically binding to two, three, or four antigenic epitopes, respectively. As used herein, the terms "epitope" or "antigenic determinant" refer to a region of an antigen that specifically binds to the antigen-binding site of an antibody. An antigenic epitope is typically composed of chemically active surface groups (e.g., amino acids or sugar side chains) of an antigen and typically has specific three-dimensional structural properties as well as specific charge properties. The second antigen may be an antigen other than DLL3. The second antigen may also be DLL3, which binds to a different epitope on DLL3 than the anti-DLL3 antibody or antigen-binding fragment thereof of the present invention. Whether the two antibodies bind to the same epitope can be determined using conventional methods in the art, such as by measuring competitive binding of the two antibodies to the same epitope using ELISA, flow cytometry, or surface plasmon resonance.

[0626] Multispecific antibodies can be multivalent (e.g., 2, 3, 4-valent) antibodies, i.e., they have multiple antigen binding sites. Methods for constructing multispecific antibodies using antibodies or antigen-binding fragments of interest are well known to those skilled in the art (see, e.g., WO 93 / 08829; Suresh et al., (1986) Methods in Enzymology, 121: 210; and Traunecker et al., (1991) EMBO, 10: 3655-3659). Multispecific antibodies can be produced and isolated using various techniques known in the art. For example, a polynucleotide encoding a multispecific antibody can be obtained by recombinant DNA technology, optionally cloned into an expression vector, and then transformed into a host cell with the polynucleotide or expression vector, cultured under appropriate conditions to allow expression of the polynucleotide or expression vector, and finally isolated and purified from the host cell or culture medium. The various portions of the multispecific antibody can also be obtained separately, for example, the first antigen-binding portion and the second antigen-binding portion as described herein can be obtained separately, and then the various portions can be coupled by enzymatic or chemical coupling techniques, optionally through a linker, to obtain a multispecific antibody that specifically binds to DLL3 and other antigens.

[0627] As used herein, "first antigen-binding portion" and "second antigen-binding portion" refer to amino acid sequences that contain an antigen-binding site and are capable of binding to an antigen epitope, and are defined within the meaning of an antibody or an antigen-binding fragment.

[0628] The first antigen-binding moiety can be any form of antibody or antigen-binding fragment, including but not limited to Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', and F(ab')2. In some embodiments, the first antigen-binding moiety comprises a DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody of the invention, or an antigen-binding fragment thereof). In one embodiment, the first antigen-binding moiety comprises a VH comprising the HCDR1, HCDR2, and HCDR3 of a DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody of the invention, or an antigen-binding fragment thereof), as described above.

[0629] The second antigen binding portion can be an antibody or antigen binding fragment that binds to any antigenic epitope of interest. In one embodiment, the second antigen is an antigen other than DLL3. Antigens that the second antigen binding portion can specifically bind to may include: tumor antigens (e.g., tumor-associated antigens and tumor-specific antigens), immunomodulatory receptors, and immune checkpoint molecules. As used herein, "tumor-associated antigen" refers to an antigen that is highly expressed in tumor cells and also present in healthy cells but at a lower expression level. As used herein, "tumor-specific antigen" refers to an antigen that is specifically expressed in tumor cells and hardly expressed in healthy cells. Non-limiting examples of tumor antigens can include CD19, CD20, EGFR, GPC3, HER-2, and FOLR1. Non-limiting examples of immune checkpoint molecules can include CTLA-4, LAG-3, PD-1, PD-L1, and TIM-3. Immunomodulatory receptors can include, for example, immune-activating receptors (e.g., CD27, CD137, CD40, GITR, and OX40) and immunoinhibitory receptors (e.g., BTLA, CTLA4, LAG-3, and PD-1). For example, the second antigen binding portion can be an agonist antibody for an immune activating receptor or an antagonist antibody for an immune inhibitory receptor. In one embodiment, the antigen to which the second antigen binding portion can specifically bind can include: an antigen on a lymphocyte such as a T cell or a natural killer (NK) cell. In one embodiment, the antigen on the lymphocyte is CD3, CD3ε (CD3 epsilon), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195 or NKG2C.

[0630] In one embodiment, the second antigen binding portion specifically binds to a tumor antigen, an immunomodulatory receptor, and an immune checkpoint molecule. In one embodiment, the second antigen binding portion specifically binds to an antigen on a lymphocyte such as a T cell or a natural killer (NK) cell.

[0631] In one embodiment, the second antigen binding portion specifically binds SIRPα, PD-1, PD-L1, LAG3, TIM-3, CTLA-4, VISTA, GPC3, EGFR, HER-2, CD19, CD20, CD33, CD40, CD73, OX40, CD3, TIP-1, folate receptor alpha (FOLR1) and / or other antigens.

[0632] The second antigen-binding portion can be any form of antibody or antigen-binding fragment, including but not limited to a single variable domain of an immunoglobulin (e.g., comprising a VHH from alpaca or an IgNAR variable domain from shark), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', and F(ab')2.

[0633] The first antigen binding portion and the second antigen binding portion can optionally be connected by a linker. In some embodiments, the first antigen binding portion and the second antigen binding portion are not connected by a linker. In other embodiments, the first antigen binding portion and the second antigen binding portion are connected by a linker, such as a peptide linker or a chemical bond. Preferably, the first antigen binding portion and the second antigen binding portion are connected by a peptide linker. Exemplary peptide linkers can include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S), or a combination thereof, such as GGAS, GGGS, GGGSG, or (G4S) n , where n is an integer from 1 to 20.

[0634] CAR and CAR-expressing immune effector cells

[0635] In a third aspect, the present invention also provides a chimeric antigen receptor (CAR) comprising a DLL3 binding molecule of the present invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention). As used herein, the term "chimeric antigen receptor (CAR)" refers to a polypeptide comprising at least an extracellular domain, a transmembrane domain, and an intracellular T cell receptor activation signaling domain, wherein the extracellular domain binds to or specifically binds to a target on a monospecific or multispecific antibody through a monospecific or multispecific antibody. CAR is a recombinant receptor that simultaneously provides antigen binding and activates T cell function. CAR structure and engineering are described, for example, in Dotti G. et al., (2014) Immunol Rev. 257(1):107-126, which is incorporated herein by reference.

[0636] In an exemplary embodiment, the CAR of the present invention comprises, from N-terminus to C-terminus:

[0637] (a) an extracellular region comprising a DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody of the invention or an antigen-binding fragment thereof);

[0638] (b) a transmembrane region that connects the extracellular region and the intracellular signaling region and anchors the CAR to the cell membrane; and

[0639] (c) Intracellular signaling region.

[0640] The extracellular region comprises a DLL3 binding molecule of the invention (e.g., an anti-DLL3 antibody of the invention or an antigen-binding fragment thereof), such that the CAR of the invention binds to cells expressing DLL3 (e.g., cancer cells).

[0641] The transmembrane region connects the extracellular region and the intracellular signaling region and anchors the CAR to the cell membrane.

[0642] When the extracellular region of CAR binds to the antigen it recognizes, the intracellular signaling region transmits T cell receptor (TCR)-like signals into the cell and activates the immune effector cells expressing CAR to exert their effector functions.

[0643] In some embodiments, the intracellular signaling region further comprises at least one co-stimulatory domain. The co-stimulatory domain can promote the activation of CAR-expressing immune effector cells after binding to the antigen targeted by the extracellular region.

[0644] In some embodiments, the CAR of the present invention further comprises a spacer connecting the extracellular region and the transmembrane region.In one embodiment, the spacer is derived from the heavy chain constant region of an immunoglobulin.

[0645] In yet another aspect, the present invention also relates to a polynucleotide encoding the CAR of the present invention and an expression vector comprising the polynucleotide.

