DLL3-targeted chimeric antigen receptor and binder

Abstract

Provided herein are chimeric antigen receptors (CARs) comprising DLL3-binding agents and DLL3-binding molecules that specifically bind to DLL3, as well as immune cells, such as CAR-T cells, comprising these DLL3-specific CARs. Also provided are methods for producing and using DLL3-specific CARs, and immune cells comprising DLL3-specific CARs.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 812,585, filed on March 1, 2019, and U.S. Provisional Patent Application No. 62 / 969,976, filed on February 4, 2020, the entire contents of which are hereby incorporated by reference in their entirety.

[0002] The present disclosure relates to chimeric antigen receptors (CARs) comprising DLL3 binders and antigen - binding molecules that bind to DLL3, polynucleotides encoding the same, and methods of using the same to treat cancer in a patient.

[0003] Sequence Listing This application has been electronically filed via EFS - Web and includes a sequence listing that was electronically submitted in.txt format. The.txt file was created on February 4, 2020, and contains a sequence listing entitled "AT - 019_03US_SL" having a size of approximately 1,026,798 bytes. The sequence listing contained in this.txt file is part of this specification and is hereby incorporated by reference in its entirety.

Background Art

[0004] Small cell lung cancer (SCLC) is an aggressive form of lung cancer with a poor prognosis and limited treatment options. SCLC represents approximately 10 - 15% of all newly diagnosed lung cancers. The American Cancer Society estimates that approximately 234,000 new cases of lung cancer will be diagnosed in 2018. The estimated 5-year relative survival rates for SCLC are 31% (for stage I), 19% (for stage II), 8% (for stage III), and 2% (for stage IV). The survival rate for SCLC has remained low for decades, mainly because there are no new therapies to fight this form of lung cancer. Conventional therapeutic treatments for cancer include chemotherapy and radiation therapy. Patients typically respond well to current first-line therapies, which include etoposide and cisplatin, but always relapse soon as chemoresistant disease. The prognosis in the refractory situation of recurrence is very poor, with rapid disease progression and a short median survival of less than 6 months. Therefore, there remains a great need to develop more targeted and potent therapies for proliferative disorders.

[0005] Adoptive transfer of immune cells genetically modified to recognize malignant tumor-associated antigens has shown promise as a new approach for treating cancer (see, for example, Brenner et al., Current Opinion in Immunology, 22(2):251-257(2010), Rosenberg et al., Nature Reviews Cancer, 8(4):299-308(2008)). Immune cells can be genetically modified to express a chimeric antigen receptor (CAR), a fusion protein composed of a DLL3 antigen recognition moiety, and a T cell activation domain (see, for example, Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724(1993), and Sadelain et al., Curr. Opin. Immunol, 21(2):215-223(2009)). Immune cells containing a CAR, such as CAR-T cells (CAR-T), are engineered to confer antigen specificity to them while retaining or enhancing their ability to recognize and kill target cells.

[0006] DLL3 is a non-canonical Notch ligand that functions cell-autonomously to inhibit Notch signaling, thereby blocking cell-cell interactions and Notch internalization in target cells. Delta-like ligand 3 (DLL3) is an SCLC tumor marker and has been found to be associated with cancer stem cells. Other indications related to DLL3 include melanoma, low-grade glioma, glioblastoma, medullary thyroid cancer, carcinoid, disseminated neuroendocrine tumors in the pancreas, bladder, and prostate, testicular cancer, and lung adenocarcinoma with neuroendocrine features. There is a need for the treatment of cancer, particularly malignancies associated with abnormal expression of DLL3. Methods and compositions for addressing this need are provided herein.

Summary of the Invention

[0007] Chimeric antigen receptors (CARs) comprising a DLL3 antigen-binding domain that specifically binds to DLL3, and immune cells comprising these DLL3-specific CARs, such as CAR-T cells, are provided herein. Methods for making and using these DLL3-specific CARs, as well as immune cells comprising the DLL3-specific CARs, are also provided. The DLL-3-targeted CAR T cells described herein exhibit good transduction efficiency, in vitro phenotype, and potent in vitro and in vivo antitumor activity.

[0008] In one aspect, the present disclosure provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises (a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 10, 19, 28, 37, 46, 55, 64, 73, 82, 91, 100, 109, 118, 127, 136, 145, 154, 163, 172, 181, 190, 199, 208, 217, 226, 235, 244, 253, 262, 271, 280, 289, 298, 307, 316, 325, 334, 343, 352, 361, 370, 379, 388, 397, 406, 415, 424, 433, 442, 451, and 460; (b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 11, 20, 38, 47, 56, 65, 74, 83, 92, 101, 110, 119, 128, 137, 146, 155, 164, 173, 182, 191, 200, 209, 218, 227, 236, 245, 254, 263, 272, 281, 290, 299, 308, 317, 326, 335, 344, 353, 362, 371, 380, 389, 398, 407, 416, 425, 434, 443, 452, 461, and 695; (c) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75, 84, 93, 102, 111, 120, 129, 138, 147, 156, 165, 174, 183, 192, 201, 210, 219, 228, 237, 246, 255, 264, 273, 282, 291, 300, 309, 318, 327, 336, 345, 354, 363, 372, 381, 390, 399, 408, 417, 426, 435, 444, 453, and 462; (d) a variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 13, 22, 31, 40, 49, 58, 67, 85, 94, 103, 112, 121, 130, 139, 148, 157, 166, 175, 184, 193, 202, 211, 220, 229, 238, 247, 256, 265, 274, 283, 292, 301, 310, 319, 328, 337, 346, 355, 364, 373, 382, 391, 400, 409, 418, 427, 436, 445,A variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of 454, 463, and 696, (e) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 14, 23, 32, 41, 50, 59, 68, 77, 86, 95, 104, 113, 122, 131, 140, 149, 158, 167, 176, 185, 194, 203, 212, 221, 230, 239, 248, 257, 266, 275, 284, 293, 302, 311, 320, 329, 338, 347, 356, 365, 374, 383, 392, 401, 410, 419, 428, 437, 446, 455, and 464, and (f) at least one of a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 15, 24, 33, 42, 51, 60, 69, 78, 87, 96, 105, 114, 123, 132, 141, 150, 159, 168, 177, 186, 195, 204, 213, 222, 231, 240, 249, 258, 267, 276, 285, 294, 303, 312, 321, 330, 339, 348, 357, 366, 375, 384, 393, 402, 411, 420, 429, 438, 447, 456, and 465.

[0009] In another aspect, the present disclosure provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises (a) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 10, 19, 28, 37, 46, 55, 64, 73, 82, 91, 100, 109, 118, 127, 136, 145, 154, 163, 172, 181, 190, 199, 208, 217, 226, 235, 244, 253, 262, 271, 280, 289, 298, 307, 316, 325, 334, 343, 352, 361, 370, 379, 388, 397, 406, 415, 424, 433, 442, 451, and 460; (b) a variable heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 11, 20, 38, 47, 56, 65, 74, 83, 92, 101, 110, 119, 128, 137, 146, 155, 164, 173, 182, 191, 200, 209, 218, 227, 236, 245, 254, 263, 272, 281, 290, 299, 308, 317, 326, 335, 344, 353, 362, 371, 380, 389, 398, 407, 416, 425, 434, 443, 452, and 695461; and (c) a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75, 84, 93, 102, 111, 120, 129, 138, 147, 156, 165, 174, 183, 192, 201, 210, 219, 228, 237, 246, 255, 264, 273, 282, 291, 300, 309, 318, 327, 336, 345, 354, 363, 372, 381, 390, 399, 408, 417, 426, 435, 444, 453, and 462.

[0010] In one aspect, the present disclosure provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises (a) a variable light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 13, 22, 31, 40, 49, 58, 67, 85, 94, 103, 112, 121, 130, 139, 148, 157, 166, 175, 184, 193, 202, 211, 220, 229, 238, 247, 256, 265, 274, 283, 292, 301, 310, 319, 328, 337, 346, 355, 364, 373, 382, 391, 400, 409, 418, 427, 436, 445, 454, 463, and 696; (b) a variable light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 14, 23, 32, 41, 50, 59, 68, 77, 86, 95, 104, 113, 122, 131, 140, 149, 158, 167, 176, 185, 194, 203, 212, 221, 230, 239, 248, 257, 266, 275, 284, 293, 302, 311, 320, 329, 338, 347, 356, 365, 374, 383, 392, 401, 410, 419, 428, 437, 446, 455, and 464; and (c) a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 15, 24, 33, 42, 51, 60, 69, 78, 87, 96, 105, 114, 123, 132, 141, 150, 159, 168, 177, 186, 195, 204, 213, 222, 231, 240, 249, 258, 267, 276, 285, 294, 303, 312, 321, 330, 339, 348, 357, 366, 375, 384, 393, 402, 411, 420, 429, 438, 447, 456, and 465.

[0011] In another aspect, the present disclosure provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises (a) a variable heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 16, 25, 34, 43, 52, 61, 70, 79, 88, 97, 106, 115, 124, 133, 142, 151, 160, 169, 178, 187, 196, 205, 214, 223, 232, 241, 250, 259, 268, 277, 286, 295, 304, 313, 322, 331, 340, 349, 358, 367, 376, 385, 394, 403, 412, 421, 430, 439, 448, 457, 466, and (b) a variable light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 17, 26, 35, 44, 53, 62, 71, 80, 89, 98, 107, 116, 125, 134, 143, 152, 161, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, 287, 296, 305, 314, 323, 332, 341, 350, 359, 368, 377, 386, 395, 404, 413, 422, 431, 440, 449, 458, and 467, and the variable heavy chain and the variable light chain are linked by at least one linker.

[0012] In a further aspect, the present disclosure provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises (a) a variable heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 16, 25, 34, 43, 52, 61, 70, 79, 88, 97, 106, 115, 124, 133, 142, 151, 160, 169, 178, 187, 196, 205, 214, 223, 232, 241, 250, 259, 268, 277, 286, 295, 304, 313, 322, 331, 340, 349, 358, 367, 376, 385, 394, 403, 412, 421, 430, 439, 448, 457, and 466, and (b) a variable light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 17, 26, 35, 44, 53, 62, 71, 80, 89, 98, 107, 116, 125, 134, 143, 152, 161, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, 287, 296, 305, 314, 323, 332, 341, 350, 359, 368, 377, 386, 395, 404, 413, 422, 431, 440, 449, 458, and 467, and the variable heavy chain and the variable light chain are linked by at least one linker.

[0013] In one aspect, the present disclosure provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises a sequence selected from the group consisting of the scFvs presented in Table 1d.

[0014] In another aspect, the present disclosure provides a chimeric antigen receptor that specifically binds to DLL3, and the chimeric antigen receptor comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 482-533 and SEQ ID NOs: 632-683. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs: 482-533 and 632-683.

[0015] In some embodiments, the present disclosure provides a chimeric antigen receptor that specifically binds to DLL3, and the chimeric antigen receptor comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 482-533 and SEQ ID NOs: 632-683, with or without a signal sequence. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs: 482-533 and 632-683, with or without a signal sequence.

[0016] In some embodiments, the intracellular domain of the chimeric antigen receptor comprises at least one co-stimulatory domain.

[0017] In some embodiments, the co-stimulatory domain of the chimeric antigen receptor is a signaling domain that specifically binds to CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1(CD11a / CD18)), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or a ligand that specifically binds to any combination thereof, or a signaling region thereof.

[0018] In some embodiments, the co-stimulatory domain comprises the signaling region of CD28.

[0019] In some embodiments, the CD28 co-stimulatory domain comprises SEQ ID NO: 550.

[0020] In some embodiments, the co-stimulatory domain comprises the signaling region of 4-1BB / CD137.

[0021] In some embodiments, the 4-1BB / CD137 co-stimulatory domain comprises SEQ ID NO: 480.

[0022] In some embodiments, the intracellular domain comprises at least one activation domain.

[0023] In some embodiments, the activation domain comprises CD3.

[0024] In some embodiments, CD3 comprises CD3 zeta.

[0025] In some embodiments, CD3 zeta comprises SEQ ID NO: 481.

[0026] In some embodiments, the chimeric antigen receptor is encoded by any one of the polynucleotide sequences of SEQ ID NOs: 571-621 and 631.

[0027] In some embodiments, the chimeric antigen receptor further comprises a safety switch.

[0028] In some embodiments, the safety switch comprises a CD20 mimotope or a QBEND-10 epitope.

[0029] In some embodiments, the safety switch comprises one or more CD20 mimotopes or one or more QBEND-10 epitopes, or a combination thereof.

[0030] In some embodiments, the chimeric antigen receptor comprises one or more safety switches in the format of QR3, SR2, RSR, or R2S.

[0031] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 622 - 628, 474 - 476, 565, and 684 - 694.

[0032] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 622 - 628, 474 - 476, 565, and 684 - 694, with or without a signal sequence.

[0033] In some aspects, the disclosure provides an isolated polynucleotide encoding any one of the chimeric antigen receptors described herein.

[0034] In another aspect, the disclosure provides a vector comprising a polynucleotide encoding any one of the chimeric antigen receptors described herein.

[0035] In some embodiments, the vector is a retroviral vector, DNA vector, plasmid, RNA vector, adenoviral vector, adeno - associated vector, lentiviral vector, or any combination thereof.

[0036] In another aspect, the disclosure provides an engineered immune cell that expresses a chimeric antigen receptor described herein.

[0037] In some aspects, the disclosure provides an engineered immune cell that expresses a polynucleotide or vector encoding any one of the chimeric antigen receptors described herein.

[0038] In some embodiments, the engineered immune cells are T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells.

[0039] In some embodiments, the engineered immune cells are autologous T cells.

[0040] In some embodiments, the engineered immune cells are allogeneic T cells.

[0041] In some embodiments, the engineered immune cells are knockout TCRs (e.g., TCRα, TCRβ).

[0042] In one aspect, the present disclosure provides a pharmaceutical composition comprising engineered immune cells expressing a chimeric antigen receptor described herein.

[0043] In some aspects, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising engineered immune cells or engineered immune cells expressing a chimeric antigen receptor described herein.

[0044] In some embodiments, the disease or disorder is cancer.

[0045] In some embodiments, the disease or disorder is small cell lung cancer.

[0046] In some aspects, the present disclosure provides a product comprising a pharmaceutical composition comprising engineered immune cells or engineered immune cells expressing a chimeric antigen receptor described herein.

[0047] In some aspects, the present disclosure provides an anti-DLL3 binding agent disclosed herein.

[0048] In some embodiments, the anti-DLL3 binding agent is an antibody, an antibody conjugate, or an antigen-binding fragment thereof, optionally an F(ab’)2 fragment, a Fab’ fragment, a Fab fragment, an Fv fragment, a scFv fragment, a dsFv fragment, or a dAb fragment.

[0049] In some embodiments, the binding agent is a monoclonal antibody that includes an IgG constant region.

[0050] In some embodiments, the anti-DLL3 binding agent includes a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the VH sequences provided in Table 1b.

[0051] In some embodiments, the anti-DLL3 binding agent includes a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the VL sequences provided in Table 1c.

[0052] In some embodiments, the anti-DLL3 binding agent includes a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the scFv sequences presented in Table 1d.

[0053] In some embodiments, the anti-DLL3 binding agent is a fusion protein that includes an scFv fragment fused to an Fc constant region.

[0054] In some aspects, the disclosure provides a pharmaceutical composition that includes an anti-DLL3 binding agent disclosed herein and a pharmaceutically acceptable excipient.

[0055] In some aspects, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, including administering to the subject an anti-DLL3 binding agent, or a pharmaceutical composition including an anti-DLL3 binding agent, as disclosed herein.

[0056] In some embodiments, the disease or disorder is cancer.

[0057] In some embodiments, the disease or disorder is small cell lung cancer.

Brief Description of the Drawings

[0058]

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Mode for Carrying Out the Invention

[0059] DLL3-specific antibodies and chimeric antigen receptors (CARs) are provided herein. The DLL-3 specific CARs described herein include an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain includes a DLL3 antigen binding domain that specifically binds to DLL3, and a polynucleotide encoding these CARs. Immune cells comprising these DLL3 specific CARs, such as CAR-T cells, and pharmaceutical compositions comprising these immune cells are also provided. For example, methods of making and using these DLL3 specific CARs and immune cells comprising these DLL3 specific CARs for the treatment of cancer are also disclosed.

[0060] I. DLL-3 Binders The present disclosure provides DLL-3 binding agents (e.g., molecules comprising a DLL3 antigen-binding domain, a DLL-3 antibody, or fragments thereof) that specifically bind to DLL-3. As used herein, the term "antibody" refers to a polypeptide comprising canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen (e.g., DLL-3). As is known in the art, intact antibodies produced in nature are generally about 150 kD tetramers composed of two identical heavy chain polypeptides (each about 50 kD) and two identical light chain polypeptides (each about 25 kD) that associate with one another in what is generally referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains (each about 110 amino acids in length) - an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CHI, CH2, and a carboxy-terminal CH3 (located at the base of the stem of the Y). A short region, known as the "switch," connects the heavy chain variable and constant regions. The "hinge" connects the CH2 and CH3 domains to the remainder of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is composed of two domains, an amino-terminal variable (VL) domain and a subsequent carboxy-terminal constant (CL) domain, separated from one another by another "switch." Those skilled in the art are familiar with antibody structure and sequence elements, recognize the "variable" and "constant" regions in the provided sequences, and understand that there can be some flexibility in the definition of the "boundaries" between such domains, and that different presentations of the same antibody chain sequence can indicate such boundaries at positions shifted by one or several residues relative to different presentations of the same antibody chain sequence.

[0061] The assignment of amino acids to each of the framework, CDR, and variable domains typically follows the Kabat numbering (see, e.g., Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda Md. 1991), Chothia numbering (see, e.g., Chothia & Lesk, (1987), J Mol Biol 196:901-917, Al-Lazikani et al., (1997) J Mol Biol 273:927-948, Chothia et al., (1992) J Mol Biol 227:799-817, Tramontano et al., (1990) J Mol Biol 215(1):175-82, and U.S. Patent No. 7,709,226), contact numbering, or the numbering scheme of the AbM scheme (Antibody Modeling program, Oxford Molecular).

[0062] Thus, in some embodiments, the CDRs of the DLL3 binders presented herein are numbered according to the Kabat numbering scheme. In other embodiments, the CDRs of the DLL3 binders presented herein are numbered according to the Chothia numbering scheme. In other embodiments, the CDRs of the DLL3 binders presented herein are numbered according to the contact numbering scheme. In other embodiments, the CDRs of the DLL3 binders presented herein are numbered according to the AbM numbering scheme.

[0063] An intact antibody tetramer is composed of two heavy-chain-light-chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond, and two other disulfide bonds connect the heavy-chain hinge regions to each other, such that the dimers are connected to each other and a tetramer is formed. Naturally produced antibodies are also typically glycosylated, typically on the CH2 domain. Each domain in a native antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., three-, four-, or five-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity-determining regions" (CDR1, CDR2, and CDR3), and four relatively invariant "framework" regions (FR1, FR2, FR3, and FR4). When a native antibody folds, the FR regions form beta sheets that provide the structural framework of the domain, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space such that a single hypervariable antigen-binding site is created at the tip of the Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also binds to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity of the Fc region for an Fc receptor and / or other binding attributes can be modulated by glycosylation or other modifications. In some embodiments, the antibodies produced and / or utilized in accordance with the present invention include a glycosylated Fc domain that has such modified or engineered glycosylation of the Fc domain.

[0064] For the purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that contains a sufficient immunoglobulin domain sequence as found in natural antibodies can be referred to and / or used as an "antibody" regardless of whether such polypeptide is produced naturally (e.g., produced by an organism that reacts to an antigen) or by recombinant manipulation, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal and in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has a constant region sequence characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art.

[0065] Furthermore, as used herein, the term "antibody" refers, in suitable embodiments (unless otherwise stated or apparent from the context), to either a construct or format known or developed in the art for exploiting the structural and functional features of an antibody in alternative presentations. For example, in some embodiments, the antibodies utilized in accordance with the present invention are intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®), small modular immunopharmaceuticals ("SMIP®); single-chain or tandem diabodies (e.g., TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®; Centyrins®; and KALBITOR®, but are not limited to formats selected from these. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it would have if naturally produced. In some embodiments, the antibody may contain covalent modifications (e.g., glycan attachment, payload (e.g., detectable moiety, therapeutic moiety, catalytic moiety, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0066] Antibodies include antibody fragments. Antibodies are polyclonal monoclonal, chimeric dAb (domain antibody), single-chain, F ab, F a , F (ab)2 fragments, scFv, and F ab also includes, but is not limited to, expression libraries. The antibody can be a whole antibody, or an immunoglobulin, or an antibody fragment.

[0067] As detailed above, a whole antibody consists of two pairs of a "light chain" (LC) and a "heavy chain" (HC) (such a light chain (LC) / heavy chain pair is abbreviated as LC / HC herein). The light chain and heavy chain of such an antibody are polypeptides consisting of several domains. In a whole antibody, each heavy chain contains a heavy chain variable region (abbreviated as HCVR or VH herein) and a heavy chain constant region. The heavy chain constant region includes heavy chain constant domains CHI, CH2, and CH3 (antibody classes IgA, IgD, and IgG), and optionally heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain contains a light chain variable domain VL and a light chain constant domain CL. The variable domains VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Janeway, C.A., Jr, et al, (2001) Immunobiology., 5th ed., Garland Publishing, and Woof, J., Burton, D., Nat Rev Immunol 4 (2004) 89 - 99). The two pairs of heavy and light chains (HC / LC) can specifically bind to the same antigen. Thus, the whole antibody is a bivalent monospecific antibody. Such "antibodies" include, for example, mouse antibodies, human antibodies, chimeric antibodies, humanized antibodies, and genetically engineered antibodies (variant or mutant antibodies) as long as their characteristic properties are retained. In some embodiments, the antibody or binder is a humanized antibody, particularly a recombinant human or humanized antibody.

[0068] In some embodiments, the antibody or binder can be "symmetric." "Symmetric" means that the antibody or binder has the same type of Fv region (e.g., the antibody has two Fab regions). In some embodiments, the antibody or binder can be "asymmetric." "Asymmetric" means that the antibody or binder has at least two different types of Fv regions (e.g., the antibody has a Fab and a scFv region, a Fab and a scFv2 region, or a Fab-VHH region). Various asymmetric antibody or binder structures are known in the art (Brinkman and Kontermann et al. 2017 Mabs (9)(2):182-212).

[0069] As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, the antibody agent may contain one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody agent may contain one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art for exploiting the structural and functional characteristics of antibodies in alternative presentations. For example, antibody agents utilized in accordance with the present invention include intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®), small modular immunopharmaceuticals ("SMIPs(™)); single-chain or tandem diabodies (e.g., TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®; Centyrins®; and KALBITOR®; but are not limited to formats selected from these.

[0070] In some embodiments, the antibody may lack covalent modifications (e.g., attachment of glycans) that it would have if produced naturally. In some embodiments, the antibody may contain covalent modifications (e.g., attachment of glycans, payloads [e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.], or other pendant groups [e.g., polyethylene glycol, etc.]). In many embodiments, the antibody agent is a polypeptide whose amino acid sequence comprises or contains one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs). In some embodiments, the antibody agent is a polypeptide whose amino acid sequence comprises or contains at least one CDR (e.g., at least one heavy-chain CDR and / or at least one light-chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, the antibody agent is a polypeptide whose amino acid sequence comprises or contains a structural element recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or mostly homologous to an immunoglobulin binding domain.

[0071] The encoded antibody or antigen-binding molecule of the invention can be single-stranded or double-stranded. In some embodiments, the antibody or antigen-binding molecule is single-stranded. In certain embodiments, the antigen-binding molecule is selected from the group consisting of scFv, Fab, Fab’, Fv, F(ab’)2, dAb, and any combination thereof.

[0072] In some embodiments, the anti-DLL-3 antibody agent is isolated. In some embodiments, the antibody agent can be purified to a purity of greater than 95% or 99% as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) (see, e.g., Flatman et al., J. Chromatogr., B 848:79-87 (2007)). In some aspects, the present disclosure provides a composition comprising a DLL-3 binder (e.g., a DLL3-specific antibody) and a pharmaceutically acceptable carrier.

[0073] In some embodiments, the anti-DLL-3 antibody agent comprises an Fc. The Fc domain can interact with cell surface receptors, which can enable the antibody to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody, and the IgM and IgE Fc regions contain three heavy chain constant domains (C H domains 2-4) in each polypeptide chain. The Fc region of IgG can carry a highly conserved N-glycosylation site (N297). Glycosylation of the Fc fragment can be essential for Fc receptor-mediated activities. The N-glycan attached to this site can mainly be a complex type of core-fucosylated biantennary structure.

[0074] While the constant regions of the light and heavy chains may not be directly involved in the binding of the antibody to the antigen, the constant regions can affect the orientation of the variable regions. The constant regions can also exhibit various effector functions, such as involvement in antibody-dependent complement-mediated lysis or antibody-dependent cell cytotoxicity through interaction with effector molecules and cells.

[0075] The disclosed anti-DLL-3 antibody agent can be an antibody of any isotype, including isotype IgA, isotype IgD, isotype IgE, isotype IgG, or isotype IgM. In some embodiments, the anti-DLL-3 antibody contains an IgG1, IgG2, IgG3, or IgG4 constant domain.

[0076] DLL3 binders (e.g., antibodies) that can bind to various regions or domains of the DLL3 target are provided herein. The epitope can be, for example, adjacent amino acids of the DLL3 target (linear or contiguous epitope) or can be formed together from two or more non-adjacent regions of the DLL3 target (conformation, non-linear, discontinuous, or non-adjacent epitope). The epitope to which the DLL3 antigen-binding domain binds can be determined by various assays, such as NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange combined with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, flow cytometry, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping).

[0077] Representative DLL3 regions or domains are shown in Figure 2. The exemplary DLL3 antibodies described herein bind to the DLL3 domains provided in Table 1a. [Table 1]

[0078] In some embodiments, the DLL3 binder comprises a variable heavy chain (VH), and the amino acid sequence of the VH is selected from the VH sequences presented in Table 1b. In some embodiments, the anti-DLL-3 binder comprises an immunoglobulin heavy chain having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences presented in Table 1b. [Table 2-1]

Table 2-2

Table 2-3

[0079] In some embodiments, the DLL3 binder comprises a variable light chain (VL), and the amino acid sequence of the VL is selected from the VL sequences presented in Table 1c. In some embodiments, the anti-DLL-3 binder comprises an immunoglobulin light chain having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence presented in Table 1c.

Table 3-1

Table 3-2

Table 3-3

[0080] Provided herein are DLL3 binders (e.g., antibodies) wherein the DLL3 antigen-binding domain comprises a variable heavy chain (VH) and a variable light chain, the amino acid sequence of the VH is selected from the VH sequences presented in Table 1b, and the amino acid sequence of the VL is selected from the VL sequences presented in Table 1c.

[0081] In some embodiments, the DLL-3 binder comprises heavy chain CDR1, CDR2, and CDR3. In some embodiments, the heavy chain CDR1, CDR2, and CDR3 sequences are selected from the heavy chain CDRs presented in Table 1e.

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

[0082] In some embodiments, the DLL-3 binder comprises light chain CDR1, CDR2, and CDR3. In some embodiments, the light chain CDR1, CDR2, and CDR3 sequences are selected from the light chain CDRs presented in Table 1f.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

[0083] The present disclosure encompasses modifications to DLL3 antibody agents that include the sequences shown in Tables 1b - 1e, and functionally equivalent DLL3 antibody agents having modifications that do not significantly affect their properties, as well as variants having enhanced or decreased activity and / or affinity. For example, the amino acid sequence can be mutated to obtain a DLL3 antigen - binding agent having a desired binding affinity for DLL3. Modification of polypeptides is routine in the art and need not be described in detail herein. Examples of modified polypeptides include those having conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly and detrimentally change the functional activity, or that mature (enhance) the affinity of the polypeptide for its ligand, or polypeptides involving the use of chemical analogs.

[0084] Amino acid sequence insertions include amino - terminal and / or carboxyl - terminal fusions ranging from a single residue to polypeptides containing 100 or more residues, as well as in - sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N - terminal methionyl residue or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include fusions of an enzyme or polypeptide that increases the half - life of the antibody in blood circulation to the N or C terminus of the antibody.

[0085] Substitution variants have at least one amino acid residue removed from the antigen - binding domain and a different residue inserted in its place. In some embodiments, the sites of interest for substitution mutagenesis include the hypervariable regions / CDRs, although FR modifications are also contemplated. Conservative substitutions are shown in Table 2 under the heading "Conservative Substitutions". If such substitutions result in a change in biological activity, more substantial changes, referred to as "Exemplary Substitutions" in Table 2 or further described below in reference to amino acid classes, can be introduced and the products screened.

Table 6

[0086] i. Antibody fragments In one aspect, the anti-DLL-3 antibody agent according to any of the above embodiments can be an antibody fragment. An antibody fragment includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, diabody, linear antibody, multispecificity formed from antibody fragment antibodies and scFv fragments, and other fragments described below. In some embodiments, the antibody is a full-length antibody, for example, an intact IgG1 antibody, or another antibody class or isotype as described herein. (See, for example, Hudson et al., Nat. Med., 9:129-134 (2003), Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, pp. 269-315 (1994), Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), WO93 / 01161, and U.S. Patent Nos. 5,571,894, 5,869,046, 6,248,516, and 5,587,458). A full-length antibody, intact antibody, or whole antibody is an antibody having a structure substantially similar to the native antibody structure or having a heavy chain containing an Fc region as defined herein. Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic digestion of an intact antibody and production by recombinant host cells (e.g., E. coli or phage) as known in the art.

[0087] The Fv antibody fragment contains the complete antigen recognition and antigen binding site. This fragment can contain a dimer of one heavy chain and one light chain variable region domain in a tight non-covalent association. From the folding of these two domains, six hypervariable loops (three loops each from the H chain and the L chain) are generated that contribute to the amino acid residues of antigen binding and give the antibody antigen binding specificity. However, even a single variable region (or half of the Fv containing only the three CDRs specific for the antigen), although having a lower affinity than the entire binding site, has the ability to recognize and bind the antigen.

[0088] A diabody is a small antibody fragment prepared by constructing an sFv fragment with a short linker (e.g., about 5 - 10 residues) between the V H domain and the V L domain, such that intermolecular pairing rather than intramolecular pairing of the V domains is achieved, resulting in a bivalent fragment. A bispecific diabody is a heterodimer of two crossover sFv fragments, where the V H domains and the V L domains are present on different polypeptide chains (see, for example, EP404,097, WO93 / 11161, and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444 - 6448 (1993)).

[0089] Domain antibodies (dAbs), which can be produced in a fully human form, are the smallest known antigen-binding fragments of antibodies and are in the range of about 11 kDa to about 15 kDa. DAbs consist of the robust variable regions of the immunoglobulin heavy and light chains (V H and V L) They are highly expressed in microbial cell cultures and exhibit favorable biophysical properties, including but not limited to solubility and temperature stability, and are well - suited for selection by in vitro selection systems such as phage display and affinity maturation. dAbs are biologically active as monomers and, due to their small size and inherent stability, can be formatted into larger molecules to create agents with extended serum half - lives or other pharmacological activities. (See, e.g., W09425591 and US2003 / 0130496).

[0090] Fvs and scFvs are species that have intact binding sites without constant regions. Thus, they may be suitable for reduced non - specific binding during in vivo use. Single - chain Fv (sFv or scFv) is an antibody fragment containing V H and V L antibody domains connected by a single polypeptide chain. The sFv polypeptide can further contain a polypeptide linker between the V H domain and the V L domain that allows the sFv to form the desired structure for antigen binding (see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer - Verlag, New York, pp. 269 - 315 (1994), Borrebaeck 1995, see below). The scFv fusion protein can be constructed to result in the fusion of an effector protein at either the amino or carboxy terminus of the sFv. Antibody fragments can also be "linear antibodies" (see, e.g., U.S. Patent No. 5,641,870). Such linear antibody fragments can be monospecific or bispecific. An exemplary DLL3 - specific scFv is provided in Table 1d.

Table 7 - 1

Table 7 - 2

Table 7-3

Table 7-4

Table 7-5

[0091] In some embodiments, the DLL3 antigen-binding domain comprises a single-chain variable fragment (scFv) comprising the variable light (VL) and variable heavy (VH) domains of a DLL3-specific monoclonal antibody linked by a flexible linker. The single-chain variable region fragment can be made by linking the light and / or heavy chain variable regions by using a linking peptide. An example of a linking peptide is a GS linker having the amino acid sequence (GGGGS) x where x is 1, 2, 3, 4, or 5 (SEQ ID NO: 470). In some embodiments, x is any integer from 6 to 15 or less than about 20. In some embodiments, the linker is (GGGGS)4 (SEQ ID NO: 478). Generally, the linker can be a short, flexible polypeptide and, in some embodiments, is composed of about 20 or fewer amino acid residues. Next, the linker can be modified for additional functionality such as attachment of a drug or attachment to a solid support. The single-chain variant can be produced either recombinantly or synthetically. For synthetic production of the scFv, an automated synthesizer can be used. For recombinant production of the scFv, a suitable plasmid containing the polynucleotide encoding the scFv can be introduced into a suitable host cell, either a eukaryote such as yeast, plant, insect, or mammalian cells, or a prokaryote such as E. coli. The polynucleotide encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0092] In exemplary embodiments, provided herein is a DLL3 antigen-binding domain comprising a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequences shown in Table 1b, and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequences shown in Table 1c. In some embodiments, VH and VL are linked together by a linker. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 478). In some embodiments, the linker can be encoded by a DNA sequence comprising GGCGGTGGAGGCTCCGGAGGGGGGGGCTCTGGCGGAGGGGGCTCC (SEQ ID NO: 564). In some embodiments, the linker can be encoded by a DNA sequence comprising ggcggcggcggctctggaggaggaggcagcggcggaggaggctccggaggcggcggctct (SEQ ID NO: 630). In some embodiments, the linker comprises the amino acid sequence GGGGGSGGGGSGGGGS (SEQ ID NO: 534). In some embodiments, the linker is a scFv Whitlow linker that can comprise the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 535). The scFv Whitlow linker can be encoded by a DNA sequence comprising GGGTCTACATCCGGCTCCGGGAAGCCCGGAAGTGGCGAAGGTAGTACAAAGGGG (SEQ ID NO: 566). In some embodiments, the VH and VL sequences of the disclosed scFv can be oriented with the VH sequence located at the N-terminus of the ScFv, followed by the linker, and then the VL sequence, and in other embodiments, the scFv can be oriented with the VL sequence at the N-terminus, followed by the linker, and then the VH sequence.

[0093] ii. Chimeric and Humanized Antibodies In some embodiments, the anti-DLL-3 antibody agent is a monoclonal antibody that comprises, or that includes, a chimeric antibody, a humanized antibody, or a human antibody.

[0094] In some embodiments, the anti-DLL-3 antibody agents provided herein can be chimeric antibodies (see, e.g., U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). A chimeric antibody can be an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In one example, a chimeric antibody can include a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody can be a “class switch” antibody in which the class or subclass has been changed from that of the parental antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0095] In some embodiments, the chimeric antibody can be a humanized antibody (see, for example, Almagro and Fransson, Front. Biosci., 13:1619-1633 (2008), Riechmann et al., Nature, 332:323-329 (1988), Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005), Padlan, Mol. Immunol, 28:489-498 (1991), Dall’Acqua et al., Methods, 36:43-60 (2005), Osbourn et al., Methods, 36:61-68 (2005), and Klimka et al., Br. J. Cancer, 83:252-260 (2000)). A humanized antibody is a chimeric antibody that contains amino acid residues from non-human variable regions and amino acid residues from human FRs. In certain embodiments, the humanized antibody comprises substantially all of at least one, typically two variable domains, with all or substantially all of the hypervariable regions (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. The humanized antibody can optionally comprise at least a portion of the antibody constant region derived from a human antibody.