[0646] In a fourth aspect, the present invention also provides an immune effector cell that expresses the CAR of the present invention on the cell surface. Preferably, the immune effector cell is selected from T lymphocytes (e.g., cytotoxic T cells (CTL)), macrophages, natural killer cells (NK) and natural killer T cells (NKT). Immune effector cells can target DLL3-positive diseased cells (e.g., DLL3-positive cancer cells) and be activated to start effector functions, for example, causing the death of DLL3-positive cancer cells.

[0647] Antibody conjugates

[0648] In a fifth aspect, the present invention further provides an antibody conjugate comprising a DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof) or a multispecific antigen-binding construct of the invention conjugated to at least one therapeutic agent. An antibody-drug conjugate (ADC) is a typical antibody conjugate, wherein the therapeutic agent can be, for example, a cytotoxic agent.

[0649] As used herein, "conjugation" refers to the attachment of two or more moieties to each other through covalent or non-covalent interactions. In a preferred embodiment, the conjugation is covalent conjugation.

[0650] The therapeutic agent can be selected from cytotoxic agents, therapeutic antibodies (e.g., antibodies or antigen-binding fragments thereof that specifically bind to another antigen), radioactive isotopes, oligonucleotides and their analogs (e.g., interfering RNA), biologically active peptides, protein toxins (e.g., diphtheria toxin, ricin toxin), and enzymes (e.g., urease).

[0651] Cytotoxic agents refer to substances that inhibit or reduce the activity, function and / or kill cells. Examples of cytotoxic agents may include, but are not limited to, maytansinoids (e.g., maytansine), auristatins (e.g., MMAF, MMAE, MMAD), duostatins, cryptophycins, vinca alkaloids (e.g., vinblastine, vincristine), colchicines, dolastatins, taxanes, paclitaxel, docetaxel, cabazitaxel, enediyne antibiotics, cytochalasins, camptothecins, anthracyclines (e.g., daunorubicin, dihydroxyanthracindione, doxorubicin), cytotoxic antibiotics (e.g., mitomycin, actinomycin), dactinomycin, dactin, dactycin, dactin ...

[0013] Examples of the present invention include, but are not limited to, chloramphenicol, chlortetracycline ...

[0652] The radioisotope may be selected from, for example 212 Bi, 213 Bi, 131 I. 125 I. 111 In, 177 Lu, 186 Re、 188 Re、 153 Sm, 90 Y.

[0653] In some embodiments, the therapeutic agent is selected from cytotoxic agents, chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, antibodies targeting tumor-specific antigens, and other anti-tumor drugs. In a preferred embodiment, the therapeutic agent is a cytotoxic agent. In another preferred embodiment, the therapeutic agent is a radioisotope. In another preferred embodiment, the therapeutic agent is an immune checkpoint inhibitor.

[0654] The therapeutic agent can be conjugated to the DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof) or the multispecific antigen-binding construct of the invention via a linker using any technique known in the art. The linker can comprise a reactive group for covalent conjugation, such as an amine, hydroxylamine, maleimido, carboxyl, phenyl, thiol, sulfhydryl, or hydroxyl group.

[0655] Pharmaceutical composition

[0656] In a sixth aspect, the present invention also provides a pharmaceutical composition comprising a DLL3 binding molecule of the present invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention), a multispecific antigen-binding construct, an immune effector cell or an antibody conjugate, and a pharmaceutically acceptable carrier.

[0657] Pharmaceutically acceptable carriers may include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitan,

[0014] Examples of the present invention include, but are not limited to, sucrose alcohols, tartaric acid, phosphoric acid, etc.), solubilizers, gelling agents, emollients, solvents (e.g., water, alcohol, acetic acid, and syrup), buffers (e.g., phosphate buffers, histidine buffers, and acetate buffers), surfactants (e.g., non-ionic surfactants such as polysorbate 80, polysorbate 20, poloxamer, or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delaying agents, chelating agents, and emulsifiers. For pharmaceutical compositions comprising a DLL3 binding molecule (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention), a multispecific antigen-binding construct, or an antibody conjugate, suitable carriers can be selected from a buffer (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), an isotonic agent (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), a non-ionic surfactant (e.g., polysorbate 80, polysorbate 20, poloxamer), or a combination thereof.

[0658] The pharmaceutical compositions provided herein may be in a variety of dosage forms, including but not limited to solid, semisolid, liquid, powder, or lyophilized forms. For pharmaceutical compositions comprising DLL3-binding molecules (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof of the present invention) or multispecific antigen-binding constructs, preferred dosage forms include, for example, injection solutions and lyophilized powders.

[0659] The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or topical administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavitary), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical). Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Methods of administration can be, for example, injection or infusion.

[0660] It will be understood by those skilled in the art that the exact dosage administered will depend on various factors, such as the metabolic kinetic properties of the pharmaceutical composition, the duration of treatment, the excretion rate of the specific compound, the purpose of the treatment, the route of administration, and the condition of the subject, such as the patient's age, health status, weight, sex, diet, medical history, and other factors well known in the medical field.

[0661] As a general guide, the DLL3-binding molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, or antibody conjugates can be administered at a dosage ranging from about 0.0001 to 100 mg / kg, more typically 0.01 to 20 mg / kg of the subject's body weight. For example, the dosage can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, or 20 mg / kg, or within the range of 1-20 mg / kg. Exemplary treatment regimens entail dosing once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, once every three to six months, or initially with a shorter dosing interval followed by a longer dosing interval.

[0662] treat

[0663] In a seventh aspect, the present invention relates to use of a DLL3 binding molecule (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof), a multispecific antigen-binding construct, an immune effector cell, an antibody conjugate, or a pharmaceutical composition of the invention in the preparation of a medicament for treating a disease in a subject.

[0664] The present invention also relates to the DLL3 binding molecules of the present invention (eg, the anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates or pharmaceutical compositions of the present invention for use in treating diseases.

[0665] The present invention also provides a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof), a multispecific antigen-binding construct, an immune effector cell, an antibody conjugate, or a pharmaceutical composition.

[0666] As used herein, a "therapeutically effective dose" refers to a dose that results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevents damage or disability caused by the suffering of the disease. For example, a therapeutically effective dose is used for anti-proliferative effects, preventing further tumor development, reducing tumor size, reducing tumor vascularity, reducing the number of cancer cells, inhibiting, delaying or reducing tumor and / or malignant cell growth and / or metastasis in cancer patients, and / or reducing one or more symptoms associated with the disease that can be observed. The therapeutically effective dose can vary depending on many different factors, including the mode of administration, the target site, the patient's physiological state, whether the patient is human or other animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. In certain embodiments, the patient is human, but non-human mammals, including transgenic animals, can also be treated. The therapeutic dose can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0667] A "therapeutically effective dose" of a DLL3 binding molecule (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof), multispecific antigen-binding construct, immune effector cell, antibody conjugate, or pharmaceutical composition of the invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%. The ability to inhibit tumor growth can be evaluated in animal model systems predictive of efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro using assays known to those skilled in the art. An effective amount of a DLL3 binding molecule (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof), multispecific antigen-binding construct, immune effector cell, antibody conjugate, or pharmaceutical composition of the invention is capable of reducing tumor size or otherwise alleviating symptoms in a subject, such as preventing and / or treating metastasis or recurrence. Such an amount can be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0668] In some embodiments, the disease is associated with abnormal DLL3 expression. The term "abnormal expression" refers to excessively high or low protein expression levels in a sample compared to a normal sample (or a standard sample, such as a sample from a subject not suffering from a disease associated with abnormal DLL3 expression). Preferably, the disease is characterized by high DLL3 expression. For example, DLL3 is highly expressed in tissues (e.g., tumor tissue and adjacent tissues) of subjects suffering from or suspected of suffering from a disease (e.g., small cell lung cancer), while DLL3 is low in the corresponding tissues of subjects not suffering from the disease.