[0096] Non-human antibodies can be humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. A humanized antibody can include one or more variable domains that include one or more CDRs, or portions thereof, derived from a non-human antibody. A humanized antibody can include one or more variable domains that include one or more FRs, or portions thereof, derived from a human antibody sequence. A humanized antibody can optionally include at least a portion of a human constant region. In some embodiments, one or more FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve the specificity or affinity of the antibody.

[0097] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method, framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions, human mature (somatic mutated) framework regions or human germline framework regions, and framework regions derived from screening FR libraries (see, e.g., Sims et al., J. Immunol, 151:2296 (1993), Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992), Presta et al., J. Immunol, 151:2623 (1993), Baca et al., J. Biol. Chem., 272:10678-10684 (1997), and Rosok et al., J. Biol. Chem., 271:22611-22618 (1996)).

[0098] iii. Human antibodies In some embodiments, the anti-DLL-3 antibody agents provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art (see, e.g., van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5:368-74 (2001), and Lonberg, Curr. Opin. Immunol, 20:450-459 (2008)). A human antibody can be one produced by a human or human cell, or one having an amino acid sequence corresponding to the amino acid sequence of an antibody derived from a non-human source that utilizes a human antibody repertoire or a sequence encoding other human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be prepared by administering an immunogen (e.g., DLL-3 protein) to a transgenic animal modified to produce intact antibodies having human variable regions in response to an antigen challenge (see, e.g., Lonberg, Nat. Biotech., 23:1117-1125 (2005), U.S. Patent Nos. 6,075,181, 6,150,584, 5,770,429, and 7,041,870, and U.S. Patent Application Publication No. US2007 / 0061900). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0099] Human antibodies can also be made by hybridoma-based methods. For example, human antibodies can be produced from human myeloma and mouse-human heteromyeloma cell lines using human B cell hybridoma technology and other methods (see, e.g., Kozbor, J. Immunol, 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (1987), Boerner et al., J. Immunol, 147:86 (1991), Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), U.S. Patent No. 7,189,826, Ni, Xiandai Mianyixue, 26(4):265-268 (2006), Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005), and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005)). Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant regions.

[0100] Modification of the oligosaccharides in an antibody can be performed, for example, to generate antibody variants with certain improved properties. For example, antibody glycosylation variants can have improved CDC function. In some embodiments, the present disclosure contemplates antibody variants that have some, but not all, effector functions and are desirable candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC activity.

[0101] iv. Antibody derivatives In some embodiments, the antibody agents provided herein can be further modified to contain additional non - proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of the antibody can include, but are not limited to, water - soluble polymers. Non - limiting examples of water - soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly - 1,3 - dioxolane, poly - 1,3,6 - trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n - vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, oxidized polypropylene / oxidized ethylene copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof, but are not limited thereto. Polyethylene glycol propionaldehyde may have advantages in manufacture due to its stability in water.

[0102] The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they can be the same or different molecules.

[0103] In some embodiments, conjugates of antibodies and non - proteinaceous moieties that can be selectively heated by exposure to radiation are provided. In some embodiments, the non - proteinaceous moiety can be a carbon nanotube (see, e.g., Kam et al., Proc. Natl. Acad. Sci. USA, 102:11600 - 11605 (2005)). The radiation can be of any wavelength and can include wavelengths that do not harm normal cells but heat the non - proteinaceous moiety to a temperature at which proximal cells of the antibody - non - proteinaceous moiety conjugate are killed, but are not limited thereto.

[0104] A DLL3 binder (e.g., a molecule comprising an antigen-binding domain) is said to "specifically bind" to its target antigen (e.g., human, cynomolgus monkey, or mouse DLL3) when the dissociation constant (Kd) is about 1 nM. The antigen-binding domain "binds with high affinity" when the Kd is 1 - 5 nM and "binds with very high affinity" when the Kd is 0.1 - 0.5 nM, and specifically binds to the antigen. In one embodiment, the antigen-binding domain has a Kd of about 1 nM. In one embodiment, the off-rate is <1×10 -5 . In other embodiments, the antigen-binding domain binds to human DLL3 with a Kd of about 1×10 -7 M - 1×10 -12 M, and in yet another embodiment, the antigen-binding domain will bind with a Kd of about 1x10 -5 M - 1x10 -12 M.

[0105] As provided herein, the antigen-binding domains of the disclosure specifically bind to mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3, or mouse DLL3). In certain embodiments, the DLL3 antigen-binding domains of the disclosure bind to mammalian DLL3 with a Kd of less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, or less than 1×10 -9 M. In one particular embodiment, the DLL3 antigen-binding domain binds to mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3, or mouse DLL3) with a Kd of less than 1×10 -7 M. In another embodiment, the DLL3 antigen-binding domain binds to mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3, or mouse DLL3) with a Kd of less than 1×10 -8 M. In some embodiments, the DLL3 antigen-binding domain binds to mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3) with a Kd of about 1×10 -7 M, about 2×10 -7 M, about 3×10 -7 M, about 4×10 -7 M, about 5×10 -7M, approximately 6×10 -7 M, approximately 7×10 -7 M, approximately 8×10 -7 M, approximately 9×10 -7 M, approximately 1x10 -8 M, approximately 2×10 -8 M, approximately 3×10 -8 M, approximately 4×10 -8 M, approximately 5×10 -8 M, approximately 6×10 -8 M, approximately 7×10 -8 M, approximately 8×10 -8 M, approximately 9×10 -8 M, approximately 1×10 -9 M, approximately 2×10 -9 M, approximately 3×10 -9 M, approximately 4×10 -9 M, approximately 5×10 -9 M, approximately 6×10 -9 M, approximately 7×10 -9 M, approximately 8×10 -9 M, approximately 9×10 -9 M, approximately 1×10 -10 M, or approximately 5×10 -10 M binds with a Kd. In certain embodiments, the Kd is calculated as the quotient of K off / K on and K on and K off are determined using a monovalent antibody, such as a Fab fragment, measured, for example, by BIAcore® surface plasmon resonance technology. In other embodiments, the Kd is calculated as the quotient of K off / K on and K on and K off are determined using a divalent antibody, such as a Fab fragment, measured, for example, by BIAcore® surface plasmon resonance technology.

[0106] In some embodiments, the DLL3 antigen-binding domain binds to mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3, or mouse DLL3) at less than 1×10 -4 M -1 s- 1 less than 2×10 -4 M -1 s- 1 less than 3×10-4 M -1 s- 1 less than 4×10 -4 M -1 s- 1 less than 5×10 -4 M -1 s- 1 less than 7×10 -4 M -1 s- 1 less than 8×10 -4 M -1 s- 1 less than 9×10 -4 M -1 s- 1 less than 1×10 -5 M -1 s- 1 less than 2×10 -5 M -1 s- 1 less than 3×10 -5 M -1 s- 1 less than 4×10 -5 M -1 s- 1 less than 5×10 -5 M -1 s- 1 less than 6×10 -5 M -1 s- 1 less than 7×10 -5 M -1 s- 1 less than 8×10 -5 M -1 s- 1 less than 9×10 -5 M -1 s- 1 less than 1×10 -6 M -1 s- 1 less than 2×10 -6 M -1 s- 1 less than 3×10 -6 M -1 s- 1 less than 4×10 -6 M -1 s- 1 less than 5×10 -6 M -1 s- 1 less than 6×10 -6 M-1 s- 1 less than 7×10 -6 M -1 s- 1 less than 8×10 -6 M -1 s- 1 less than 9×10 -6 M -1 s- 1 less than, or 1×10 -7 M -1 s- 1 less than the association rate (k on ) binds. In certain embodiments, k on is determined using a monovalent antibody, such as a Fab fragment, as measured by, for example, BlAcore® surface plasmon resonance technology. In other embodiments, k on is determined using a bivalent antibody such as that measured by, for example, BlAcore® surface plasmon resonance technology.

[0107] In some embodiments, the DLL3 antigen-binding domain is to mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3, or mouse DLL3) at 1×10 -2 s -1 less than 2×10 -2 s -1 less than 3×10 -2 s -1 less than 4×10 -2 s -1 less than 5×10 -2 s -1 less than 6×10 -2 s -1 less than 7×10 -2 s -1 less than 8×10 -2 s -1 less than 9×10 -2 s -1 less than 1×10 -3 s -1 less than 2×10 -3 s -1 less than 3×10 -3 s -1 less than 4×10 -3 s -1 less than 5×10 -3 s-1 less than 6×10 -3 s -1 less than 7×10 -3 s -1 less than 8×10 -3 s -1 less than 9×10 -3 s -1 less than 1×10 -4 s -1 less than 2×10 -4 s -1 less than 3×10 -4 s -1 less than 4×10 -4 s -1 less than 5×10 -4 s -1 less than 6×10 -4 s -1 less than 7×10 -4 s -1 less than 8×10 -4 s -1 less than 9×10 -4 s -1 less than 1×10 -5 s -1 less than or 5×10 -4 s -1 less than dissociation rate (k off ). It binds in certain embodiments, k off is determined using a monovalent antibody such as a Fab fragment, as measured by, for example, BlAcore® surface plasmon resonance technology. In other embodiments, k off is determined using a bivalent antibody such as that measured by, for example, BlAcore® surface plasmon resonance technology.

[0108] II. Chimeric Antigen Receptor As used herein, a chimeric antigen receptor (CAR) is a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell). When bound to the target antigen, the CAR can activate immune cells to attack and destroy the cells bearing that antigen (e.g., cancer cells). The CAR can also incorporate a co-stimulatory domain or a signaling domain to enhance their potency. See Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626), Finney et al., Journal of Immunology, 1998, 161:2791-2797, Song et al., Blood 119:696-706 (2012), Kalos et al., Sci. Transl. Med. 3:95 (2011), Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016), U.S. Patent No. 7,741,465, and No. 6,319,494.

[0109] The chimeric antigen receptor described herein includes an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain includes a DLL3 antigen-binding domain that specifically binds to DLL3. In some embodiments, the DLL-3 specific CAR includes the following elements from 5' to 3': a signal sequence, a DLL3 antigen-binding domain (e.g., an anti-DLL3 scFv), a hinge and transmembrane region, and one or more contiguous signaling domains. In certain embodiments, the DLL-3 specific CAR includes the following elements from 5' to 3': a CD8α signal sequence, a DLL3 scFv comprising a DLL3 variable heavy chain and / or variable light chain described herein, a CD8α hinge and transmembrane region, a 41BB cytoplasmic signaling domain, and a CD3ζ cytoplasmic signaling domain. (Figure 4, Table 7).

[0110] In some embodiments, the DLL-3 specific CAR further includes a safety switch and / or a monoclonal antibody-specific epitope.

[0111] a. Antigen-binding domain As discussed above, the DLL3 CAR described herein includes an antigen-binding domain. As used herein, "antigen-binding domain" means any polypeptide that binds to a specific target antigen. For example, the specific target antigen can be the DLL3 (DLL-3) protein or a fragment thereof (alternatively referred to herein as "DLL3 antigen", "DLL3 target antigen", or "DLL3 target"). In some embodiments, the antigen-binding domain binds to the DLL3 antigen on tumor cells. In some embodiments, the antigen-binding domain binds to the DLL3 antigen on cells involved in proliferative diseases.

[0112] In some embodiments, the antigen-binding domain includes a variable heavy chain, a variable light chain, and / or one or more CDRs described herein. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) that includes light chain CDRs, CDR1, CDR2, and CDR3, as well as heavy chain CDRs, CDR1, CDR2, and CDR3.

[0113] In some embodiments, the DLL-3 specific CAR includes the VH shown in Table 1b. In some embodiments, the DLL-3 specific CAR includes the VL shown in Table 1c. In some embodiments, the DLL-3 specific CAR includes the heavy chain CDR1, CDR2, CDR3 shown in Table 1e. In some embodiments, the DLL-3 specific CAR includes the light chain CDR1, CDR2, CDR3 shown in Table 1f.

[0114] Variants of the antigen-binding domain (e.g., variants of CDR, VH, and / or VL) are also within the scope of the present disclosure. For example, each has at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or more than 99% identity with the amino acid sequence of the antigen-binding domain sequences described herein. Variable light chains and / or variable heavy chains. In some examples, such molecules include at least one heavy chain and one light chain, while in other examples, the variant form contains two variable light chains and two variable heavy chains (or subparts thereof). One of ordinary skill in the art would be able to determine suitable variants of the antigen-binding domains described herein using well-known techniques. In certain embodiments, one of ordinary skill in the art can identify suitable regions of a molecule that can be altered without disrupting activity by targeting regions that are not considered important for activity.

[0115] In certain embodiments, the polypeptide structure of the antigen-binding domain is antibody-based and includes, but is not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. In some embodiments, the antigen-binding domain comprises or consists of an avimer.

[0116] The DLL3 antigen-binding domain is said to be "selective" if it binds more closely to one target than to a second target.

[0117] In some embodiments, the DLL3 antigen-binding domain is a scFv. In some embodiments, the DLL3-specific CAR comprises the scFv provided in Table 1d.

[0118] In some embodiments, the DLL3-specific CAR comprises a leader or signal peptide, and in some embodiments, the leader peptide is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 477). In some embodiments, the leader peptide comprises the amino acid sequence of SEQ ID NO: 477. In some embodiments, the leader peptide is encoded by a nucleic acid sequence comprising ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCGCACGCCCG (SEQ ID NO: 555).

[0119] In other embodiments, the disclosure relates to an isolated polynucleotide encoding any one of the DLL3 antigen-binding domains described herein. In some embodiments, the disclosure relates to an isolated polynucleotide encoding the DLL3 CAR described in Table 10. Vectors comprising the polynucleotide, and methods of making the same, are also provided herein.

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

Table 8-19

Table 8-20

Table 8-21

Table 8-22

Table 8-23

Table 8-24

Table 8-25

Table 8-26

Table 8-27

[0120] b. Safety switch and monoclonal antibody specific epitope Safety switch It will be understood that by transducing a suicide gene into immune cells (containing one or more CARs), adverse events can be minimized. In some cases, it may be desirable to incorporate an inducible "on" or "promoter" switch into immune cells. Suitable techniques include the use of inducible caspase-9 (U.S. Patent Application No. 2011 / 0286980) or thymidine kinase before, after, or simultaneously with the cells being transduced with the CAR constructs of the present disclosure. Additional methods for introducing suicide genes and / or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.

[0121] According to the present disclosure, additional on-off or other types of control switch techniques can be incorporated herein. These techniques can employ the use of dimerization domains and any activator of such domain dimerization. These techniques include, for example, those described by Wu et al., Science 2014 350(6258) which utilize the FKBP / rapalog dimerization system in certain cells, the content of which is hereby incorporated by reference in its entirety. Additional dimerization techniques are described, for example, in Fegan et al. Chem. Rev. 2010, 110, 3315-3336 and U.S. Patent Nos. 5,830,462, 5,834,266, 5,869,337, and 6,165,787, the content of which are also hereby incorporated by reference in their entirety. Additional dimerization pairs can include cyclosporin-A / cyclophilin, receptors, estrogen / estrogen receptor (optionally using tamoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, vitamin D / vitamin D receptor. Further examples of dimerization techniques can be found, for example, in WO2014 / 127261, WO2015 / 090229, US2014 / 0286987, US2015 / 0266973, US2016 / 0046700, U.S. Patent No. 8,486,693, US2014 / 0171649, and US2012 / 0130076, the content of which are hereby further incorporated by reference in their entirety.

[0122] In some embodiments, the CAR immune cells (e.g., CAR-T cells) of the present disclosure include a polynucleotide encoding a suicide polypeptide, such as, for example, RQR8. See, for example, WO2013 / 153391A, which is hereby incorporated by reference in its entirety. In CAR immune cells (e.g., CAR-T cells) containing the polynucleotide, the suicide polypeptide is expressed on the surface of the CAR immune cells (e.g., CAR-T cells). In some embodiments, the suicide polypeptide has the sequence number 552: It contains the amino acid sequence shown in CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 552).

[0123] The suicide polypeptide may also contain a signal peptide at the amino terminus, for example, MGTSLLCWMALCLLGADHADA (SEQ ID NO: 553). In some embodiments, the suicide polypeptide contains the amino acid sequence shown in SEQ ID NO: 554, which is SEQ ID NO: 553: MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 554) including the signal sequence.

[0124] When the suicide polypeptide is expressed on the surface of CAR immune cells (CAR-T cells), the binding of rituximab to the R epitope of the polypeptide causes cell lysis. Two or more molecules of rituximab may bind to each polypeptide expressed on the cell surface. Each R epitope of the polypeptide may bind to a separate molecule of rituximab. The depletion of DLL3-specific CAR immune cells (e.g., CAR-T cells) can occur in vivo, for example, by administering rituximab to a patient. The decision to deplete the transplanted cells can result from undesirable effects detected in the patient due to the transplanted cells, such as when unacceptable levels of toxicity are detected.

[0125] In some embodiments, the suicide polypeptide is expressed on the surface of the cell. In some embodiments, the suicide polypeptide is included in the CAR construct. In some embodiments, the suicide polypeptide is not part of the DLL3 CAR construct.

[0126] In some embodiments, the extracellular domain of any one of the DLL3-specific CARs disclosed herein may include one or more epitopes that are specific for (i.e., specifically recognized by) a monoclonal antibody. These epitopes are also referred to herein as mAb-specific epitopes. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No. WO2016 / 120216, which is hereby incorporated by reference in its entirety. In these embodiments, the extracellular domain of the CAR includes an antigen-binding domain that specifically binds to DLL3 and one or more epitopes that bind to one or more monoclonal antibodies (mAbs). A CAR that includes an mAb-specific epitope can be single-chain or multi-chain.