[0669] In one embodiment, the disease as described above is cancer. In one embodiment, the cancer is a DLL3-positive tumor. In a preferred embodiment, the cancer is selected from small cell lung cancer (SCLC); large cell neuroendocrine carcinoma (LCNEC); neuroendocrine tumors of various tissues including the kidney, genitourinary tract (e.g., bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (e.g., stomach, colon), thyroid (e.g., medullary thyroid carcinoma), pancreas, and lung; glioma or pseudoneuroendocrine tumor (pNET).

[0670] The DLL3-binding molecules of the present invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates, or pharmaceutical compositions can be administered in combination with at least one or more therapeutic agents described herein. The manner of combined administration is not limited. For example, all of the above therapeutic agents can be administered at once or separately.

[0671] For cancer treatment, the DLL3-binding molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates, or pharmaceutical compositions of the invention can be used in combination with other treatment methods, including but not limited to surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition, and palliative care.

[0672] In certain embodiments, the DLL3 binding molecules of the present invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates, or pharmaceutical compositions are further used in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen-targeted drugs. Chemotherapeutic agents may include, for example, antimetabolites, alkylating agents, cytotoxic agents, topoisomerase inhibitors, and microtubule inhibitors. Tumor antigen-targeted drugs include, but are not limited to, drugs that target tumor-associated antigens and tumor-specific antigens. Other non-limiting examples of therapeutic agents may include, for example, angiogenesis inhibitors, heparin deacetylase (HDAC) inhibitors, hedgehog signaling pathway blockers, mTOR inhibitors, p53 / mdm2 inhibitors, PARP inhibitors, proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib, marizomib, oprozomib), and tyrosine kinase inhibitors (e.g., BTK inhibitors).

[0673] In some embodiments, the DLL3 binding molecules (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates, or pharmaceutical compositions of the present invention are used in combination with a chemotherapeutic agent. In some embodiments, the DLL3 binding molecules (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates, or pharmaceutical compositions of the present invention are used in combination with an immune checkpoint inhibitor. In some embodiments, the DLL3 binding molecules (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates, or pharmaceutical compositions of the present invention are used in combination with a radioisotope. In some embodiments, the DLL3 binding molecules (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof), multispecific antigen-binding constructs, immune effector cells, antibody conjugates, or pharmaceutical compositions of the present invention are used in combination with a tumor antigen-targeted drug.

[0674] Polynucleotides, vectors and host cells

[0675] In an eighth aspect, the present invention provides a polynucleotide encoding a DLL3 binding molecule of the present invention (eg, an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention) or a multispecific antigen-binding construct.

[0676] The polynucleotides of the present invention can be obtained using methods known in the art. For example, the polynucleotides of the present invention can be isolated from phage display libraries, yeast display libraries, immune animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells) or chemically synthesized. Polynucleotides encoding binding molecules (e.g., antibodies or their antigen-binding fragments) or multispecific antigen-binding constructs can be prepared by separation from immune animals or chemical synthesis, and then expression vectors can be constructed using the polynucleotides. The polynucleotides of the present invention can be codon-optimized for the host cells used for expression.

[0677] In a ninth aspect, the present invention also provides an expression vector comprising a polynucleotide of the present invention. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The polynucleotide of the present invention may be present in one or more expression vectors. In one embodiment, the polynucleotide of the present invention is prepared as a recombinant nucleic acid. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR) technology), etc.

[0678] In a tenth aspect, the present invention further provides a host cell comprising a polynucleotide or expression vector of the present invention. The polynucleotide or expression vector of the present invention can be introduced into a suitable host cell using various methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.

[0679] In preferred embodiments, host cells are used to express the DLL3-binding molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof) or multispecific antigen-binding constructs of the invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as E. coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for antibody expression include, but are not limited to, human cervical carcinoma cells (HeLa cells), human embryonic kidney cells (HEK cells, such as HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

[0680] In an eleventh aspect, the present invention further provides a method for producing a DLL3-binding molecule of the present invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof of the present invention) or a multispecific antigen-binding construct of the present invention, the method comprising:

[0681] a) culturing the host cells of the invention under suitable conditions to express the DLL3-binding molecules of the invention (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof) or multispecific antigen-binding constructs of the invention; and

[0682] b) isolating the DLL3 binding molecule (eg, an anti-DLL3 antibody or antigen-binding fragment thereof) or multispecific antigen-binding construct from the host cell or culture thereof.

[0683] Reagent test kit

[0684] The present invention also provides kits comprising a DLL3-binding molecule of the invention (e.g., an anti-DLL3 antibody or antigen-binding fragment thereof), a multispecific antigen-binding construct, an immune effector cell, an antibody conjugate, or a pharmaceutical composition, and instructions for use. The kit may also comprise a suitable container. In certain embodiments, the kit further comprises a device for administration. Kits generally include a label indicating the intended use and / or instructions for use of the kit contents. The term "label" includes any written or recorded material provided on or with the kit, or otherwise accompanying the kit. Beneficial effects

[0685] The DLL3-binding molecules of the present invention can exhibit excellent effects, such as, but not limited to: 1) The DLL3 antibody of the present invention is a novel fully human antibody containing only a "heavy chain" and has binding activity to both human DLL3 and cynomolgus monkey DLL3. This heavy chain antibody is only half the size of a traditional IgG antibody. Due to the fact that it does not contain a light chain, it can be advantageously used in bispecific antibodies and solves the problems of light chain mispairing and heterodimerization. (2) It can bind to DLL3 proteins derived from humans and monkeys or DLL3 proteins from mice. (3) It can be internalized into DLL3-positive cells. (4) The binding epitope is the same as or different from that of the reference antibodies PR012621 and PR012622.

[0686] Example

[0687] A further understanding of the present invention may be obtained by reference to some of the specific examples provided herein, which are intended to illustrate the present invention only and are not intended to limit the scope of the present invention in any way. Obviously, various modifications and variations may be made to the present invention without departing from the spirit of the present invention, and therefore, such modifications and variations are also within the scope of the present application. Ratios used herein include percentages, and unless otherwise specified, are by weight.

[0688] Example 1. Acquisition of fully human anti-DLL3 HCAb antibodies

[0689] Antibodies specifically binding to DLL3 can be obtained by immunizing experimental animals with DLL3 antigens. These experimental animals can include mice, rats, rabbits, sheep, camels, and others. Typically, the resulting antibodies are non-human. After obtaining non-human antibodies, these molecules need to be humanized using antibody engineering techniques to reduce immunogenicity and improve drugability. However, the humanization process is technically complex, and humanized molecules often have reduced affinity for the antigen. On the other hand, advances in transgenic technology have enabled the creation of genetically engineered mice that carry a human immunoglobulin repertoire and lack the endogenous murine immune repertoire. The antibodies produced by these transgenic mice possess fully human sequences, eliminating the need for further humanization, significantly improving the efficiency of therapeutic antibody development. The Harbour HCAb mouse (Harbour Antibodies BV, WO2002 / 085945A3) is a transgenic mouse carrying a human immunoglobulin repertoire that produces novel heavy chain-only antibodies that are half the size of traditional IgG antibodies. The antibodies it produces only have human antibody "heavy chain" variable domains and mouse Fc constant domains. Due to the absence of light chains, the antibodies almost solve the problems of light chain mispairing and heterodimerization, making this technology platform capable of developing products that are difficult to achieve with traditional antibody platforms.