[0127] The inclusion of an epitope specific for a monoclonal antibody in the extracellular domain of the CARs described herein enables the selection and depletion of engineered immune cells expressing the CAR. In some embodiments, this feature also promotes the recovery of endogenous DLL3-expressing cells depleted by the administration of engineered immune cells expressing the CAR. In some embodiments, enabling depletion provides a safety switch, for example, in the case of adverse effects upon administration to a subject.

[0128] Accordingly, in some embodiments, the disclosure relates to methods for selecting and / or depleting engineered immune cells endowed with a CAR that includes an mAb-specific epitope, as well as methods for promoting the recovery of endogenous DLL3-expressing cells.

[0129] Using several epitope-monoclonal antibody couples, CARs can be generated that target monoclonal antibody-specific epitopes, particularly those already approved for medical use, including, by way of non-limiting example, CD20 epitopes / Rituximab, etc.

[0130] The present disclosure also encompasses a method for selecting engineered immune cells endowed with a DLL3-specific CAR that expresses an mAb-specific epitope, and a therapeutic method in which the activation of these engineered immune cells endowed with these CARs is regulated by depleting the cells using an antibody that targets the extracellular ligand-binding domain of the CAR. Table 4 provides exemplary mimotope sequences that can be inserted into the extracellular domain of any one of the CARs of the present disclosure. [Table 9]

[0131] In some embodiments, the extracellular binding domain of the CAR has the following sequences: -V1-L1-V2-(L) x -epitope 1-(L) x - -V1-L1-V2-(L) x -epitope 1-(L) x -epitope 2-(L) x - -V1-L1-V2-(L) x -epitope 1-(L) x -epitope 2-(L) x -epitope 3-(L) x - -(L) x -epitope 1-(L) x -V1-L1-V2 -(L) x -epitope 1-(L) x -epitope 2-(L) x -V1-L1-V2 -epitope 1-(L) x -epitope 2-(L) x -epitope 3-(L)x -V1-L1-V2, -(L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x , -(L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x , -(L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x , -(L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2-(L) x -Epitope 3-(L) x , -(L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x , -V1-(L) x -Epitope 1-(L) x -V2, -V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x , -V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x , -V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x 、 -(L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2, or -(L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2-(L) x -Epitope 3-(L) x comprising -wherein -V1 is V L and V2 is V H or V1 is V H and V2 is V L and -L1 is a linker suitable for linking the V H chain to the V L chain, -Epitope 1, Epitope 2, Epitope 3, and Epitope 4 are mAb-specific epitopes, which are identical or

[0132] c. Hinge domain The extracellular domain of the CARs of the present disclosure may include a "hinge" domain (or hinge region). The term generally refers to any polypeptide that functions to link the transmembrane domain in a CAR to the extracellular antigen-binding domain in the CAR. In particular, the hinge domain can be used to provide more flexibility and accessibility to the extracellular antigen-binding domain.

[0133] The hinge domain may contain up to 300 amino acids, in some embodiments 10 to 100 amino acids, or in some embodiments 25 to 50 amino acids. The hinge domain may be derived in whole or in part from all or part of the extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (in particular, the hinge region of IgG, it will be understood that the hinge region may contain some or all of a member of the immunoglobulin family such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof), or all or part of the constant region of the antibody heavy chain, etc. Alternatively, the A domain may be a synthetic sequence corresponding to a naturally occurring A sequence or a completely synthetic A sequence. In some embodiments, the A domain is part of the human CD8α chain (e.g., NP_001139345.1). In another specific embodiment, the hinge and transmembrane domains include part of the human CD8α chain. In some embodiments, the hinge domain of the CAR described herein includes a sub-sequence of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα, in particular, the hinge region of any of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain includes the human CD8α hinge, the human IgG1 hinge, human IgG4, human PD-1, or the human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein includes a scFv, a human CD8α hinge and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. Table 5 provides exemplary amino acid sequences of hinges provided herein.

Table 10

[0134] In certain embodiments, the hinge region comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the extracellular domain amino acid sequence described in Table 5 herein.

[0135] d. Transmembrane domain The CARs of the present disclosure are designed with transmembrane domains fused to the extracellular domain of the CAR. Similarly, they can be fused to the intracellular domain of the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to minimize interaction with other members of the receptor complex and to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins. In some embodiments, short linkers can form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0136] Transmembrane domains suitable for the CARs disclosed herein have (a) the ability to be expressed on the surface of immune cells such as, for example, without limitation, T helper (T h ) cells, cytotoxic T (T c ) cells, T regulatory (T reg ) cells, or natural killer (NK) cells, and / or (b) the ability to interact with the extracellular antigen-binding domain and the intracellular signaling domain to induce a cellular response of the immune cell against the target cell.

[0137] The transmembrane domain can be derived from either a natural or synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0138] The transmembrane regions for specific uses in the present disclosure can be derived from (including or corresponding to) ligands that specifically bind to CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0139] As a non-limiting example, the transmembrane region can be derived from or be a part of a T cell receptor, such as the α, β, γ, or δ polypeptide that constitutes the CD3 complex, the IL-2 receptor p55 (chain), p75 (β chain) or γ chain, a subunit chain of an Fc receptor, particularly Fcγ receptor III or a CD protein. Alternatively, the transmembrane domain can be synthetic and can contain mainly hydrophobic residues such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8α chain (e.g., NP_001139345.1).

[0140] In some embodiments, the transmembrane domain in the CAR of the present disclosure is the CD8α transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD8α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 549). In some embodiments, the CD8α transmembrane domain comprises a nucleic acid sequence encoding the transmembrane amino acid sequence of SEQ ID NO: 549. In some embodiments, the hinge and transmembrane domain in the CAR of the present disclosure is a CD8α hinge and transmembrane domain comprising the amino acid sequence of SEQ ID NO: 479.

[0141] In some embodiments, the transmembrane domain in the CAR of the present disclosure is the CD28 transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the present disclosure is a CD28 transmembrane domain comprising the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 550). In some embodiments, the CD28 transmembrane domain comprises a nucleic acid sequence encoding the transmembrane amino acid sequence of SEQ ID NO: 550.

[0142] e. Intracellular domain The intracellular (cytoplasmic) domain of the CAR of the present disclosure can provide activation of at least one of the normal effector functions of immune cells comprising the CAR, such as signal 1 / activation and / or signal 2 / co-stimulation. For example, the effector function of a T cell can refer to cytolytic activity or helper activity, including the secretion of cytokines. In some embodiments, the activating intracellular signaling domain for use in a CAR can be, for example, without limitation, the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction upon ligation of the antigen receptor, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional ability.

[0143] Suitable (e.g., activating) intracellular domains include, but are not limited to, CD3 zeta, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, and signal transduction domains derived from (or corresponding to) ligands that specifically bind to them, or any combination thereof.

[0144] The intracellular domain of the CARs of the present disclosure can incorporate co-stimulatory signaling domains (also interchangeably referred to herein as co-stimulatory molecules) in addition to the activation domains described above to enhance their potency. The co-stimulatory domains can provide signals in addition to the primary signal provided by the activation molecules described herein.

[0145] Suitable costimulatory domains within the scope of the present disclosure can be derived from (or corresponding to), for example, CD28, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1(CD11a / CD18)), CD247, CD276(B7-H3), LIGHT (tumor necrosis factor superfamily member 14, TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNFR, integrin, signal transduction lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or fragments or combinations thereof.Additional costimulatory molecules, or fragments thereof, not listed above will be understood to be within the scope of the present disclosure.

[0146] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the 41BB / CD137 domain, either alone or in combination with any other desired intracellular domain useful in the context of the CARs of the present disclosure. The complete native amino acid sequence of 41BB / CD137 is set forth in NCBI reference sequence: NP_001552.2. The complete native 41BB / CD137 nucleic acid sequence is set forth in NCBI reference sequence: NM_001561.5.

[0147] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the CD28 domain, either alone or in combination with any other desired intracellular domain useful in the context of the CARs of the present disclosure. The complete native amino acid sequence of CD28 is set forth in NCBI reference sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is set forth in NCBI reference sequence: NM_006139.1.

[0148] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the CD3 zeta domain, either alone or in combination with any other desired intracellular domain useful in the context of the CARs of the present disclosure. In some embodiments, the intracellular signaling domain of the CAR can include a CD3ζ signaling domain having an amino acid sequence with at least about 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown by SEQ ID NO: 481 in Table 7. For example, the intracellular domain of the CAR can include a CD3 zeta chain moiety and a portion of a co-stimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CARs of the present disclosure can be linked to each other in a random or specific order. In some embodiments, the intracellular domain is designed to include the activation domain of CD3 zeta and the signaling domain of CD28. In some embodiments, the intracellular domain is designed to include the activation domain of CD3 zeta and the co-stimulatory / signaling domain of 4-1BB.

[0149] In some embodiments, 4-1BB (intracellular domain) has the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 480). In some embodiments, 4-1BB (intracellular domain) is encoded by the nucleic acid sequence: AAGCGCGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTATGAGGCCCGTACAGACAACACAGGAGGAAGATGGCTGTAGCTGCAGATTTCCCGAGGAGGAGGAAGGTGGGTGCGAGCTG (SEQ ID NO: 568).

[0150] In some embodiments, the intracellular domain in the CAR is designed to include a portion of CD28 and CD3 zeta, and the intracellular CD28 includes the nucleic acid sequence shown by SEQ ID NO: 567. AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC (SEQ ID NO: 567).

[0151] In some embodiments, the intracellular domain in the CAR is designed to include the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 551). The CD3 zeta amino acid sequence may include SEQ ID NO: 481 or 469, and the nucleic acid sequence may include SEQ ID NO: 569: AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCCACCTAGG (SEQ ID NO: 569).

[0152] In some embodiments, the intracellular signaling domain of the CARs of the present disclosure includes the domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain of the CARs of the present disclosure includes a portion of a costimulatory molecule selected from the group consisting of fragments of 4-1BB (GenBank: AAA53133) and CD28 (NP_006130.1). In some embodiments, the intracellular signaling domain of the CARs of the present disclosure includes an amino acid sequence having at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 480 and SEQ ID NO: 551. In some embodiments, the intracellular signaling domain of the CARs of the present disclosure includes an amino acid sequence having at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 480, and / or at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 551.

[0153] In an exemplary embodiment, the CARs of the present disclosure include, from the N-terminus to the C-terminus, a (cleavable) CD8α signal sequence, a DLL3 scFv, a CD8α hinge and transmembrane region, a 4-1BB cytoplasmic (costimulatory) signaling domain, and a CD3ζ cytoplasmic (stimulatory) signaling domain.

[0154] III. Immune Cells Comprising CARs a. Immune Cells Engineered immune cells (e.g., CAR-T cells) expressing the CARs of the present disclosure are provided herein.

[0155] In some embodiments, the engineered immune cells include a population of CARs, and each CAR includes a different extracellular antigen-binding domain. In some embodiments, the immune cells include a population of CARs, and each CAR includes the same extracellular antigen-binding domain.

[0156] The engineered immune cells can be allogeneic or autologous.

[0157] In some embodiments, the engineered immune cells are T cells (e.g., inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes (Tregs), helper T lymphocytes, tumor infiltrating lymphocytes (TILs)), natural killer T cells (NKTs), TCR-expressing cells, dendritic cells, killer dendritic cells, mast cells, or B cells. In some embodiments, the cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. In some exemplary embodiments, the engineered immune cells are T cells. In some exemplary embodiments, the engineered immune cells are gamma delta T cells. In some exemplary embodiments, the engineered immune cells are macrophages. In some exemplary embodiments, the engineered immune cells are natural killer (NK) cells.

[0158] In some embodiments, the engineered immune cells may be derived from, for example, without limitation, stem cells. The stem cells can be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0159] In some embodiments, the cells are obtained or prepared from peripheral blood. In some embodiments, the cells are obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells are obtained or prepared from bone marrow. In some embodiments, the cells are obtained or prepared from umbilical cord blood. In some embodiments, the cells are human cells.

[0160] In some embodiments, the cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, particle guns (e.g., gene guns), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV), or polyplexes.

[0161] In some embodiments, engineered immune cells expressing the disclosed DLL3-specific CAR on their cell surface membranes comprise a percentage of stem cell memory and central memory cells that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 100%. In some embodiments, engineered immune cells expressing the disclosed DLL3-specific CAR on their cell surface membranes comprise from about 10% to about 100%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 20%, from about 15% to about 100%, from about 15% to about 90%, from about 15% to about 80%, from about 15% to about 70%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 20% to about 100%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 30% to about 100%, from about 30% to about 90%, from about 30% to about 80%, from about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50%, from about 30% to about 40%, from about 40% to about 100%, from about 40% to about 90%, from about 40% to about 80%, from about 40% to about 70%, from about 40% to about 60%, from about 40% to about 50%, from about 50% to about 100%, from about 50% to about 90%, from about 50% to about 80%, from about 50% to about 70%, from about 50% to about 60%, from about 60% to about 100%, from about 60% to about 90%, from about 60% to about 80%, from about 60% to about 70%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 100%, from about 80% to about 90%, from about 90% to about 100%, from about 25% to about 50%, from about 75% to about 100%, or from about 50% to about 75% of stem cell memory and central memory cells.

[0162] In some embodiments, the immune cell is an inflammatory T lymphocyte that expresses any one of the CARs described herein. In some embodiments, the immune cell is a cytotoxic T lymphocyte that expresses any one of the CARs described herein. In some embodiments, the immune cell is a regulatory T lymphocyte that expresses any one of the CARs described herein. In some embodiments, the immune cell is a helper T lymphocyte that expresses any one of the CARs described herein.

[0163] Prior to expansion and genetic modification, the source of the cells can be obtained from a subject through a variety of non-limiting methods. The cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines that are available to and known by those of skill in the art can be used. In some embodiments, the cells can be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infectious disease. In some embodiments, the cells can be a portion of a mixed population of cells that exhibit different phenotypic characteristics.

[0164] Also provided herein are cell lines obtained from immune cells (e.g., T cells) transformed according to any of the methods described above. Also provided herein are modified cells that are resistant to immunosuppressive therapy. In some embodiments, the isolated cells according to the disclosure comprise a polynucleotide encoding a CAR.

[0165] The immune cells of the present disclosure can be activated and proliferated either before or after genetic modification of the immune cells using generally known methods. Generally, the engineered immune cells of the present disclosure can proliferate, for example, by contacting with an agent that stimulates the CD3 TCR complex and co-stimulatory molecules on the surface of T cells to create an activation signal for the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to create an activation signal for the T cells.

[0166] In some embodiments, the T cell population can be stimulated in vitro by contact with an anti-CD3 antibody, such as the OKT3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) combined with a calcium ionophore. For co-stimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody (e.g., the OKT3 antibody) and an anti-CD28 antibody under conditions appropriate for stimulating T cell proliferation. The anti-CD3 antibody and the anti-CD28 antibody can be disposed on beads such as plastic or magnetic beads, or on plates or other substrates. Conditions suitable for T cell culture include a suitable medium (e.g., Minimal Essential Media or RPMI Media 1640, or X-vivo 15 (Lonza)) containing factors necessary for growth and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGF beta, and TNF, or any other additive for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium can be supplemented with amino acids, sodium pyruvate, and vitamins, and can either be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines (e.g., IL-7 and / or IL-15) in an amount sufficient for T cell growth and proliferation, and can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells to be injected into a subject.Target cells are maintained under conditions necessary to support growth, such as at an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air and 5% CO2). T cells that have been exposed to different stimulation times can exhibit different characteristics. In some embodiments, the cells of the present disclosure can be expanded by co-culturing with tissue or cells. The cells can also be expanded in vivo in the blood of a subject, for example, after administering the cells into the subject.

[0167] In some embodiments, the engineered immune cells according to the present disclosure may comprise one or more disrupted or inactivated genes. In some embodiments, the engineered immune cells according to the present disclosure comprise one disrupted or inactivated gene selected from the group consisting of CD52, DLL3, GR, PD-1, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα, and TCRβ, and / or express a CAR, a multi-chain CAR, and / or a pTα transgene. In some embodiments, the isolated cells comprise a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, the isolated cells according to the present disclosure comprise two disrupted or inactivated genes selected from the group consisting of CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, DLL3 and CD52, DLL3 and TCRα, DLL3 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, TIM3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and / or express a CAR, a multi-chain CAR, and a pTα transgene. In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing into the cell an endonuclease capable of selectively inactivating genes by selective DNA cleavage.In some embodiments, the endonuclease can be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease, a transcription activator-like effector nuclease (TALE-nuclease / TALEN), or a CRISPR (e.g., Cas9) endonuclease.

[0168] In some embodiments, the TCR becomes non-functional in the cell according to the present disclosure by disrupting or inactivating the TCRα gene and / or the TCRβ gene. In some embodiments, a method of obtaining modified cells from an individual is provided, and the cells can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can proliferate independently of the MHC signaling pathway and are readily obtained by this method are included within the scope of the present disclosure. The modified cells disclosed herein can be used to treat patients in need of treatment for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), and thus, a method of treating a patient in need of treatment for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), the method comprising treating the patient by administering to the patient an effective amount of modified cells comprising a disrupted or inactivated TCRα and / or TCRβ gene is within the scope of the present disclosure.

[0169] In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapeutic agents. The chemotherapeutic agent can be, for example, a purine nucleotide analog (PNA), and thus render the immune cells suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, cladribine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) into mono-, di-, and tri-phosphate PNAs. Their tri-phosphate forms compete with ATP for DNA synthesis, act as apoptosis promoters, and are potent inhibitors of ribonucleotide reductase (RNR) involved in trinucleotide production. DLL3-specific CAR-T cells containing disrupted or inactivated dCK genes are provided herein. In some embodiments, the dCK knockout cells are generated by transfection of T cells using a polynucleotide encoding a specific TAL nuclease directed to the dCK gene, for example, by electroporation of mRNA. The dCK knockout DLL3-specific CAR-T cells are resistant to PNAs containing, for example, cladribine and / or fludarabine, and maintain the cytotoxic activity of the T cells against DLL3-expressing cells.

[0170] In some embodiments, the isolated cells or cell lines of the present disclosure can include pTα or a functional variant thereof. In some embodiments, the isolated cells or cell lines can be further genetically modified by disrupting or inactivating the TCRα gene.

[0171] The present disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein. In some embodiments, the CAR can be introduced into immune cells as a transgene via a plasmid vector. In some embodiments, the plasmid vector can also include a selectable marker, for example, to provide identification and / or selection of the vector-receiving cells.

[0172] The CAR polypeptide can be synthesized in situ in a cell after introduction of the polynucleotide encoding the CAR polypeptide into the cell. Alternatively, the CAR polypeptide can be produced outside the cell and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, a stable transformation method (e.g., using a lentiviral vector) can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, a transient transformation method can be used to transiently express the polynucleotide construct and polynucleotide constructs that are not integrated into the genome of the cell. In other embodiments, virus-mediated methods can be used. The polynucleotide can be introduced into the cell by any suitable means such as, for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, without limitation, microinjection, electroporation, or particle bombardment. The polynucleotide can be contained in a vector such as, for example, a plasmid vector or a viral vector.

[0173] In some embodiments, there is provided an isolated nucleic acid comprising a promoter operably linked to a first polynucleotide encoding a DLL3 antigen-binding domain, at least one co-stimulatory molecule, and an activation domain. In some embodiments, the nucleic acid construct is contained within a viral vector. In some embodiments, the viral vector is selected from the group consisting of a retroviral vector, a murine leukemia virus vector, an SFG vector, an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, and a vaccinia virus vector. In some embodiments, the nucleic acid is contained within a plasmid.

[0174] b. Method of production Methods for making the CARs and CAR-containing immune cells of the present disclosure are provided herein.

[0175] When preparing polynucleotides, polypeptides, vectors, antigen-binding domains, immune cells, compositions, etc. according to the present disclosure, various known techniques can be utilized.

[0176] Prior to the in vitro manipulation or genetic modification of the immune cells described herein, the cells can be obtained from a subject. Cells expressing the DLL3 CAR can be derived from allogeneic or autologous processes.

[0177] i. Source material In some embodiments, the immune cells include T cells. T cells can be obtained from a number of sources including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL™ separation.

[0178] Cells can be obtained from the circulating blood of an individual by apheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing.

[0179] In certain embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, using centrifugation on a PERCOLL™ gradient. Certain subpopulations of T cells (e.g., CD28+, CD4+, CD8+, CD45RA−, CD45RO+, CD8+, CD62−, CD95−, CD95+, IL2Rβ+, IL2Rβ−, CCR7+, CCR7−, CD1−, CD62L+, and combinations thereof) can be further isolated by positive or negative selection techniques known in the art. In one example, a subpopulation of T cells is CD45RA+, CD95−, IL-2Rβ−, CCR7+, CD62L+. In one example, a subpopulation of T cells is CD45RA+, CD95+, IL-2Rβ+, CCR7+, CD62L+. In one example, a subpopulation of T cells is CD45RO+, CD95+, IL-2Rβ+, CCR7+, CD62L+. In one example, a subpopulation of T cells is CD45RO+, CD95+, IL-2Rβ+, CCR7−, CD62L−. In one example, a subpopulation of T cells is CD45RA+, CD95+, IL-2Rβ+, CCR7−, CD62L−. For example, enrichment of a T cell population by negative selection can be achieved with a combination of antibodies directed to surface markers specific for the negatively selected cells. One method for use herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate a cell population of interest for use in the present disclosure.

[0180] PBMC can be used directly for genetic modification by immune cells (such as CAR or TCR) using the methods described herein. In certain embodiments, after isolating PBMC, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets either before or after gene modification and / or expansion.

[0181] In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying characteristic cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the phenotypic markers expressed by central memory T cells include CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and are negative for granzyme B. In some embodiments, stem cell memory T cells are CD45RO−, CD62L+, CD8+ T cells. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and are positive for granzyme B and perforin.

[0182] In certain embodiments, CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying a cell population having characteristic cell surface antigens.

[0183] iii. Immune cells derived from stem cells In some embodiments, the immune cells can be derived from stem cells such as progenitor cells, bone marrow stem cells, induced pluripotent stem cells, iPSCs, hematopoietic stem cells, and mesenchymal stem cells. iPS cells and other types of stem cells can be cultured immortal cell lines or can be isolated directly from a patient. Various methods for isolating, developing, and / or culturing stem cells are known in the art and can be used to practice the present invention.

[0184] In some embodiments, the immune cells are induced pluripotent stem cells (iPSCs) derived from reprogrammed T cells. In some embodiments, the source material can be induced pluripotent stem cells (iPSCs) derived from T cells or non-T cells. Alternatively, the source material can be B cells, or any other cells from a peripheral blood mononuclear cell isolate, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, or any other somatic cell type.

[0185] ii. Genetic modification of isolated cells Immune cells such as T cells can be genetically modified after isolation using known methods, or the immune cells can be activated and expanded (or differentiated in the case of progenitor cells) in vitro prior to being genetically modified. In some embodiments, the isolated immune cells are genetically modified to reduce or eliminate the expression of endogenous TCRα and / or CD52. In some embodiments, the cells are genetically modified using gene editing techniques (e.g., CRISPR / Cas9, CRISPR / CAS12, zinc finger nucleases (ZFNs), TALENs, MegaTALs, meganucleases) to reduce or eliminate the expression of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, immune cells such as T cells are optionally further genetically modified with a chimeric antigen receptor as described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and / or expanded in vitro.

[0186] Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent No. 6,905,874, U.S. Patent No. 6,867,041, U.S. Patent No. 6,797,514, and PCT WO2012 / 079000, the contents of which are hereby incorporated by reference in their entirety. Generally, such methods involve contacting PBMCs or isolated T cells with stimulatory and co-stimulatory molecules, such as anti-CD3 and anti-CD28 antibodies, generally attached to plastic or magnetic beads or other surfaces, in a culture medium containing appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same beads function as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other embodiments, T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent No. 6,040,177, U.S. Patent No. 5,827,642, and WO2012 / 129514, the contents of which are hereby incorporated by reference in their entirety.

[0187] Certain methods for making the constructs and engineered immune cells of the present disclosure are described in PCT application PCT / US15 / 14520, the contents of which are hereby incorporated by reference in their entirety.

[0188] It will be understood that PBMCs can further include other cytotoxic lymphocytes such as NK cells or NKT cells. Expression vectors carrying the coding sequences of chimeric receptors as disclosed herein can be introduced into populations of human donor T cells, NK cells, or NKT cells. Successfully transduced T cells carrying the expression vector are sorted using flow cytometry to isolate CD3-positive T cells, which are then further expanded to increase the number of these CAR-expressing T cells in addition to cell activation using anti-CD3 antibody and IL-2 or other methods known in the art described elsewhere herein. Standard procedures are used for cryopreservation of T cells expressing CAR for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells is performed in the absence of products derived from non-human animals such as fetal calf serum (fetal calf serum and fetal bovine serum). In one embodiment, cryopreservation can include freezing in a suitable medium such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions).

[0189] For cloning of polynucleotides, the vector is introduced into a host cell (an isolated host cell) to enable replication of the vector itself, thereby amplifying copies of the polynucleotide contained therein. Cloning vectors can contain sequence components including, but not limited to, an origin of replication, a promoter sequence, a transcription start sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by those skilled in the art. For example, the origin of replication can be selected to facilitate autonomous replication of the vector in the host cell.

[0190] In certain embodiments, the present disclosure provides an isolated host cell containing the vectors provided herein. A host cell containing a vector can be useful for the expression or cloning of the polynucleotide contained in the vector. Suitable host cells can include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells, and more specifically human cells.

[0191] The vector can be introduced into the host cell using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, and delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for viral transfection of cells and transformation for the expression of the vector of interest are well known in the art. In further embodiments, a mixture of different expression vectors can be used when genetically modifying a donor population of immune effector cells, and each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, and a proportion of the engineered cells express two or more different CARs.

[0192] In one embodiment, the present disclosure provides a method of preserving genetically engineered cells that express a chimeric antigen receptor (CAR) targeting DLL3 protein. In one embodiment, this involves cryopreserving immune cells such that the cells continue to survive upon thawing. In one embodiment, cryopreservation can include freezing in a suitable medium such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions). The fraction of immune cells expressing the CAR can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients afflicted with a malignancy. Optionally, the cryopreserved transformed immune cells can be thawed, grown, and expanded for more of such cells.

[0193] In some embodiments, the cells are formulated by first harvesting them from their culture medium and then washing and concentrating the cells in a medium and container system (a "pharmaceutically acceptable" carrier) suitable for administration in a therapeutically effective amount. Suitable infusion media can be any isotonic media formulation, typically normal saline, Normosol® R (Abbott), or Plasma-Lyte® A (Baxter), but 5% dextrose in water or Ringer's lactate can also be utilized. The infusion media can be supplemented with human serum albumin.

[0194] iv. Allogeneic CAR T cells Briefly, the process for manufacturing allogeneic CAR T therapies, or AlloCAR™, involves collecting healthy, selected, screened, and tested T cells from healthy donors. Allogeneic T cells are gene-edited to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. Selected T cell receptor genes (e.g., TCRα, TCRβ) are knocked out to avoid GvHD. The CD52 gene can also be knocked out to render the CAR T product resistant to anti-CD52 antibody therapy. Thus, anti-CD52 antibody therapy can be used to lymphodeplete the host immune system and keep the CAR T cells engrafted to achieve a complete therapeutic effect. Next, the T cells are engineered to express a chimeric antigen receptor (CAR) that recognizes a specific cell surface protein (e.g., DLL-3) expressed on hematologic or solid tumors. The engineered T cells then undergo a purification step and are ultimately cryopreserved in vials for delivery to a patient.

[0195] v. autologous CAR T cells Autologous chimeric antigen receptor (CAR) T cell therapy involves collecting a patient's own cells (e.g., white blood cells including T cells), genetically engineering the T cells to express a CAR that recognizes a target antigen expressed on the cell surface of one or more specific cancer cells, and killing the cancer cells. The engineered cells are then cryopreserved and subsequently administered to the patient from whom the cells were removed for engineering.

[0196] IV. Methods of Treatment The present disclosure includes methods for treating or preventing a condition associated with undesired and / or elevated DLL3 levels in a patient, comprising administering to a patient in need thereof an effective amount of at least one CAR, or an immune cell comprising a CAR disclosed herein.

[0197] Methods for treating a disease or disorder, including cancer, are provided. In some embodiments, the disclosure relates to forming a T cell-mediated immune response in a subject, comprising administering to the subject an effective amount of the engineered immune cells of the present application. In some embodiments, the T cell-mediated immune response is directed against target cells. In some embodiments, the engineered immune cells comprise a chimeric antigen receptor (CAR). In some embodiments, the target cells are tumor cells. In some aspects, the disclosure includes methods for treating or preventing a malignancy, the method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen-binding domain described herein. In some aspects, the disclosure includes methods for treating or preventing a malignancy, the method comprising administering to a subject in need thereof an effective amount of at least one immune cell, the immune cell comprising at least one chimeric antigen receptor and / or an isolated antigen-binding domain described herein. The CAR containing the immune cells of the disclosure can be used to treat malignancies associated with aberrant expression of DLL3. In some embodiments, the CAR containing the immune cells of the disclosure is used to treat malignancies such as small cell lung cancer, melanoma, low-grade glioma, glioblastoma, medullary thyroid cancer, carcinoid, pancreatic, bladder, and disseminated neuroendocrine tumors in the prostate, testicular cancer, and lung adenocarcinoma with neuroendocrine features. In an exemplary embodiment, CAR-containing immune cells, such as the anti-DLL3 CAR-T cells of the disclosure, are used to treat small cell lung cancer.

[0198] Also provided is a method for reducing the size of a tumor in a subject, comprising administering to the subject the engineered cells of the disclosure, the cells comprising a chimeric antigen receptor comprising a DLL3 antigen-binding domain that binds to the DLL3 antigen on the tumor.

[0199] In some embodiments, the subject has a solid tumor, or a hematologic malignancy such as lymphoma or leukemia. In some embodiments, the engineered cells are delivered to a tumor bed, such as a tumor bed found in small cell lung cancer. In some embodiments, the cancer is present in the bone marrow of the subject. In some embodiments, the engineered cells are autologous immune cells, such as autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, such as allogeneic T cells. In some embodiments, the engineered cells are xenogeneic immune cells, such as xenogeneic T cells. In some embodiments, the engineered cells are transfected or transduced ex vivo. As used herein, the term "in vitro cell" refers to any cell cultured ex vivo.

[0200] A "therapeutically effective amount", "effective dose", "effective amount", or "therapeutically effective dosage" of a therapeutic agent, such as engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the development of a disease, or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or a prevention of functional or disability impairments due to the pain of the disease. The ability of a therapeutic agent to promote regression of a disease can be evaluated using various methods known to skilled practitioners (e.g., physicians or clinicians), such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0201] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as subjects with a formally diagnosed disorder, subjects without a formally recognized disorder, subjects undergoing a medical treatment, subjects at risk of developing a disorder, and the like.

[0202] The terms "treating" and "treatment" include therapeutic treatment, prophylactic treatment, and uses for reducing the risk that a subject will develop a disorder or other risk factor. Treatment does not require complete cure of the disorder and encompasses embodiments for reducing symptoms or potential risk factors. The term "preventing" does not require 100% exclusion of the likelihood of an event. Rather, it indicates that the likelihood of the event occurring is reduced in the presence of a compound or method.

[0203] The desired total amount of cells in the composition comprises at least two cells (e.g., at least one CD8+ T cell and at least one CD4+ T cell, or two CD8+ T cells, or two CD4+ T cells), or more typically 2 more than 10 6 cells, and up to 10 8 or 10 9 and up to 10 10 or 10 12 or more than 10 6 cells, and may be 10 7 or more than 10 8 cells / ml. The number of cells will depend on the desired use of the composition and the type of cells included therein. The desired cell density is typically greater than 10 5 cells / ml, generally greater than 10 6 cells / ml, and generally 10 7 or more cells / ml. A clinically relevant number of immune cells can be distributed over multiple infusions that cumulatively equal or exceed 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells, or 10 12 cells. In some embodiments of the disclosure, particularly where all infused cells are redirected to a specific target antigen (e.g., DLL3), 10 6 / kilogram (10 6 to 10 11A lower number of cells within the range of () can be administered. The CAR therapy can be administered multiple times at dosages within these ranges. The cells can be autologous, allogeneic, or xenogeneic to the patient receiving the therapy.

[0204] In some embodiments, the therapeutically effective amount of CAR T cells is about 1X10 5 cells / kg, about 2X10 5 cells / kg, about 3X10 5 cells / kg, about 4X10 5 cells / kg, about 5X10 5 cells / kg, about 6X10 5 cells / kg, about 7X10 5 cells / kg, about 8X10 5 cells / kg, about 9X10 5 cells / kg, 2X10 6 cells / kg, about 3X10 6 cells / kg, about 4X10 6 cells / kg, about 5X10 6 cells / kg, about 6X10 6 cells / kg, about 7X10 6 cells / kg, about 8X10 6 cells / kg, about 9X10 6 cells / kg, about 1X10 7 cells / kg, about 2X10 7 cells / kg, about 3X10 7 cells / kg, about 4X10 7 cells / kg, about 5X10 7 cells / kg, about 6X10 7 cells / kg, about 7X10 7 cells / kg, about 8X10 7 cells / kg, or about 9X10 7 cells / kg.

[0205] In some embodiments, the target dosage of CAR+ / CAR-T+ cells is about 1×10 6 ~ about 1×10 10 cells / kg, for example, about 1×10 6 cells / kg, about 1×10 7 cells / kg, about 1×10 8 cells / kg, about 1×10 9cells / kg, or about 1×10 10 cells / kg. It will be understood that dosages above and below this range may be appropriate for a particular subject and that the appropriate dosage level may be determined by a medical provider as needed. In addition, multiple dosages of the cells can be provided in accordance with the present disclosure.

[0206] In some aspects, the present disclosure includes a pharmaceutical composition comprising at least one antigen-binding domain described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0207] The CAR-expressing cell population of the present disclosure can be administered alone or as a pharmaceutical composition combined with a diluent and / or other components such as IL-2 or other cytokines or cell populations. The pharmaceutical compositions of the present disclosure can comprise a CAR-expressing cell population such as the T cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present disclosure are preferably formulated for intravenous administration.

[0208] Pharmaceutical compositions (solutions, suspensions, etc.) include the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol or other solvents that can serve as a solvent or suspension medium; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate, and one or more agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. For therapeutic use, injectable pharmaceutical compositions are preferably sterile.

[0209] In some embodiments, upon administration to a patient, engineered immune cells that express any one of the DLL3-specific CARs described herein on their cell surface may reduce, kill, or lyse the patient's endogenous DLL3-expressing cells. In one embodiment, the percentage reduction or lysis of cells of a DLL3-expressing endogenous cell or cell line expressing DLL3 by engineered immune cells expressing any one of the DLL3-specific CARs described herein is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more. In one embodiment, the percentage reduction or lysis of cells of a DLL3-expressing endogenous cell or cell line expressing DLL3 by engineered immune cells expressing any one of the DLL3-specific CARs described herein is from about 5% to about 95%, from about 10% to about 95%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 25% to about 75%, or from about 25% to about 60%. In one embodiment, the endogenous DLL3-expressing cells are endogenous DLL3-expressing myeloid cells.