[0690] Example 1.1 Immunization of mice

[0691] Multiple rounds of immunization of 6-8 week old Harbour human antibody transgenic mice Harbour HCAb V2.1 mice were performed using recombinant human DLL3-his tag protein (Acro, #DL3-H52H4). In the first immunization, each mouse was injected with 50 μg of immunogen protein and 0.2 mL of complete Freund's adjuvant (CFA, Sigma, #F5881) in the abdominal cavity, axillary lymph nodes, and inguinal lymph nodes. To enhance the immune response, two weeks after the first immunization, 25 μg of immunogen protein was injected into the abdominal cavity and subcutaneous lymph nodes of each mouse together with 200 μL of Ribi (Sigma adjuvant system, Sigma, #S6322). Subsequently, each mouse was injected with 25 μg of immunogen and 200 μL of Ribi adjuvant every 2 weeks, for a total of 5 times, including the first immunization. Immunizations were performed on days 0, 14, 28, 42, and 56, and serum antibody titers were measured on days 21, 35, 49, and 61. Five days before HCAb mouse spleen B cell isolation, a final booster immunization was performed at a dose of 25 μg antigen protein per mouse.

[0692] Example 1.2 Serum titer detection

[0693] At specific time points, mouse serum was collected and diluted 10-fold to six concentrations (1:100, 1:1000, 1:10,000, 1:100,000, and 1:1,000,000). Anti-DLL3 titers were determined by ELISA using ELISA plates coated with huDLL3-his protein (Acro, #DL3-H52H4), cynoDLL3-his protein (Acro, #DL3-C52H9), or proteins unrelated to DLL3. Flow cytometry was used to analyze the specific reactivity of two concentrations of mouse serum (1:100 and 1:1000) against CHO-K1 / humanDLL3 cells, CHO-K1 / cynoDLL3 cells, and CHO-K1 blast cells expressing high DLL3 expression. A blank control (PB) consisted of serum from pre-immunized mice.

[0694] In the ELISA method, 100 μL / well of 1 μg / mL huDLL3-his protein (Acro, #DL3-H52H4) or 1 μg / mL cynoDLL3-his protein (Acro, #DL3-C52H9) or 1 μg / mL of an irrelevant his protein was coated on an ELISA plate (corning, #9018) and incubated overnight at 4°C; after rinsing twice, the plate was blocked with PBST containing 1% BSA at 37°C for 1 hour; 100 μL / well of serially diluted serum was added and incubated at 37°C for 1 hour; after rinsing three times, 100 μL / well of 1:5000 diluted anti-mouse-HRP (Bethyl, #A90-231P) was added and incubated at 37°C for 30 minutes. After washing three times, 100 μL / well TMB substrate was added and incubated for about 10 minutes. 50 μL / well 1N HCl was added to stop color development, and the absorbance at 450 nm was read (Molecular Devices, Plus 384).

[0695] For FACS analysis, CHO-K1 / humanDLL3, CHO-K1 / cynoDLL3, or CHO-K1 cells were incubated with serially diluted mouse serum for 1 hour at 4°C. After washing twice, the cells were incubated with a secondary antibody, Alexa Fluor™ 488 goat anti-mouse IgG Fcγspecific (Jackson, #115-545-071), for 1 hour at 4°C. After washing twice, the cells were resuspended and analyzed by flow cytometry (BD Canto II). CHO-K1 cells served as a background control.

[0696] Example 1.3 Obtaining anti-DLL3 HCAb antibody sequences

[0697] When the DLL3-specific antibody titer in mouse serum reached a certain level, splenocytes were removed from the mice to isolate B cells. CD138-positive plasma cells and human DLL3-positive B cells were sorted using a BD FACS AriaII Cell Sorter. RNA from the B cells was extracted and reverse-transcribed into cDNA (SuperScript IV First-Strand Synthesis System, Invitrogen, 18091200). The human VH gene was then amplified using PCR using specific primers: 5'-GGTGTCCAGTGTSAGGTGCAGCTG-3' (SEQ ID NO: 282) and 5'-AATCCCTGGGCACT GAAGAGACGGTGACC-3' (SEQ ID NO: 283). The amplified VH gene fragment was then inserted into the mammalian cell expression plasmid pCAG, which encodes the Fc domain sequence of the human IgG1 antibody heavy chain.

[0698] The constructed plasmid was transfected into mammalian host cells (such as human embryonic kidney HEK293 cells) for expression to obtain HCAb antibodies. Flow cytometry was used to analyze the binding of HCAb-expressing supernatants to CHO-K1 / huDLL3 stable cell lines overexpressing human DLL3 or CHO-K1 / cynoDLL3 stable cell lines overexpressing cynomolgus monkey DLL3. ELISA was also used to analyze the binding of HCAb-expressing supernatants to huDLL3-his or cynoDLL3-his proteins. Conventional sequencing was used to determine the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence. After removing repetitive sequences, 168 fully human DLL3 monoclonal antibodies with unique sequences that bound to both CHO-K1 / huDLL3 and CHO-K1 / cynoDLL3 were obtained. Based on the results of human and monkey cell binding, the top 44 antibodies were selected for recombinant expression. Germline gene analysis and post-translational modification (PTM) analysis of the 44 antibodies are shown in Table 1.

[0699] Table 1. Germline gene analysis and post-translational modification (PTM) analysis of HCAb antibody sequences

[0700] Example 1.4 Expression and purification of fully human recombinant anti-DLL3 antibodies

[0701] The plasmid encoding the HCAb antibody is transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and the purified anti-DLL3 recombinant heavy chain antibody can be obtained using conventional recombinant protein expression and purification techniques. Specifically, the plasmid was purified using a large extraction kit (Macherey-Nagel, The plasmid was extracted with Xtra Midi to improve the purity and quality of the plasmid, and the plasmid was filtered through a 0.22 μm filter (millpore). The purified plasmid was used for transfection. HEK293 cells were cultured in FreeStyle TM Before transient transfection, adjust the cell density to 6×10 5 cells / ml, and cultured in a shaker at 37°C with 8% CO2 for 24 hours. The cell concentration was 1.2×10 6 Cells / mL. Prepare 30mL of cultured cells, dissolve 30μg of the plasmid encoding the HCAb heavy chain in 1.5ml Opti-MEM reduced serum medium (Thermo, #31985088), then take 1.5ml Opti-MEM and dissolve 120μl of 1mg / mL PEI (Polysciences, Inc, #23966-2), and let it stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drip the plasmid PEI mixed solution while shaking the culture bottle, and culture in a 37℃ 8% CO2 shaker for 5 days. Observe the cell viability after 5 days. Collect the culture, centrifuge at 3300G for 10 minutes, and then take the supernatant; then centrifuge the supernatant at high speed to remove impurities. Use PBS (pH7.4) to balance the culture medium containing MabSelect TM A gravity column (Bio-Rad, #7311550) (GE Healthcare Life Science, #71-5020-91AE) was used, with 2-5 column volumes of washes. The supernatant sample was passed through the column. The column was rinsed with 5-10 column volumes of PBS. The target protein was then eluted with 0.1 M glycine (pH 3.5), then adjusted to neutral with Tris-HCl (pH 8.0). Finally, the column was concentrated using an ultrafiltration tube (Millipore, UFC901024) and exchanged with PBS buffer to obtain a purified anti-DLL3 heavy chain antibody solution. Antibody concentration was determined by absorbance at 280 nm using a NanoDrop flow cytometer, and antibody purity was determined by SEC-HPLC and SDS-PAGE. Through these experiments, a total of 44 DLL3 monoclonal antibodies with unique sequences were obtained, all of which were of the IgG1 subtype.

[0702] In addition, two comparative antibodies were prepared for use in subsequent examples, one being the comparative example 1 antibody PR0012621 (prepared in-house), and the other being the comparative example 2 antibody PR0012622 (prepared in-house). Specifically, the comparative example 1 antibody Tab (PR012621) was prepared based on Rovalpituzumab tesirine (Rova-T) from ABBVie, and the amino acid sequences of its heavy chain and light chain are shown in SEQ NO ID: 220 and SEQ NO ID: 266, respectively.

[0703] Comparative Example 2 Antibody Tab (PR012622) is a comparative example antibody prepared based on AMG757 from Amgen. The amino acid sequences of its heavy chain and light chain are shown in SEQ NO ID: 221 and SEQ NO ID: 267, respectively.

[0704] Example 1.5 Analysis of protein purity and aggregates using SEC-HPLC

[0705] This example uses analytical size exclusion chromatography (SEC) to analyze the purity and polymeric form of protein samples. An analytical chromatographic column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 μm, 7.8 mm × 30 cm) is connected to a high pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated at room temperature with PBS buffer for at least 1 hour. An appropriate amount of protein sample (at least 10 μg) is filtered through a 0.22 μm filter membrane and injected into the system. The HPLC program is set to flow the sample through the chromatographic column at a flow rate of 1.0 mL / min with PBS buffer for a maximum of 25 minutes. The HPLC will generate an analytical report reporting the retention times of the different molecular size components in the sample.

[0706] Example 1.6 Anti-DLL3 Antibody Sequences and Numbering

[0707] In this article, the amino acid sequence of CDR is all shown according to the Chothia definition rule. However, it is well known to those skilled in the art that the CDR of antibody can be defined in this area by a variety of methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Immunological Protein Sequences, 5th edition, National Institutes of Health, Bethesda, Maryland (1991)) and the Chothia definition rule based on structural loop region position (see J Mol Biol 273:927-48,1997). In the technical scheme of the present invention, the Combined definition rule comprising the Kabat definition and the Chothia definition can also be used to determine the amino acid residues in the variable domain sequence. Wherein the Combined definition rule is to combine the scope of the Kabat definition and the Chothia definition, based on which a larger scope is taken, see Table 2 for details. It will be understood by those skilled in the art that, unless otherwise specified, the term "CDR" and "complementary determining region" of a given antibody or its district (such as variable region) should be understood to encompass the complementary determining region defined as defined in any of the above-mentioned known schemes described by the present invention. Although the scope of protection requested in the claims of the present invention is based on the sequences shown in the Chothia definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of protection of the present invention.

[0708] Table 2. Kabat definition, Chothia definition and Combined definition

[0709] Here, Haa-Hbb may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and starting from position aa (Chothia numbering rules) to position bb (Chothia numbering rules). For example, H26-H35 may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and starting from position 26 to position 35 according to the Chothia numbering rules. It should be understood by those skilled in the art that when using Chothia to encode CDRs, some positions may have insertion sites (see http: / / bioinf.org.uk / abs / ).

[0710] The amino acid sequences of 44 candidate antibodies and two comparative antibodies are shown in Tables 3 and 4. The CDR sequences were determined using the Chothia definition of CDR.

[0711] Table 3. Amino acid sequences of 44 candidate antibodies (SEQ ID NO:)

[0712] Table 4. Amino acid sequences of two comparative antibodies (SEQ ID NO:)

[0713] Example 2 ELISA detection of the binding ability of anti-human DLL3 HCAb monoclonal antibody protein level

[0714] This example investigates the in vitro binding activity of anti-human DLL3 HCAb monoclonal antibodies to human, monkey, and mouse DLL3. Protein-level antibody binding experiments were performed using human DLL3-his-tagged protein (Acro, #DL3-H52H4), monkey DLL3-his-tagged protein (Acro, #DL3-C52H9), and mouse DLL3-his-tagged protein (Acro, #DL3-M52H9). Briefly, human, monkey, and mouse DLL3 were diluted to 1 mg / mL in PBS and added to 96-well plates (Corning, #9018), 100 mL per well, and incubated overnight at 4°C. After discarding the liquid, the plates were washed three times with PBST buffer (pH 7.4, containing 0.05% Tween-20), and 250 mL of 2% BSA blocking solution was added, followed by incubation at 37°C for 1 hour. The blocking buffer was discarded, and the plate was washed three times with PBST buffer (pH 7.4, containing 0.05% Tween-20). Then, 100 mL of the antibody to be tested was added to each well in a 5-fold serial dilution, and the cells were incubated at 37°C for 1 hour. An isotype antibody was used as a control. After washing three times with PBST buffer (pH 7.4, containing 0.05% Tween-20), a 5000-fold diluted goat anti-human HRP secondary antibody (Jackson ImmunoResearch, #109-035-098) was added and incubated at 37°C for 1 hour in the dark. After washing three times with PBST buffer (pH 7.4, containing 0.05% tween-20), 100 mL / well of TMB (Biopanda, #TMB-S-003) was added and the cells were incubated at room temperature in the dark for approximately 30 minutes. 50 mL / well of stop solution (BBI life sciences, #E661006-0200) was added to each well to terminate the reaction, and the absorbance at 450 nm (OD450) was measured using a microplate reader (MDC, Spectramax Plus384).

[0715] The data of antibody binding to human DLL3, monkey DLL3, and mouse DLL3 are summarized in Table 5. The results of 44 monoclonal antibodies binding to human DLL3, monkey DLL3, and mouse DLL3 on the cell surface are shown in Figures 1A-1D, 2A-2D, and 3A-3D, respectively.

[0716] The results show:

[0717] 1) 18 antibodies such as PR013234, PR013235, PR013236, PR013239, PR013240, PR013241, PR013244, PR013246, PR013254, PR013256, PR013257, PR013258, PR013259, PR013261, PR013364, PR013377, PR013392, and PR013402 have excellent binding activity to human DLL3, monkey DLL3, and mouse DLL3, especially PR013364, PR013377, and PR013402, with maximum values ​​and EC50 values ​​comparable to those of the reference antibody PR012621.

[0718] 2) 11 antibodies have binding activity to human DLL3 and monkey DLL3, but do not bind to mouse DLL3, such as PR013363, PR013367, PR013368, PR013370, PR013371, PR013376, PR013378, PR013382, PR013384, PR013387, and PR013399.

[0719] 3) PR013372 and PR013394 only bind to human DLL3.

[0720] Table 5. Binding activity of DLL3 antibodies to human / monkey / mouse DLL3 proteins “\” means not detected, below the detection limit.

[0721] Example 3 FACS detection of the binding ability of anti-DLL3 HCAb monoclonal antibody at the cellular level

[0722] This example is to study the activity of anti-DLL3 HCAb monoclonal antibody binding to human / cynomolgus monkey DLL3 in vitro. CHO-K1 cell line (hamster ovary cell subline) overexpressing human DLL3 (CHO-K1 / humanDLL3, also referred to as CHO-K1-humanDLL3, Nona Bio), CHO-K1 cell line overexpressing cynomolgus monkey DLL3 (CHO-K1 / cynoDLL3, also referred to as CHO-K1-cynoDLL3, Nona Bio), CHO-K1 mother cell line ( CCL-61) or the cell line SHP-77 (human small cell lung cancer cells) that highly expresses human DLL3 ( CRL-2195), DMS79 (human small cell lung cancer cells) CRL-2049) was used for cell-based antibody binding experiments. Briefly, CHO-K1 / humanDLL3 cells, CHO-K1 / cynoDLL3 cells, CHO-K1 cells, SHP-77 cells, or DMS79 cells were digested and harvested and resuspended in PBS containing 2% FBS. The cell density was adjusted to 1×10 6 100 μL of cells / well were seeded into a 96-well V-bottom plate (Corning, #3894), followed by the addition of 100 μL / well of the test antibody, diluted 5-fold to 2 times the final concentration. The cells were incubated at 4°C in the dark for 2 hours. Afterwards, the cells were rinsed twice with 100 μL / well of pre-chilled PBS containing 2% FBS, centrifuged at 500g for 5 minutes at 4°C, and the supernatant discarded. Then, 100 μL / well of fluorescent secondary antibody (Alexa Fluor 647-labeled affinity-purified goat anti-human IgG secondary antibody, specifically reactive with Fcγ fragments, Jackson, #109-605-098, 1:1000 dilution) was added and incubated at 4°C in the dark for 1 hour. The cells were washed twice with 100 μL / well of pre-chilled PBS containing 2% FBS, centrifuged at 500g for 5 minutes at 4°C, and the supernatant discarded. Finally, resuspend the cells in 200 μL / well of pre-chilled PBS containing 2% FBS, and read the fluorescence signal using a BD CantoII flow cytometer.