[0210] In one embodiment, the percent reduction or lysis of target cells, e.g., a cell line expressing DLL3, by engineered immune cells expressing the DLL3-specific CAR of the present disclosure on their cell surface membrane can be measured using the assays disclosed herein.

[0211] The method can further comprise administering to the patient one or more chemotherapeutic agents prior to administering the engineered cells provided herein. In certain embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapeutic agent. For example, a method of conditioning a patient in need of T cell therapy is to administer to the patient a specific beneficial dose of cyclophosphamide (200 mg / m 2 / day to 2000 mg / m 2 / day, about 100 mg / m2 / day to approximately 2000 mg / m 2 / day, for example, approximately 100 mg / m 2 / day, approximately 200 mg / m 2 / day, approximately 300 mg / m 2 / day, approximately 400 mg / m 2 / day, approximately 500 mg / m 2 / day, approximately 600 mg / m 2 / day, approximately 700 mg / m 2 / day, approximately 800 mg / m 2 / day, approximately 900 mg / m 2 / day, approximately 1000 mg / m 2 / day, approximately 1500 mg / m 2 / day, or approximately 2000 mg / m 2 / day), and a specific dose of fludarabine (20 mg / m 2 / day to 900 mg / m 2 / day, approximately 10 mg / m 2 / day to approximately 900 mg / m 2 / day, for example, approximately 10 mg / m 2 / day, approximately 20 mg / m 2 / day, approximately 30 mg / m 2 / day, approximately 40 mg / m 2 / day, approximately 40 mg / m 2 / day, approximately 50 mg / m 2 / day, approximately 60 mg / m 2 / day, approximately 70 mg / m 2 / day, approximately 80 mg / m 2 / day, approximately 90 mg / m 2 / day, approximately 100 mg / m 2 / day, approximately 500 mg / m 2 / day, or approximately 900 mg / m 2 / day). An exemplary dosing regimen involves administering approximately 30 mg / m 2 / day of fludarabine in combination with, or before or after, administering approximately 300 mg / m 2 / day of cyclophosphamide to the patient daily for 3 days prior to administration of the engineered T cells in a therapeutically effective amount to the patient.

[0212] In some embodiments, particularly where the engineered cells provided herein are gene edited to remove or minimize surface expression of CD52, lymphodepletion further comprises administration of an anti-CD52 antibody such as alemtuzumab. In some embodiments, the CD52 antibody is administered at a dose of about 1 to 20 mg / day IV, for example, about 13 mg / day IV, for 1, 2, 3 or more days. The antibody can be administered in combination with, before, or after administration of other elements of the lymphodepletion regimen (e.g., cyclophosphamide and / or fludarabine).

[0213] In other embodiments, the antigen-binding domain, the transduced (or otherwise engineered) cells, and the chemotherapeutic agent are each administered in an amount effective to treat a disease or condition in a subject.

[0214] In certain embodiments, the compositions containing the CAR-expressing immune effector cells disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as actinomycin, aclarubicin, aclacinomycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, calminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, promycin, quelamycin, rhodomycin, streptozocin, streptonigrin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioguanine, thiamiprine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens such as calusterone, drostanolone propionate, epithioestanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; dexamethasone; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark); razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’-trichloroethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids such as paclitaxel (TAXOL (trademark), Bristol-Myers Squibb) and docetaxel (TAXOTERE (registered trademark), Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RF S2000; difluoromethylornithine (DMFO); retinoid acid derivatives such as Targretin (trademark) (bexarotene), Panretin (trademark), (alitretinoin); ONTAK (trademark) (denileukin diftitox); esperamicin; capecitabine;and pharmaceutically acceptable salts, acids, or derivatives thereof as described above. This definition includes, for example, anti-estrogens such as tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-hormonal agents that act to modulate or inhibit the hormonal action on tumors such as anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives thereof as described above. Optionally, CHOP, i.e., a combination of chemotherapeutic agents including cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, but not limited thereto, is also administered.;

[0215] In some embodiments, the chemotherapeutic agent is administered simultaneously with, or within 1 week after, the administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered about 1 to 7 days, about 1 to about 4 weeks, or about 1 week to about 1 month, about 1 week to about 2 months, about 1 week to about 3 months, about 1 week to about 6 months, about 1 week to about 9 months, or about 1 week to about 12 months after the administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administering the cell, polypeptide, or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0216] A variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0217] Additional therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), imatinib (Gleevec®), cetuximab (Erbitux®), panitumumab (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tiboxanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, luxolutinib, pacritinib, cobimetinib, selumetinib, trametinib, vinimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as CDK inhibitor (palbociclib).

[0218] In some embodiments, a composition comprising CAR-containing immune cells can be administered with a treatment regimen to prevent or reduce cytokine release syndrome (CRS) or neurotoxicity. Treatment regimens for preventing cytokine release syndrome (CRS) or neurotoxicity can include ranizumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (e.g., L-NIL or 1400W). In additional embodiments, a composition comprising CAR-containing immune cells can be administered with an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, triamcinolone), aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF pharmaceuticals, cyclophosphamide, and non-steroidal anti-inflammatory drugs (NSAIDs) including mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialates. Exemplary analgesics include acetaminophen, oxycodone, and tramadol of propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, or prednisolone. Exemplary biological response modifiers include molecules directed to cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, e.g., TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0219] In certain embodiments, the compositions described herein are administered in combination with a cytokine. Examples of cytokines are lymphokines, monokines, and conventional polypeptide hormones. Among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian duct inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF) such as NGF-beta; platelet growth factor; transforming growth factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factors-I and -II; erythropoietin (EPO); osteogenic factor; interferons such as interferon-alpha, beta, and gamma; colony stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-21; tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines.

[0220] V. Methods of Sorting and Depletion In some embodiments, a method for in vitro selection of a population of immune cells is provided, wherein a subset of the population of immune cells comprises engineered immune cells expressing any one of DLL3-specific CARs comprising an epitope specific for a monoclonal antibody (e.g., an exemplary mimotope sequence). The method comprises contacting a population of immune cells with a monoclonal antibody specific for the epitope and selecting immune cells that bind to the monoclonal antibody to obtain a population of cells enriched in engineered immune cells expressing DLL3-specific CARs.

[0221] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a fluorophore. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be performed by fluorescence-activated cell sorting (FACS).

[0222] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to magnetic particles. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be performed by magnetic-activated cell sorting (MACS).

[0223] In some embodiments, the mAb used in the method for selecting immune cells expressing CAR is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, and / or ustekinumab. In some embodiments, the mAb is rituximab. In another embodiment, the mAb is QBEND-10.

[0224] In some embodiments, when using the method for in vitro selection of the CAR-expressing immune cells described above, the population of CAR-expressing immune cells obtained comprises at least 70%, 75%, 80%, 85%, 90%, 95% of the CAR-expressing immune cells. In some embodiments, when using the method for in vitro selection of the CAR-expressing immune cells described above, the population of CAR-expressing immune cells obtained comprises at least 85% of the CAR-expressing immune cells.

[0225] In some embodiments, when using the method for in vitro selection of the CAR-expressing immune cells described above, the population of CAR-expressing immune cells obtained exhibits increased cytotoxic activity in vitro compared to the initial (unselected) cell population. In some embodiments, the cytotoxic activity in vitro increases by 10%, 20%, 30%, or 50%. In some embodiments, the immune cells are T cells.

[0226] In some embodiments, the mAb is pre-bound to a support or surface. Non-limiting examples of solid supports can include beads, agarose beads, plastic beads, magnetic beads, plastic well plates, glass well plates, ceramic well plates, columns, or cell culture bags.

[0227] The CAR-expressing immune cells administered to the recipient can be enriched in vitro from a source population. Methods for expanding the source population can include using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting to select cells expressing an antigen such as the CD34 antigen.

[0228] Flow cytometry can be used to quantify specific cell types within a cell population. Generally, flow cytometry is a method for quantifying the components or structural features of cells mainly by optical means. Different cell types can be distinguished by quantifying the structural features, so flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[0229] Flow cytometry analysis includes two main steps: 1) labeling the selected cell type with one or more labeled markers, and 2) determining the number of labeled cells relative to the total number of cells in the population. In some embodiments, the method of labeling the cell type includes binding a labeled antibody to a marker expressed by a specific cell type. The antibody can be either directly labeled with a fluorescent compound or indirectly labeled, for example, using a fluorescently labeled second antibody that recognizes the first antibody.

[0230] In some embodiments, the method used to sort T cells expressing a CAR is magnetic-activated cell sorting (MACS). Magnetic-activated cell sorting (MACS) is a method for the separation of various cell populations according to their surface antigens (CD molecules) by using superparamagnetic nanoparticles and columns. MACS can be used to obtain a pure cell population. Cells in a single cell suspension can be magnetically labeled with microbeads. The sample is applied to a column composed of strongly magnetic spheres covered with a cell-friendly coating that allows for a quick and gentle separation of the cells. While the magnetically labeled cells are retained within the column, the unlabeled cells pass through. The flow-through can be collected as the unlabeled cell fraction. After a washing step, the column is removed from the separator and the magnetically labeled cells are eluted from the column.

[0231] Detailed protocols for the purification of specific cell populations such as T cells can be found in Basu S et al. (2010). (Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J Vis Exp. (41):1546).

[0232] In some embodiments, the disclosure provides methods for depleting DLL3-specific CAR-expressing immune cells by in vivo depletion. In vivo depletion can include the administration of a treatment (e.g., a molecule that binds to an epitope on the CAR) to a mammalian organism aimed at arresting the proliferation of CAR-expressing immune cells by inhibition or elimination.

[0233] One aspect of the invention relates to a method for in vivo depletion of engineered immune cells expressing a DLL3 CAR comprising an mAb-specific epitope, the method comprising contacting the engineered immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb. Another aspect of the invention relates to a method for in vivo depletion of CAR-expressing immune cells comprising a chimeric scFv (e.g., formed by insertion of an mAb-specific epitope) by contacting the engineered immune cells with an epitope-specific antibody. In some embodiments, the immune cells are T cells and / or the antibody is monoclonal.

[0234] According to one embodiment, in vivo depletion of the engineered cells is performed on the engineered immune cells previously selected using the in vitro method of the present invention. In this case, the same injected mAb can be used. In some embodiments, the mAb-specific antigen is the CD20 antigen, and the epitope-specific mAb is rituximab. In some embodiments, the present invention relates to a method for in vivo depletion of engineered immune cells (CAR-expressing immune cells) expressing a CAR comprising an mAb-specific epitope, comprising contacting the CAR-expressing immune cells with at least one epitope-specific mAb.

[0235] In some embodiments, the step of contacting the engineered immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb comprises injecting the patient with an epitope-specific mAb (e.g., rituximab). In some embodiments, the amount of the epitope-specific mAb administered to the patient is sufficient to eliminate at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the CAR-expressing immune cells in the patient.

[0236] In some embodiments, the step of contacting the engineered immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb comprises injecting the patient with about 375 mg / m 2 of rituximab one or more times. In some embodiments, the mAb (e.g., rituximab) is administered once a week.

[0237] In some embodiments, when immune cells expressing a CAR that includes an mAb-specific epitope (CAR-expressing immune cells) are depleted in a complement-dependent cytotoxicity (CDC) assay using an epitope-specific mAb, the amount of viable CAR-expressing immune cells decreases. In some embodiments, the amount of viable CAR-expressing immune cells decreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the mAb-specific epitope is a CD20 epitope or mimotope, and / or the epitope-specific mAb is rituximab.

[0238] In certain embodiments, in vivo depletion of CAR-engineered immune cells is effected by injecting a bispecific antibody. By definition, a bispecific monoclonal antibody (BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigens. These BsAbs and their use in immunotherapy are reviewed in Muller D and Kontermann R.E. (2010) Bispecific Antibodies for Cancer Immunotherapy, BioDrugs 24(2):89-98.

[0239] According to another specific embodiment, the injected bispecific mAb can bind to both an mAb-specific epitope on engineered immune cells expressing a chimeric scFv and a surface antigen on effector and cytotoxic cells (e.g., immune cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), cytotoxic T lymphocytes (CTL)). By doing so, depletion of the engineered immune cells caused by the BsAb can occur through antibody-dependent cell cytotoxicity (ADCC). (Deo Y M, Sundarapandiyan K, Keler T, Wallace PK, and Graziano RF, (2000), Journal of Immunology, 165(10):5954-5961]).

[0240] In some embodiments, the cytotoxic drug is conjugated to an epitope-specific mAb that can be used to deplete CAR-expressing immune cells. By combining the targeting ability of the monoclonal antibody with the cancer-killing ability of the cytotoxic drug, an antibody-drug conjugate (ADC) enables a sensitive distinction between healthy and diseased tissue when compared to the use of the drug alone. Market approval has been obtained for several ADCs, and the technology for making them, especially on the linker, is described in (Payne, G. (2003) Cancer Cell 3:207-212, Trail et al (2003) Cancer Immunol. Immunother. 52:328-337, Syrigos and Epenetos (1999) Anticancer Research 19:605-614, Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172, U.S. Patent No. 4,975,278).

[0241] In some embodiments, the injected epitope-specific mAb is pre-conjugated to a molecule that can promote complement-dependent cytotoxicity (CDC). Thus, the complement system helps or complements the ability of antibodies to remove pathogens from an organism. When stimulated, the activation cascade is triggered as a large-scale amplification of the response and activation of the cell-killing membrane attack complex. Different molecules such as glycans can be used to conjugate the mAb (Courtois, A, Gac-Breton, S., Berthou, C, Guezennec, J., Bordron, A. and Boisset, C. (2012), Complement dependent cytotoxicity activity of therapeutic antibody fragments may be acquired by immunogenic glycan coupling, Electronic Journal of Biotechnology ISSN:0717-3458, http: / / www.ejbiotechnology.info DOI:10.2225 / voll5-issue5).

[0242] VI. Kits and Products The present application provides a kit comprising any one of the DLL3-containing CARs or DLL3 CAR-containing immune cells described herein, and a pharmaceutical composition thereof. In one embodiment of the kit, the engineered CAR cells are frozen in a suitable medium such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions).

[0243] In some exemplary embodiments, the kits of the disclosure include allogeneic DLL3 CAR-containing T cells, as well as a CD52 antibody for administering a lymphodepletion regimen and a CAR-T regimen to a subject.

[0244] The present application also provides a product comprising any one of the therapeutic compositions or kits described herein. Examples of the product include vials (e.g., sealed vials).

Examples

[0245] Example 1: Generation and Testing of DLL3-Targeted Antibodies The monoclonal antibodies used in accordance with the present invention can be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Anti-DLL3 antibodies were first screened by Flag-DLL3 (Adipogen) ELISA and then by FACS to determine binding to HEK-293T cells with or without human DLL3 expression.

[0246] To test whether a DLL3-specific antibody can recognize cells expressing endogenous DLL3, DMS 273 (Sigma, catalog number 95062830), DMS 454 (Sigma, catalog number 95062832), and SHP-77 (ATCC, catalog number CRL-2195) cells were stained with 2 μg / ml of purified DLL3 antibody or control mIgG2a antibody containing mouse IgG2A backbone (mIgG2a) in PBS supplemented with 1% BSA. The bound DLL3 antibody was detected with a PE-labeled anti-mouse IgG antibody (Biolegend, catalog number 405307). Samples were analyzed by flow cytometry. Representative images showing the binding of the DLL3 antibody to DMS 273, DMS 454, and SHP-77 cells are included in Figure 1.

[0247] Example 2: Determination of the kinetics and affinity of anti-DLL3 antibodies for DLL3 This example determines the binding kinetics and affinity of various anti-DLL3 antibodies at 37°C, both as full-length monoclonal antibodies (IgG) and as scFvs against human, cynomolgus (cyno), and mouse DLL3. For the scFvs, the variable regions of the anti-DLL3 antibodies derived from their respective hybridomas were cloned adjacent to a (GGGGS)3 (SEQ ID NO: 472) or (GGGGS)4 (SEQ ID NO: 478) linker, followed by a hinge and a portion of the Fc from the human IgG2 sequence, resulting in scFv-Fc fusions, which were expressed using Expi293. The extracellular domains (ECDs) from human, cynomolgus, and mouse DLL3 were fused to a C-terminal 8xHis epitope tag (SEQ ID NO: 473) and an Avi tag, expressed using Expi293, and then purified by immobilized metal affinity chromatography (IMAC), followed by size exclusion chromatography (SEC).

[0248] The antibody binding rate was determined by surface plasmon resonance (BiacoreTM surface plasmon resonance (SPR) system, GE Healthcare Bio-Sciences, Pittsburg PA). Antibodies diluted in HBS-T+ running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% volume / volume Tween20, 1 mg / mL BSA) were captured on a CM4 chip immobilized with an antibody specific for the anti-DLL3 antibody constant domain. Purified DLL3 was serially diluted with HBS-T+ and injected at 30 μL / min for 2 minutes. After a dissociation time of 10 minutes, the surface was regenerated with either 10 mM glycine-HCl pH 1.7 or phosphoric acid between injections. The kinetic association rate (kon) and dissociation rate (koff) were obtained simultaneously by fitting the data globally to a 1:1 Langmuir binding model (Karlsson, R., Roos, H., Fagerstam, L., Petersson, B. (1994). Methods Enzymology 6.99-110) using the BIA evaluation program. The equilibrium dissociation constant (K d ) value is calculated as k off / k on .

[0249] The rate and affinity parameters of the anti-DLL3 antibodies tested are shown in Table 8. Specifically, Table 8 shows the affinity of anti-DLL3 antibodies (either IgG or scFv-Fc fusions) for human, cynomolgus monkey, and mouse DLL3. The last column indicates which extracellular domain of human DLL3 each anti-DLL3 antibody recognizes.