[0723] The data for antibody binding to human DLL3, cynomolgus monkey DLL3, CHO-K1 mother cells, and DLL3 on the surface of tumor cells SHP-77 and DMS79 are summarized in Tables 6 and 7. The results of 44 monoclonal antibodies binding to CHO-K1 / human DLL3, CHO-K1 / cyno DLL3, CHO-K1 cells, and tumor cells SHP-77 and DMS-79 are shown in Figures 4A-4D, 5A-5D, 6A-6D, 7A-7D, and 8A-8D.

[0724] The results show:

[0725] 1) Nine antibodies, including PR013236, PR013239, PR013240, PR013254, PR013256, PR013371, PR013377, PR013393, and PR013406, showed excellent binding ability, with maximum values ​​and EC50 values ​​comparable to those of the reference antibody PR012622, whether binding to recombinant cells overexpressing DLL3 or to tumor cells.

[0726] 2) PR013250 has binding activity to CHO-K1 mother cells, showing non-specific binding.

[0727] Table 6. Binding activity of anti-DLL3 antibodies to CHO-K1 / humanDLL3 and CHO-K1 / cynoDLL3 cells

[0728] Table 7. Binding activity of anti-DLL3 antibodies to tumor cells SHP-77 and DMS79

[0729] Example 4 Physicochemical Properties of Anti-DLL3 Antibodies

[0730] Example 4.1 Analysis of protein purity and hydrophobicity using HIC-HPLC

[0731] Analytical hydrophobic interaction chromatography (HIC) was used to analyze the purity and hydrophobicity of protein samples. An analytical column, TSKgel Buty1-NPR (Tosoh Bioscience, 14947, 4.6 mm × 3.5 cm), was connected to a high-pressure liquid chromatograph (HPLC) (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. A linear gradient from 100% mobile phase A (20 mM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0) over 16 minutes was used at a flow rate of 0.7 mL / min. The protein concentration was 1 mg / mL, the injection volume was 20 μl, and the detection wavelength was 280 nm. After acquisition, the chromatograms were integrated and the relevant data were calculated using ChemStation software. An analysis report was generated, reporting the retention times of different size components within the sample.

[0732] Example 4.2 Determination of thermal stability of protein molecules using DSF

[0733] Differential Scanning Fluorimetry (DSF) is a commonly used high-throughput method for determining protein thermal stability. It uses a real-time fluorescence quantitative PCR instrument to monitor the changes in the fluorescence intensity of the dye bound to the unfolded protein molecule to reflect the process of protein denaturation, thereby reflecting the thermal stability of the protein molecule. This example uses the DSF method to determine the thermal denaturation temperature (Tm) of the protein molecule. 10 μg of protein was added to a 96-well PCR plate (Thermo, #AB-0700 / W), followed by the addition of 2 μL of 100× diluted dye SYPRO™ (Invitrogen, #2008138), and then buffer was added to make the final volume 40 μL per well. The PCR plate was sealed and placed in a real-time fluorescence quantitative PCR instrument (Bio-Rad CFX96 PCR System). The plate was first incubated at 25°C for 5 minutes, then gradually heated from 25°C to 95°C in a gradient of 0.2°C / 0.2 minutes. The temperature was lowered to 25°C at the end of the test. The FRET scanning mode was used and Bio-Rad CFX Maestro software was used for data analysis and calculation of the Tm of the samples.

[0734] The physicochemical properties of the antibodies are shown in Table 8. The results showed that all 12 antibodies had excellent SEC-HPLC purity. PR013236, PR013377, and PR013397 had good hydrophilicity and thermal stability.

[0735] Table 8. Physicochemical properties of antibodies "Quantity" refers to the amount of antibody obtained in 100 mL of purification system. Tm1 is the thermal stability of the VH region, and Tm2 is the thermal stability of the CH2 region. " / " indicates that the antibody was not detected in this experiment.

[0736] Example 5 Determination of Binding Affinity and Dissociation Constant of Anti-DLL3 Antibodies to Recombinant Human or Monkey DLL3 Protein

[0737] Affinity was measured using an Octet RED96 instrument (Fortiebio) and an anti-human IgG Fc avidin sensor (AHC sensor, Pall FortieBio, #18-5060) according to the manufacturer's instructions. Specifically, human DLL3 protein (Acro, #DL3-H52H4) or monkey DLL3 protein (Acro, #DL3-C52H9) was diluted to 200 nM in PBS buffer (pH 7.4) containing 0.1% (w / w) BSA and 0.02% (v / v) Tween 20 and incubated with the AHC sensor. 40 nM of DLL3 antibody was incubated with the human DLL3 protein- or monkey protein-loaded AHC sensor at 30°C for 3 minutes. The reaction mixture was further incubated in PBS buffer (pH 7.4) containing 0.1% (v / w) BSA and 0.02% (v / v) Tween 20 at 30°C for 5 minutes. The binding and dissociation signals of the DLL3 antibody and the human or monkey DLL3 protein were recorded in real time using Octet Red 96. Kon, Kdis (also known as Koff), and KD values ​​(KD = Kdis (Koff) / Kon) were determined using Octet software.

[0738] The results are shown in Table 9, Table 10 and Table 11:

[0739] 1) Among the eight anti-DLL3 antibodies tested, the KD values ​​of PR013393 for human DLL3 protein and monkey DLL3 protein were lower than those of two reference antibodies (PR012621 and PR012622), indicating its stronger binding affinity to human and monkey DLL3 proteins.

[0740] 2) In terms of binding affinity to human DLL3 protein, the KD values ​​of PR013240 and PR013371 were comparable to the reference antibody PR012621. PR013397 was even superior to the reference antibody PR012622.

[0741] 3) In terms of binding affinity to cynomolgus monkey DLL3 protein, the KD values ​​of PR013240, PR013371, PR013377 and PR013397 were comparable to those of the reference antibody PR012621.

[0742] Table 9. Binding affinity determination of DLL3 antibodies to human DLL3 protein

[0743] Table 10. Binding and dissociation constants of DLL3 antibodies to monkey DLL3 protein

[0744] Table 11. Binding and dissociation constants of DLL3 antibodies to monkey DLL3 protein

[0745] Example 6 Endocytic Effect of Anti-DLL3 Antibodies on Target Cells

[0746] Anti-DLL3 antibodies can mediate the internalization of DLL3 proteins expressed on the cell surface by binding to the extracellular portion of DLL3. In this example, the internalization of anti-DLL3 antibodies was tested within 24 hours. The specific method is as follows: CHO-K1-humanDLL3 or SHP-77 cells were cultured and expanded in T-75 culture flasks using RPMI1640 (Thermo, #C11875500CP) containing 10% serum (VivaCell, fetal bovine serum, #2128194). The cells were transferred to a 15 mL sterile centrifuge tube and centrifuged at 1000 rpm at room temperature for 5 minutes to pellet the cells. The culture medium was aspirated and the cells were resuspended in culture medium. Gently pipette the cells to obtain a single cell suspension. Count the cells using a cell counting plate and adjust the cell concentration to 2×10 5 cells / mL (CHO-K1 / humanDLL3, i.e., CHO-K1-hDLL3 in Figures 9A and 9B ) or 1×10 6 50 mL of cell suspension per well of SHP-77 cells was transferred to a 96-well plate (Corning, #3599) and incubated overnight in a 37°C, 5% CO2 cell culture incubator. 50 mL of a 1:1 mixture of antibody and Zenon pHrodo (Invitrogen, #Z25611) was added to the 96-well plate. The mixture was incubated in a 37°C, 5% CO2 cell culture incubator for 24 hours. The cells were then digested and harvested in a 96-well V-shaped plate (Corning, #3894), washed twice with PBS, and resuspended in 150 mL of FACS buffer (2% FBS). Fluorescence intensity of the cells was analyzed using an ACEA Novocyte 3000 flow cytometer.