Table 11

[0250] Example 3: Generation of CHO cells expressing full-length and truncated DLL3 Using a panel of CHO cells expressing full-length and various truncated human DLL3, we determined which domain each DLL3-targeting antibody recognizes. The extracellular domain of human DLL3 can be subdivided into distinct subdomains defined by the following amino acid positions: signal peptide: 1 - 26, N-terminus (N-ter): 27 - 175, DSL: 176 - 215, EGF1: 215 - 249, EGF2: 274 - 310, EGF3: 312 - 351, EGF4: 353 - 389, EGF5: 391 - 427, and EGF6: 429 - 465.

[0251] To generate the truncated DLL3 proteins used for epitope mapping, the sequences of each of the eight extracellular domains of human DLL3 (signal peptide and N-terminus, DSL, EGF1, EGF2, EGF3, EGF4, EGF5, and EGF6) were deleted one by one from the antigen starting from the N-terminus. Table 6 shows the generated truncated DLL3 proteins (see also Figures 2A - 2D). [Table 12-1] [Table 12-2]

[0252] To establish CHO cells expressing full-length and truncated human DLL3 with an N-terminal HA tag, full-length human DLL3 (SEQ ID NO: 556, GeneBank record NM_016941) and truncated human DLL3 with a 7 HA tag (SEQ ID NOs: 557-563) were cloned into the pLVX-SFFV-Puro-P2A-TetO3G vector (Clontech). Lentiviruses encoding either full-length or truncated human DLL3 were generated by co-transfecting 293T cells with the pLVX-SFFV-Puro-P2A-TetO3G vector, the psPAX2 and pMD2G vectors. Two days after transfection, the supernatant containing virus particles was collected and used to transduce CHO cells together with 5 μg / ml polybrene.

[0253] Expression of full-length and truncated DLL3 was verified by FACS assay using a PE-conjugated anti-HA antibody (Biolegend, catalog number 901518). As a negative control, cells were incubated with an isotype-matched, PE-labeled antibody (Biolegend, catalog number 400111) instead of the anti-HA antibody. The lower panel in Figure 2A shows the expression of full-length and truncated DLL3 on CHO cells.

[0254] Example 4: Epitope mapping of DLL3-targeting antibodies CHO cells expressing full-length and truncated DLL3 were stained with hybridoma supernatants or purified DLL3 antibodies in PBS + 1% BSA. Bound DLL3 antibodies were detected with a PE-labeled anti-mouse IgG antibody (Biolegend, catalog number 405307). Samples were analyzed by flow cytometry. The binding domain of each clone was determined using a panel of CHOs expressing full-length or truncated DLL3 as described in Example 2. Flow cytometry analysis showed, for example, that if a clone binds to all truncated proteins containing EGF3 but not to any truncated protein lacking EGF3, such a clone recognizes EGF3. As shown in the representative image in Figure 2D, anti-DLL3 antibodies recognize the DSL, EGF1, and EGF3 domains, respectively. The signal from the PE channel is shown on the x-axis and the count is shown on the y-axis.

[0255] Example 5: Generation of DLL3-Specific CAR-T Cells This example describes the construction of an anti-DLL3 chimeric antigen receptor (CAR).

[0256] The anti-DLL3 antibodies listed in Table 1a were reformatted into CARs. Using the amino acid sequences of the heavy-chain variable region and light-chain variable region of these antibodies (Table 1b and Table 1c), a single-chain variable fragment (scFv) having the following general structure: heavy-chain variable region--linker--light-chain variable region was designed (Table 1d). The linker had the following amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 478).

[0257] The protein sequence encoding the chimeric antigen receptor was designed to contain the following elements from 5' to 3': CD8α signal sequence (SEQ ID NO: 477), anti-DLL3 scFv, hinge and transmembrane region of the human CD8α molecule (SEQ ID NO: 479), cytoplasmic portion of the 41BB molecule (SEQ ID NO: 291), and cytoplasmic portion of the CD3ζ molecule (SEQ ID NO: 292) (Figure 3A, Table 7).

Table 13-1

Table 13-2

Table 13-3

Table 13-4

Table 13-5

Table 13-6

Table 13-7

Table 13-8

Table 13-9

Table 13-10

Table 13-11

Table 13-12

Table 13-13

Table 13-14

Table 13-15

Table 13-16

Table 13-17

Table 13-18

Table 13-19

Table 13-20

Table 13-21

Table 13-22

Table 13-23

Table 13-24

Table 13-25

Table 13-26

Table 13-27

[0258] A schematic diagram of the CAR structure is shown in Figure 3A. Representative CAR sequences reformatted from anti-DLL3 clones are included in SEQ ID NOs: 482 to 533. The codon-optimized DLL3 CAR sequence was synthesized and subcloned into the following lentiviral vector pLVX-EF1a-DLL3 CAR (Clontech) using XmaI (5’) and MluI (3’) restriction sites.

[0259] To generate DLL3 CAR-T cells, PBMCs were first purified from buffy coat samples using Ficoll gradient density medium (Ficoll Paque PLUS / GE Healthcare Life Sciences). T cells were purified from PBMCs using a commercially available T cell isolation kit (Miltenyi Biotec, catalog number 130-096-535). Alternatively, primary human T cells can be purified directly from LeukoPak (StemCell Technologies).

[0260] To generate a lentivirus encoding DLL3 CAR, HEK-293T cells were seeded on day 0 at 400,000 cells per mL in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone or JR Scientific) per well of a 6-well plate. On day 1, the lentivirus was prepared by mixing 1.5 μg of the lentivirus packaging vector psPAX2, 0.5 μg of pMD2G, and 0.5 μg of the appropriate transfer CAR vector together in 250 μL of Opti-MEM (Gibco) per well of a 6-well plate (“DNA mix”). 10 μL of Lipofectamine 2000 (Invitrogen) in 250 μL of Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mix. The mixture was incubated at room temperature for 20 minutes and added slowly to the side of the well containing HEK-293T to a total volume of 500 μL. Purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL of human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, catalog number 130-111-160, 1:100 dilution). On day 2, the medium from each well of the 6-well plate was replaced with 2 mL of T cell transduction medium per well, i.e., X-Vivo-15 supplemented with 10% FBS. On day 3, the T cells were resuspended at 500,000 cells per mL in 1 mL of T cell transduction medium per well of a Grex-24 plate (Wilson Wolf, catalog number 80192M). The lentivirus supernatant from HEK293T cells was harvested, passed through a 0.45 micron filter (EMD Millipore) to remove cell debris, and then added to the T cells together with 100 IU / mL of human IL-2. On day 5, 4.5 mL of T cell growth medium, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), was added to each well of the Grex-24 plate.On days 9 and 13, transduction efficiency was determined by detecting the percentage of T cells recognizing recombinant DLL3 (Adipogen) using flow cytometry. Cells were grown in T cell growth medium in larger flasks or G-Rex vessels (Wilson Wolf) as needed. On day 14, DLL3 CAR-T cells were cryopreserved. The percentage of cells stained with recombinant DLL3 was normalized across clones immediately prior to cryopreservation.

[0261] To determine the percentage of T cells successfully transduced with DLL3 CAR, T cells were first incubated with 1 μg / ml of Flag-tagged recombinant DLL3 (Adipogen) in PBS + 1% BSA at 4°C for 20 minutes. Cells were then washed with PBS + 1% BSA and stained with a PE-labeled anti-Flag antibody (Biolegend, catalog number 637310) and analyzed using flow cytometry.

[0262] An example of DLL3 CAR-T cells is shown in Figure 3B. Figure 3B shows experimental data demonstrating that the anti-DLL3 CAR is expressed on the surface of primary T cells and can recognize recombinant DLL3. The plots are gated on live CD3+ cells. The numbers on the plots are the percentages of cells expressing each anti-DLL3 CAR.

[0263] Example 6: In Vitro Characterization This example describes the experiments used to determine the specificity and in vitro activity of the CAR for DLL3.

[0264] SHP-77, WM266.4, DMS 454, and DMS 273 are DLL3+ cell lines purchased from ATCC or Sigma. HEK-293T is a DLL3-negative cell line. To express human DLL3 in HEK-293T, HEK-293T cells were transduced with a lentivirus encoding full-length human DLL3.

[0265] To test for DLL3-specific killing, HEK-293T cells expressing firefly luciferase with or without human DLL3 expression were then seeded in a 96-well assay plate (Costar) at a seeding density of 5,000 cells per well. DLL3 CAR-T cells were thawed and added to seeded HEK-293T with or without human DLL3 expression at an effector:target (E:T) ratio ranging from 1:9 to 9:1 in T cell growth medium, namely X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio). Cell viability was measured after 72 hours using the one-glo assay kit (Promega). Representative DLL3 CAR-T cells showed strong killing against HEK-293T-DLL3 cells but no detectable activity against HEK-293T parental cells (Figure 4A).

[0266] To test the cytotoxic activity of DLL3 CAR-T cells against cell lines expressing endogenous DLL3, DLL3 CAR-T cells were incubated with firefly luciferase-labeled DLL3+SHP-77, WM266.4, DMS 454, or DMS 273 cells at an effector:target (E:T) ratio ranging from 1:9 to 9:1 in T cell growth medium, namely X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio). Cell viability was measured after 72 hours using the one-glo assay kit (Promega). Each condition was assayed in triplicate. Mean percentages and standard deviations of viable cells were plotted (Figure 4B and Figure 4C).

[0267] Figure 4A shows experimental data indicating that anti-DLL3 CAR-T cells specifically killed HEK-293T cells expressing human DLL3 but not parental HEK-293T cells in a 3-day cytotoxicity assay at the indicated effector:target ratios. T cells not expressing the anti-DLL3 CAR (labeled empty vector) were used as a negative control.

[0268] Figure 4B shows experimental data indicating that anti-DLL3 CAR-T cells killed SHP-77 and WM266.4 cells expressing endogenous DLL3 in a 3-day cytotoxicity assay at the indicated effector:target ratios.

[0269] Figure 4C shows experimental data indicating that anti-DLL3 CAR-T cells killed DMS 454 and DMS 273 small cell lung cancer cells expressing endogenous DLL3 in a 3-day cytotoxicity assay at the indicated effector:target ratios. For all plots in Figure 4C, target cell viability was evaluated using the One-glo assay system (n = 3).

[0270] To measure cytokines secreted from DLL3 CAR-T cells, DLL3 CAR-T cells were incubated with DLL3+SHP-77 cells at an effector:target (E:T) ratio of 1:1 or 1:9 in T cell growth medium, namely X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio). After 24 hours, tissue culture supernatants were collected and levels of three cytokines [interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-2] in the supernatants were measured using the human inflammatory panel 9 plex assay (MSD) according to the manufacturer's protocol. Figure 5 shows that anti-DLL3 CAR-T cells released cytokines after co-incubation with the DLL3-expressing SHP-77 cell line. CAR-T cells and SHP-77 cells were incubated at an effector:target ratio of 1:1 or 1:9 for 24 hours (n = 3).

[0271] Example 7: Serial killing assay The serial killing assay involves growing CAR-T cells and, in certain cases, repeated exposure of the CAR-T cells to their targets, which causes differentiation and exhaustion. This assay was used to select optimal clones with high target cell lysis and proliferation capabilities after several rounds of exposure to target cells.

[0272] On the first day of the assay, 5,000 firefly luciferase-labeled WM266.4, DMS 454, or DMS 273 cells known to express DLL3 were seeded in 100 μl of X-Vivo-15 medium with 5% human serum in a 96-well plate with white walls and a flat transparent bottom. After attaching the target cells to the bottom of the plate, the DLL3 CAR-T cells were thawed and added to the seeded target cells at a 1:1 effector:target (E:T) ratio in X-VIVO medium containing 5% human serum. Every two days thereafter, 100 μl of the medium containing the DLL3 CAR-T cells was transferred to freshly seeded target cells, and the percentage of lysis of the pre-seeded target cells was measured using the one-glo assay system or the CellTiter-glo system (Promega). Each condition was assayed in 3 - 6 replicates. The mean percentage and standard deviation of lysis were plotted (Figures 6A - 6C). The optimal clone had the highest target cell lysis throughout the assay on day 12. These data show the experimental data of the continuous killing assay, indicating that some of the clones remained active after repeated exposure of anti-DLL3 CAR-T cells to DLL3+WM266.4 cells. The One-glo assay system or CellTiter-glo was used at each indicated time point to evaluate target cell survival (n = 3 - 6).

[0273] Example 8: In Vivo Activity To test the antitumor activity of DLL3 CAR-T cells, SHP-77 tumor-bearing NSG mice were used. SHP-77 cells were obtained from a frozen stock vial, thawed, and counted according to standard procedures. The cells were diluted to 50 × 10 6 viable cells / mL in complete growth medium (RPMI + 10% FBS). The cell suspension was kept on ice until transplantation. Immediately after transplantation, the cells were mixed 1:1 with BD Matrigel Matrix (catalog number 354234) and 5 x 10 6200 μL of a cell / matrigel suspension containing the indicated number of SHP-77 cells was injected subcutaneously. Tumor growth was monitored by caliper measurements starting on day 5 after implantation. Tumor size was calculated using the formula tumor volume = (width^2 × length / 2). Mice were randomized into 5 groups based on tumor volume approximately 2 weeks after implantation. The average tumor volume per group was 314 mm 3 was as follows. One day after randomization, non-transduced T cells and DLL3 CAR-T cells were thawed and counted according to standard procedures. The cells were resuspended in RPMI + 10% FBS and injected by tail vein IV injection at a volume of 200 μL per mouse with 2 million or 5 million CAR+ cells per mouse. Tumors were continuously monitored every 3 - 4 days until the end of the study. 26C8 and 10G1-K DLL3 CAR-T cells induced tumor inhibition in a dose-dependent manner (Figures 7A - 7B). Figures 7A - 7B show experimental data indicating that anti-DLL3 CAR-T cells can eliminate established small cell lung cancer tumors.

[0274] To test the anti-tumor activity of DLL3 CAR-T cells in a model showing metastases similar to human disease, SHP-77 tumors were established by tail vein injection. Tumors were observed in the lung, liver, brain, kidney, and spleen. Specifically, SHP-77 cells were thawed and 40 x 10 6It was diluted to [[number of viable cells]] cells / mL. The cell suspension was kept on ice until transplantation, and 200 μL of the cell suspension per mouse was injected via the tail vein IV. On the 7th day after transplantation, 200 μL of luciferin (15 mg / mL) was injected, and tumor growth was monitored by IVIS imaging. On the 11th day after transplantation, the mice were randomized into groups of 5 based on the total light flux. On the 12th day after transplantation, the CAR-Ts were thawed and counted according to standard procedures. The cells were resuspended in RPMI + 10% FBS and injected via the tail vein IV at a volume of 200 μL per mouse with [[number of CAR+ cells]] or [[number of CAR+ cells]] CAR+ cells per mouse. The tumors were continuously monitored every 3 - 4 days until the end of the study. As shown in Figure 8, the 10G1-K anti-DLL3 CAR-T cells can inhibit small cell lung cancer tumors established in mice in a dose-dependent manner.

[0275] Example 9: Anti-DLL3 CAR Constructs with a Safety Switch This example describes the construction, expression, and cytotoxic activity of anti-DLL3 CARs with a safety switch. The anti-DLL3 CARs in Table 6 were reformatted to include the different safety switch structures listed below (Table 8). [Table 14]

[0276] The protein sequences encoding anti-DLL3 CAR constructs containing a safety switch are shown in Table 9. Exemplary safety switch constructs can include the CD8α signal sequence (SEQ ID NO: 477), the anti-DLL3 scFv described herein, the CD20 mimotope (SEQ ID NO: 536), the QBEND-10 epitope (SEQ ID NO: 544), the hinge and transmembrane regions of the human CD8α molecule (SEQ ID NO: 479), the cytoplasmic portion of the 4-1BB molecule (SEQ ID NO: 291), and the cytoplasmic portion of the CD3ζ molecule (SEQ ID NO: 292). [Table 15-1] [Table 15-2]

Table 15-3

Table 15-4

Table 15-5

Table 15-6

[0277] CAR-Ts were generated using the method described in Example 5, and their cytotoxic activities were investigated using the method described in Example 7. Figure 9A is a plot showing the structures of four different safety switches. Figure 9B shows experimental flow cytometry data indicating that anti-DLL3 CARs 2G1, 4H8, and 10G1-K with safety switches are expressed on the surface of primary T cells and can recognize recombinant DLL3. The plots were gated on live CD3+ cells, and the numbers on the plots are the percentages of cells expressing each anti-DLL3 CAR. Figure 9C shows experimental data indicating that anti-DLL3 CARs with safety switches are active in a continuous killing assay of the DLL3+ WM266.4 cell line.

[0278] Example 10: Cytotoxicity against Small Cell Lung Cancer PDX Models Small cell lung cancer PDX models were purchased from Crown Bioscience. To investigate the DLL3 expression on the cell surface, frozen vials of the PDX models were thawed, and 200,000 cells were used for each staining sample. The expression of DLL3 was verified by a FACS assay using a PE-conjugated anti-DLL3 antibody. Brilliant violet 421-conjugated anti-human CD45 and anti-mouse CD45 antibodies were added to the same staining samples to exclude human and mouse lymphocytes.

[0279] Figure 10A shows experimental data indicating that DLL3 is expressed on the surface of two small cell lung cancer PDX models. Figure 10B shows experimental data indicating that anti-DLL3 CAR-T cells killed the same two small cell lung PDX models in a 3-day cytotoxicity assay at the indicated effector:target ratios. T cells that did not express the anti-DLL3 CAR (labeled empty vector) were used as a negative control.