[0747] The data for the endocytosis effects of anti-DLL3 antibodies in CHO-K1 / humanDLL3 and SHP-77 cells are summarized in Tables 12 and 13. The endocytosis results of the eight monoclonal antibodies are shown in Figures 9A-9B and 10A-10B. The results show that PR013240, PR013371, and PR013397 have endocytosis effects comparable to those of the reference antibody in CHO-K1 / humanDLL3 and SHP-77 cells.

[0748] Table 12. Endocytic effects of anti-DLL3 antibodies on CHO-K1 / humanDLL3 and SHP-77 cells

[0749] Table 13. Endocytic effects of anti-DLL3 antibodies on CHO-K1 / humanDLL3 and SHP-77 cells

[0750] Example 7 Construction of HEK293 recombinant cells overexpressing the DLL3 extracellular domain and its cell surface subdomains

[0751] Based on the sequence of the full-length human DLL3 gene (Uniprot: Q9NYJ7), the coding sequences of the corresponding extracellular domains and their subdomains were cloned into the pcDNA3.1 vector, with a FLAG tag inserted between the signal peptide and the extracellular domain. Recombinant plasmids overexpressing the extracellular domains and subdomains all contained an N-terminal mouse IgG Vk leader sequence followed by a FLAG tag and the corresponding human DLL3 domain. To ensure cell surface localization of the human DLL3 domain, all recombinant plasmids were followed by a Ser / Gly linker, the transmembrane domain of human EpCAM (epithelial cell adhesion molecule) (P16422) (aa 266-288), and the intracellular domain of human EpCAM (P16422) (aa 289-314). FITC-labeled anti-FLAG antibody (clone M2, Sigma) was used to verify expression of the extracellular domains and their subdomains on the cell surface.

[0752] The extracellular domain of DLL3 consists of several distinct subdomains:

[0753] Human DLL3 signal peptide: aa 1-26

[0754] Human DLL3 N-terminal ECD region: aa 27-175

[0755] Human DLL3 DSL: aa 176-215

[0756] Human DLL3EGF 1: aa 216-249

[0757] Human DLL3EGF2: aa 274-310

[0758] Human DLL3EGF3: aa 312-351

[0759] Human DLL3EGF4: aa 353-389

[0760] Human DLL3EGF5: aa 391-427

[0761] Human DLL3EGF6: aa 429-465

[0762] Membrane-proximal peptide segment: aa 466-492

[0763] The sequences of various DLL3 subdomains overexpressed in HEK293 cells are shown in the table below.

[0764] Table 14. Sequences of various DLL3 subdomains overexpressed in HEK293 cells

[0765] Example 8 Identification of the Binding Epitope of Anti-DLL3 Antibodies to DLL3 Protein

[0766] Detailed information about recombinant plasmids overexpressing the DLL3 subdomain is shown in Table 14 and Figure 11A. These plasmids were transiently transfected into HEK239T cells. Cells were harvested 48 hours later, and the transfection efficiency of the recombinant cell lines was assessed using a FITC-labeled anti-FLAG antibody. Untransfected HEK93T cells served as a negative control for transfection efficiency. Primary antibody staining was performed using 1 mg / mL DLL3 antibody. hIgG1 was used as an isotype negative reference antibody. The binding of the anti-DLL3 antibody to the transiently transfected cells with the subdomain was measured by flow cytometry using a fluorescent secondary antibody (Alexa Fluor 647-labeled affinity-purified goat anti-human IgG, specifically reactive with Fcγ fragments, Jackson, #109-605-098, 1:1000 dilution).

[0767] Figure 11B shows that recombinant plasmids overexpressing DLL3 subdomains were transiently transfected into HEK293T cells. Figures 12A-12J show that DLL3 antibodies bind to different regions of the extracellular end of human DLL3, and the results are shown in Table 15.

[0768] Table 15. Binding epitopes of anti-DLL3 antibodies to DLL3 protein

[0769] Sequence Listing

[0770] PR012621 (anti-DLL3 rovalvituzumab hIgG1)

[0771] PR012622 (anti-DLL3 DLL3-4_CDDI hIgG1)

[0772] PR013234 (anti-DLL3 N1003P003A12 HCAb hIgG1(C220S))

[0773] PR013235 (anti-DLL3 N1003P003B12 HCAb hIgG1(C220S))

[0774] PR013236 (anti-DLL3 N1003P003D01 HCAb hIgG1(C220S))

[0775] PR013239 (anti-DLL3 N1003P005E04 HCAb hIgG1(C220S))

[0776] PR013240 (anti-DLL3 N1003P005G03 HCAb hIgG1(C220S))

[0777] PR013241 (anti-DLL3 N1003P007B07 HCAb hIgG1(C220S))

[0778] PR013242 (anti-DLL3 N1003P008A11 HCAb hIgG1(C220S))

[0779] PR013244 (anti-DLL3 N1003P008B09 HCAb hIgG1(C220S))

[0780] PR013246 (anti-DLL3 N1003P008H01 HCAb hIgG1(C220S))

[0781] PR013249 (anti-DLL3 N1003P013C05 HCAb hIgG1(C220S))

[0782] PR013250 (anti-DLL3 N1003P015H03 HCAb hIgG1(C220S))

[0783] PR013252 (anti-DLL3 N1003P017G01 HCAb hIgG1(C220S))

[0784] PR013253 (anti-DLL3 N1003P018C04 HCAb hIgG1(C220S))

[0785] PR013254 (anti-DLL3 N1003P018F06 HCAb hIgG1(C220S))

[0786] PR013256 (anti-DLL3 N1003P019B04 HCAb hIgG1(C220S))

[0787] PR013257 (anti-DLL3 N1003P020A07 HCAb hIgG1(C220S))

[0788] PR013258 (anti-DLL3 N1003P020C12 HCAb hIgG1(C220S))

[0789] PR013259 (anti-DLL3 N1003P022A02 HCAb hIgG1(C220S))

[0790] PR013260 (anti-DLL3 N1003P024E01 HCAb hIgG1(C220S))

[0791] PR013261 (anti-DLL3 N1003P024G01 HCAb hIgG1(C220S))

[0792] PR013262 (anti-DLL3 N1003P027E04 HCAb hIgG1(C220S))

[0793] PR013263 (anti-DLL3 N1003P028F03 HCAb hIgG1(C220S))

[0794] PR013264 (anti-DLL3 N1003P030F03 HCAb hIgG1(C220S))

[0795] PR013363 (anti-DLL3 N1003P041D05 HCAb hIgG1(C220S))

[0796] PR013364 (anti-DLL3 N1003P041G02 HCAb hIgG1(C220S))

[0797] PR013367 (anti-DLL3 N1003P044A10 HCAb hIgG1(C220S))

[0798] PR013368 (anti-DLL3 N1003P044G07 HCAb hIgG1(C220S))

[0799] PR013370 (anti-DLL3 N1003P046B02 HCAb hIgG1(C220S))

[0800] PR013371 (anti-DLL3 N1003P046E04 HCAb hIgG1(C220S))

[0801] PR013372 (anti-DLL3 N1003P046G02 HCAb hIgG1(C220S))

[0802] PR013376 (anti-DLL3 N1003P047C08 HCAb hIgG1(C220S))

[0803] PR013377 (anti-DLL3 N1003P047D04 HCAb hIgG1(C220S))

[0804] PR013378 (anti-DLL3 N1003P047D06 HCAb hIgG1(C220S))

[0805] PR013381 (anti-DLL3 N1003P048B11 HCAb hIgG1(C220S))

[0806] PR013382 (anti-DLL3 N1003P049A11 HCAb hIgG1(C220S))

[0807] PR013384 (anti-DLL3 N1003P051B09 HCAb hIgG1(C220S))

[0808] PR013387 (anti-DLL3 N1003P052G11 HCAb hIgG1(C220S))

[0809] PR013392 (anti-DLL3 N1003P054C06 HCAb hIgG1(C220S))

[0810] PR013393 (anti-DLL3 N1003P054C07 HCAb hIgG1(C220S))

[0811] PR013394 (anti-DLL3 N1003P054D06 HCAb hIgG1(C220S))

[0812] PR013397 (anti-DLL3 N1003P054E04 HCAb hIgG1(C220S))

[0813] PR013399 (anti-DLL3 N1003P054H03 HCAb hIgG1(C220S))

[0814] PR013402 (anti-DLL3 N1003P055D04 HCAb hIgG1(C220S))

[0815] PR013406 (anti-DLL3 N1003P058H08 HCAb hIgG1(C220S))

Claims

1. A DLL3 binding molecule comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and wherein HCDR1 comprises the sequence shown in SEQ ID NO: 18 or 32, or an amino acid sequence that differs from the sequence shown in SEQ ID NO: 18 or 32 by no more than 2 amino acids in addition, deletion or substitution; and / or HCDR2 comprises the sequence shown in SEQ ID NO: 69, 74, 79, 80, 87 or 90, or an amino acid sequence that differs from the sequence shown in SEQ ID NO: 69, 74, 79, 80, 87 or 90 by no more than 2 amino acid additions, deletions or substitutions; and / or The HCDR3 comprises the sequence of SEQ ID NO: 126, 127, 128, 129, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151 or 152, or an amino acid sequence that differs from the sequence of SEQ ID NO: 126, 127, 128, 129, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151 or 152 by amino acid addition, deletion or substitution of no more than 2 amino acids.

2. The DLL3 binding molecule of claim 1, wherein the DLL3 binding molecule is an antibody or antigen-binding fragment thereof directed against DLL3.

3. The DLL3 binding molecule of claim 1 or 2, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34; HCDR2 comprises the amino acid sequence of SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90; and / or HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 or 152.

4. The DLL3 binding molecule of any one of claims 1 to 3, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and wherein HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 60 and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 19, 60 and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 61 and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 20, 62 and 127, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 63 and 128, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 21, 64 and 129, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 22, 65 and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 23, 65 and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 66 and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 64 and 131, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 19, 68 and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 68 and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 20, 69 and 132, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 24, 66 and 126, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 70 and 133, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 70 and 134, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 71 and 134, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 72 and 135, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 18, 73 and 134, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 74 and 135, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 75 and 136, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 27, 76 and 137, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 77 and 138, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 78 and 139, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 79 and 140, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 80 and 141, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 26, 81 and 142, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 25, 82 and 143, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 28, 83 and 144, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 29, 84 and 143, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 85 and 145, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 30, 75 and 136, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 31, 85 and 146, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 32, 86 and 147, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 85 and 148, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 33, 87 and 149, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 88 and 150, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 18, 89 and 151, respectively; or HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 34, 90 and 152, respectively.

5. The DLL3 binding molecule of any one of claims 1 to 4, wherein The heavy chain variable region comprises 1) an amino acid sequence of SEQ ID NO: 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, or 217; 2) an amino acid sequence of SEQ ID NO: 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210 NO:174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 187, 188, 189, 190, 1 91, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 2 08, 209, 210, 211, 212, 213, 214, 215, 216, or 217; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 208, 209, 210, 211, 212, 213, 214, 215, 216, or 217; or 17, 180, 181, 182, 183, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216 or 217, wherein the amino acid sequence has one or more substitutions, additions and / or deletions compared to the amino acid sequence of The DLL3 binding molecule of any one of claims 1 to 5, which is a heavy chain antibody and / or a fully humanized antibody.

7. The DLL3 binding molecule of any one of claims 1 to 5, wherein the heavy chain variable region can be fused to another molecule; Preferably, the other molecule is the Fc domain of an immunoglobulin; Most preferably, the other molecule is the Fc domain of immunoglobulin G1 (IgG1).

8. The DLL3 binding molecule of any one of claims 1-7, comprising 1) an amino acid sequence of SEQ ID NO: 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, or 265; NO:222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 233, 234, 235, 236, 237, 238, 2 39, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 2 56, 257, 258, 259, 260, 261, 262, 263, 264 or 265 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity; or 3) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 56, 257, 258, 259, 260, 261, 262, 263, 264 or 265; , 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 330, 331, 332, 333, 334, 335 9. The DLL3 binding molecule of any one of claims 1 to 8, having at least one of the following characteristics: 1) Has affinity activity for DLL3 protein; 2) has affinity activity for DLL3-positive cells; 3) Ability to be internalized into DLL3-positive cells.

10. A multispecific antigen-binding construct comprising a first antigen-binding moiety that binds DLL3, wherein the first antigen-binding moiety comprises the DLL3-binding molecule of any one of claims 1 to 9; Preferably, the multispecific antigen-binding construct comprises a first antigen-binding moiety that binds DLL3 and a second antigen-binding moiety that binds a second antigen; Preferably, the second antigen binding moiety specifically binds to tumor antigens, immunomodulatory receptors and immune checkpoint molecules; Preferably, the second antigen binding moiety specifically binds an antigen on a lymphocyte.

11. A chimeric antigen receptor comprising the DLL3 binding molecule of any one of claims 1-9.

12. An immune effector cell expressing the chimeric antigen receptor of claim 11 on its surface; preferably, the immune effector cell comprises a cytotoxic T cell, a natural killer cell, a macrophage or a natural killer T cell.

13. An antibody conjugate comprising the DLL3 binding molecule of any one of claims 1 to 9 or the multispecific antigen-binding construct of claim 10 conjugated to at least one therapeutic agent.

14. A pharmaceutical composition comprising the DLL3 binding molecule of any one of claims 1 to 9, the multispecific antigen-binding construct of claim 10, the immune effector cell of claim 12 or the antibody conjugate of claim 13, and a pharmaceutically acceptable carrier.

15. Use of the DLL3 binding molecule of any one of claims 1 to 9, the multispecific antigen-binding construct of claim 10, the immune effector cell of claim 12, the antibody conjugate of claim 13, or the pharmaceutical composition of claim 14 in the preparation of a medicament for treating cancer; preferably, the cancer is a DLL3-positive tumor; more preferably, the cancer is selected from small cell lung cancer (SCLC); large cell neuroendocrine carcinoma (LCNEC); neuroendocrine tumors of various tissues including the kidney, genitourinary tract, gastrointestinal tract, thyroid, pancreas and lung; glioma or pseudoneuroendocrine tumor (pNET).

16. The use of claim 15, wherein the drug is used in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen targeting drugs.

17. A polynucleotide encoding the DLL3 binding molecule of any one of claims 1 to 9 or the multispecific antigen-binding construct of claim 10.

18. An expression vector comprising the polynucleotide of claim 17.

19. A host cell comprising the polynucleotide of claim 17 or the expression vector of claim 18.

20. A method of producing the DLL3 binding molecule of any one of claims 1 to 9 or the multispecific antigen-binding construct of claim 10, the method comprising: a) culturing the host cell of claim 19 under suitable conditions to express the DLL3 binding molecule of any one of claims 1 to 9 or the multispecific antigen-binding construct of claim 10; and b) isolating the DLL3 binding molecule or multispecific antigen-binding construct from the host cell or culture thereof.

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