[0280] Example 11: In Vitro Detection and Depletion of DLL3 CAR-T Cells Using a Rituximab-Based Safety Switch To deplete or turn off CAR T cells in the event of unwanted activity, a rituximab off-switch was developed by inserting rituximab mimotopes at various positions in the extracellular region of the CAR described in Example 9. A complement-dependent cytotoxicity assay was used to evaluate rituximab-dependent in vitro depletion of DLL3 CAR-T cells. In this assay, cryopreserved CAR-T cells were thawed and 1x10 5 cells were incubated in RPMI 1640 medium supplemented with 10% FBS in a 96-well plate. Cells were incubated for 3 hours in the absence or presence of 25% guinea pig complement (Cedarlane, CL3441-S) and rituximab antibody (manufactured in-house, 100 mg / mL). Cells were stained with recombinant DLL3 (Adipogen) and cytotoxicity was analyzed by flow cytometry. Figure 11A shows experimental data indicating that anti-DLL3 CAR-T cells can be detected by both recombinant DLL3 and rituximab staining. Figure 11B shows experimental data indicating that DLL3 CAR-T cells were depleted in vitro in a rituximab-dependent and complement-dependent manner.

[0281] Example 12: In Vivo Activity of Anti-DLL3 CAR-T Cells with a Safety Switch To test the anti-tumor activity of DLL3 CAR-T cells with a safety switch, SHP-77 tumor-bearing NSG mice were used. SHP-77 cells were thawed from cryovials, counted, and diluted. 50×10 6 viable cells / mL in RPMI medium / matrigel suspension per mouse were injected subcutaneously. Tumor growth was monitored by caliper measurements starting on day 5 after transplantation. Tumor size was calculated using the formula tumor volume = (width^2 × length / 2). Mice were randomized into 8 groups based on tumor volume at approximately 14 days after transplantation. The average tumor volume per group was 178 mm 3 . On the same day the mice were randomized, non-transduced T cells and DLL3 CAR-T cells were thawed and counted according to standard procedures. The cells were resuspended in RPMI and injected via tail vein IV at a volume of 200 μL per mouse with 5×10 6 CAR+ cells per mouse. Tumors were monitored every 3 - 4 days until the end of the study. All groups of DLL3 CAR-T cells with a safety switch induced significant tumor inhibition and complete or nearly complete elimination of detectable tumors by day 50 (Figure 12A).

[0282] To test the anti-tumor activity of DLL3 CAR-T cells in a model showing metastasis like human disease, DMS 273 small cell lung tumors expressing exogenous DLL3 (DMS 273-DLL3) were established by tail vein injection. Specifically, DMS 273-DLL3 cells were thawed and diluted to 5×10 5 viable cells per mL in RPMI medium. 200 μL of the cell suspension per mouse was injected via tail vein IV. On day 3 after transplantation, the mice were randomized into 9 groups. On the same day, DLL3 CAR-T was thawed, counted, and 5x10 6The cells were resuspended in RPMI medium and injected into the tail vein of mice at a volume of 200 μL per mouse via intravenous injection with individual CAR+ cells. Tumors were continuously monitored every 3 - 4 days using an IVIS imaging system until the end of the study. As shown in Figure 12B, multiple different DLL3 CARs with different rituximab-based safety switches were effective against metastatic tumors.

[0283] Example 13: Mouse safety study using non-tumor-bearing animals DLL3 RNA has been reported in the human brain and pituitary gland (GTex). Similarly, mouse DLL3 RNA has also been reported in the pituitary gland (Bio-GPS). To understand DLL3 RNA expression in the mouse brain, brains from 3 NSG mice were fixed in 10% neutral buffered formalin (NBF), embedded, sectioned continuously at 4 - 6 microns, and analyzed by RNAscope® LS Red ISH assay (ACD Bio). DLL3 RNA was detected at low levels in brain samples of NSG mice. Figure 13A shows a representative image of mouse DLL3 RNA staining observed in this assay.

[0284] To understand the potential toxicity liability of DLL3 RNA expression in non-transduced T cells of the brain and pituitary gland, 8x10 6 individual 10G1-K DLL3 CAR-T cells, or 8x10 6 individual 2G1 DLL3 CAR-T cells were injected intravenously into NSG mice. Seven days after injection, the spleen, brain, and pituitary gland were harvested, fixed in 10% NBF, embedded, sectioned continuously at 4 - 6 microns, and stained with an anti-human CD3 antibody (Abcam, ab52959, diluted 1:500) to detect human T cells by immunohistochemistry. T cells were detected in the spleen from all animals, but they were not detected in brain or pituitary gland samples (Figure 13B). Thus, DLL3 RNA was detected at low levels in non-tumor-bearing NSG mice, but DLL3 CAR-T cells were not detected in mouse brain or pituitary gland samples.

[0285] Example 14: Mouse safety study using animals bearing subcutaneous tumors To further evaluate the potential for brain and pituitary toxicity liability, DLL3 CAR-T cells were injected into NSG mice bearing subcutaneous LN229 tumors expressing exogenous murine DLL3 (LN229-mDLL3). In this model, activation of CAR-T by tumor cells could potentially lead to increased sensitivity and activity against normal tissues that potentially express DLL3. The experimental design is shown in Figure 14A. Three days prior to tumor implantation (-3 days), adeno-associated virus (AAV) (Vigene Biosciences) encoding IL-7 and IL-15 was injected via the tail vein to support CAR-T cell proliferation and persistence. LN229-mDLL3 cells were then thawed from frozen vials and diluted to 4.25×10 7 cells / mL in complete growth medium (RPMI + 10% FBS). The cell suspension was kept on ice until transplantation. Immediately prior to transplantation, the cells were mixed 1:1 with BD Matrigel matrix (catalog number 354234), and 200 μL of cell / matrix suspension containing 4.25x10 6 LN229-mDLL3 cells per mouse were injected subcutaneously. Tumor growth was monitored by caliper measurements starting on day 8 post-transplantation. Tumor size was calculated using the formula tumor volume = (width^2 × length / 2). On day 22 post-transplantation, mice were randomized into five groups based on tumor volume and serum concentrations of IL-7 and IL-15. On the same day (day 22), non-transduced T cells and murine cross-reactive 10G1-K DLL3 CAR-T cells were thawed, resuspended in RPMI + 10% FBS, and injected by tail vein IV injection at a volume of 200 μL per mouse with 1x10 7 CAR+ cells per mouse. Tumors were monitored every 3 - 4 days until the end of the study, and robust anti-tumor activity was observed with DLL3 CAR T treatment (Figure 14B).

[0286] On day 49, when the animals that received DLL3 CAR-T cells had no tumors, brain tissues from the animals were fixed in 10% NBF, embedded, the ventricular system including the third and fourth lateral ventricles was revealed, three sections were placed in a single block, and it was serially sectioned at 4 - 6 microns and stained with hematoxylin and eosin (H&E), or stained by immunohistochemistry to detect human-specific CD3 (hCD3). The pituitary gland was fixed in 10% NBF, processed, and stained with H&E or immunohistochemically stained to show hCD3. The H&E slides were examined under a microscope, and the histopathological findings were scored by a pathologist using a standard system. Administration of DLL3 CAR-T cells resulted in abundant hCD3-stained T cells in the intermediate and neural lobes of the pituitary gland and relatively fewer T cells in the anterior lobe (Figure 14C and data not shown). Sparsely to moderately low or moderately to moderately high hCD3 staining of T cells was present in the neural network and vascular system of the brain (as circulating T cells) (Figure 14C and data not shown). No other pituitary or brain findings were present (Figure 14C - D). To understand the functional consequences of T cell infiltration, two hormones released in the neural lobe, vasopressin and oxytocin, were stained using immunohistochemistry. For vasopressin detection, samples were stained with an anti-vasopressin antibody (ImmunoStar, 20069) at 1 / 7,000 dilution for 1 hour at room temperature and then with Rabbit-on-Rodent HRP-Polymer (Biocare Medical) for 30 minutes at room temperature. For oxytocin detection, samples were stained with an anti-oxytocin antibody (ImmunoStar, 20068) at 1 / 10,000 dilution for 15 minutes at room temperature and then with Rabbit-on-Rodent HRP-Polymer (Biocare Medical) for 30 minutes at room temperature. Both hormones could be detected in the neural lobe of the pituitary gland of animals that received non-transduced T cells or DLL3 CAR-T cells, indicating that the hormone-producing neurons in this region remained functional (Figure 14E - F). Thus, based on pathological evaluation and hormone staining, no tissue damage was seen in the samples.

[0287] Example 15: Mouse Safety Study Using Animals Bearing Intracranial Tumors To promote T cell infiltration into the brain and further understand potential brain toxicity, NSG mice with intracranial LN229 tumors expressing exogenous mouse DLL3 and human EGFRvIII (LN229-mDLL3-vIII) were used. The experimental design is shown in Figure 15A. LN229-mDLL3 cells were thawed from frozen vials and diluted in RPMI to 1×10 7 viable cells / mL. Then, a cell suspension containing 3 μL of 3×10 4 cells of LN229-mDLL3 per mouse was injected intracranially. Tumor growth was monitored by an IVIS imaging system. On day 17 post-implantation, mice were randomized into 10-animal groups based on tumor volume. On the same day (day 17), TCR knockout non-transduced T cells, 10G1-K DLL3 CAR-T cells, and EGFRvIII CAR-T cells were thawed, resuspended in RPMI, and injected by tail vein IV injection at a volume of 200 μL / mouse at 1x10 7 CAR+ cells / mouse. EGFRvIII CAR-T cells were included as a control to evaluate potential inflammation caused by tumor lysis in the brain. To support the proliferation and persistence of CAR-T cells, 0.5 μg of IL-15 (Peprotech AF-200-15) and 3 μg of IL-15Ra Fc fusion protein (R&D Systems 7194-IR) were administered to each animal twice a week starting on day 17 until the end of the study. Tumors were continuously monitored every 3 - 4 days until the end of the study, and clear anti-tumor activity was observed (Figure 15B). On days 22 and 38, brain tissues from all animals were trimmed, processed, embedded, the ventricular system including the third and fourth lateral ventricles was revealed, three sections were placed in a single block, and it was sectioned continuously at 4 - 6 microns and stained with H&E or stained by immunohistochemistry to detect human-specific CD45 (hCD45). Pituitary tissue was processed to include the neural lobe, intermediate lobe, and anterior lobe and stained with H&E or immunohistochemically stained to detect hCD45 as a marker for human T cells.

[0288] On day 22, animals that received non-transduced T cells or 10G1-K DLL3 CAR-T cells had rare / sparse hCD45-stained T cells in the brain or pituitary gland (data not shown). In contrast, for animals treated with EGFRvIII CAR-T cells, hCD45+ staining was in the range of rare / sparse to moderately low or moderately to moderately high in the areas of infiltration / glialosis or glioma, consistent with the anti-tumor activity of this group (data not shown). On day 38, animals that received non-transduced T cells or EGFRvIII CAR-T cells had rare / sparse hCD45+ staining in the brain and pituitary gland. Animals that received 10G1-K DLL3 CAR-T cells had minimal or mild mononuclear cell infiltration in the pituitary gland, mainly in the intermediate and neural lobes (Figure 15C-D). Also, these animals had slightly more (moderately low) hCD45+ staining associated with small glioma lesions compared to the rare / sparse staining in other areas of the brain (brain vasculature, choroid plexus, and meninges), consistent with the anti-tumor activity of this group shown in Figure 15B.

[0289] Example 16: In Vitro Cytotoxicity of Dissociated Mouse Pituitary Cells To directly test whether DLL3 CART is active against the pituitary gland, mouse pituitary glands from NSG mice were harvested under sterile conditions for in vitro analysis. The tissue was dissociated by incubation at 37 °C for 3 rounds in 1 mL of dissociation mix [5 mL of DMEM, high glucose, GlutaMax (Gibco, catalog number 10564), 50 uL of enzyme H, 5 uL of enzyme R, 6.25 uL of enzyme A (Miltenyi tumor dissociation kit number 130 - 095 - 929)], followed by mechanical dissociation using homogenization. Single cells were transferred to complete medium (DMEM, high glucose, GlutaMax, 20%, 1X insulin - transferrin - selenium solution, 1X MEM non - essential amino acids, 1X penicillin - streptomycin) and pooled after each round. The cells were pelleted, treated with ACK lysis buffer for 3 minutes at RT, and then neutralized in complete medium. The cell suspension was filtered through a 70u filter and centrifuged to remove the buffer. The cells were counted and seeded at 5x10 4 cells per well in a 96 - well plate and allowed to recover for 3 days before the addition of CAR - T cells. At the time of addition of CAR - T cells, the expected target density was 1×10 4 cells per well. For the control, DLL3 + cells (DMS - 273) and DLL3 - cells (293T) were seeded at the same density. 10G1 - K and 2G1 DLL3 CAR - T cells were added at E:T = 9:1, 3:1, and 1:1 and co - cultured with the target for 3 days. At the end of the 3 - day co - culture, the medium was separated from the wells and centrifuged to pellet the T cells. The target cells were treated with 50 uL / well of Cell Titer Glo (Promega, G7570) for 10 minutes and analyzed for cytotoxicity readings using a SpectraMax plate reader.

[0290] Figure 16A shows experimental data indicating that DLL3 CAR-T cells are active against the DLL3+ DMS 273 cell line, but they are not cytotoxic to mouse pituitary cells in vitro. T cells were pooled and stained for activation markers (41BB and CD25) for flow cytometry analysis. Figure 16B shows that mouse pituitary cells do not activate DLL3 CAR-T cells in vitro. The supernatant was frozen at -80 °C and then thawed for cytokine analysis using the Human TH1 / TH2 10-Plex Tissue Culture Kit (Meso Scale Discovery, K15010B). Figure 16C shows that both 10G1-K and 2G1 DLL3 CAR-T cells secrete interferon-gamma (IFNγ), tumor necrosis factor alpha (TNF-α), and IL-2 when co-cultured with the DLL3+ DMS 273 cell line, but there is no cytokine secretion after co-culturing DLL3 CAR-T cells with mouse pituitary cells. Thus, DLL3 CAR-T cells were not cytotoxic to pituitary cells in vitro.

Claims

1. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain a variable heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 109, a variable heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110, a variable heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 111, a variable light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 112, a variable light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 113, and a variable light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 114, A chimeric antigen receptor comprising the same.

2. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 115, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 116, comprising the same, A chimeric antigen receptor, wherein the variable heavy chain and the variable light chain are linked by at least one linker.

3. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

117.

4. A chimeric antigen receptor that specifically binds to DLL3, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least about 90% identical to any one of SEQ ID NOs: 494 and 644, and the amino acid sequence a variable heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 109, a variable heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110, a variable heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 111, a variable light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 112, a variable light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 113, and a variable light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 114 A chimeric antigen receptor comprising the same.

5. The chimeric antigen receptor according to Claim 4, wherein the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs: 494 and 644.

6. The chimeric antigen receptor according to any one of claims 1 to 3, wherein the intracellular domain comprises at least one co-stimulatory domain. **Claim 7** The co-stimulatory domain is a signal transduction region that specifically binds to a ligand of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18)), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof, the chimeric antigen receptor according to claim 6.

8. The chimeric antigen receptor according to claim 6, wherein the co-stimulatory domain comprises the signaling region of 4-1BB / CD137.

9. The chimeric antigen receptor according to claim 8, wherein the 4-1BB / CD137 co-stimulatory domain comprises SEQ ID NO: 480 or a fragment thereof.

10. The chimeric antigen receptor according to any one of claims 1 to 3 and 6 to 9, wherein the intracellular domain comprises at least one activation domain.

11. The chimeric antigen receptor according to claim 10, wherein the activation domain comprises CD3.

12. The chimeric antigen receptor according to claim 11, wherein the CD3 comprises CD3 zeta.

13. The chimeric antigen receptor according to claim 12, wherein the CD3 zeta comprises SEQ ID NO: 481 or a fragment thereof.

14. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor is encoded by the polynucleotide sequence of SEQ ID NO:

582.

15. The chimeric antigen receptor according to any one of claims 1 to 14, further comprising a safety switch.

16. The chimeric antigen receptor according to claim 15, wherein the safety switch comprises a CD20 mimotope or a QBEND-10 epitope.

17. The chimeric antigen receptor according to claim 16, wherein the safety switch comprises one or more CD20 mimotopes or one or more QBEND-10 epitopes, or a combination thereof.

18. The chimeric antigen receptor according to any one of claims 15 to 17, wherein the chimeric antigen receptor comprises one or more safety switches in the format of QR3, SR2, RSR, or R2S.

19. The chimeric antigen receptor according to any one of claims 15 to 18, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least about 80% identical to any one of SEQ ID NOs: 622 to 625 and 684 to 687.

20. The chimeric antigen receptor according to any one of claims 15 to 18, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least about 90% identical to any one of SEQ ID NOs: 622 to 625 and 684 to 687.

21. The chimeric antigen receptor according to any one of claims 15 to 18, wherein the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs: 622 to 625 and 684 to 687.

22. An isolated polynucleotide encoding a chimeric antigen receptor according to any one of claims 1 to 21.

23. A vector comprising the polynucleotide according to claim 22.

24. The vector according to claim 23, wherein the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated vector, a lentiviral vector, or any combination thereof.

25. An engineered immune cell expressing a chimeric antigen receptor according to any one of claims 1 to 21.

26. An engineered immune cell expressing the polynucleotide according to claim 22 or the vector according to claim 23 or 24.

27. The engineered immune cell according to claim 25 or 26, wherein the immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), an NK cell, a TCR-expressing cell, a dendritic cell, or an NK-T cell.

28. The engineered immune cell according to claim 27, wherein the cell is an autologous T cell.

29. The engineered immune cell according to claim 27, wherein the cell is an allogeneic T cell.

30. A pharmaceutical composition comprising the engineered immune cell according to any one of claims 25 to 27.

31. The pharmaceutical composition according to claim 30, for treating a disease or disorder in a subject in need thereof.

32. The pharmaceutical composition according to claim 31, wherein the disease or disorder is cancer.

33. The pharmaceutical composition according to claim 31 or 32, wherein the disease or disorder is small cell lung cancer.

34. A product comprising the engineered immune cell according to any one of claims 25 to 29, or the pharmaceutical composition according to claim 30.

35. A variable heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 109, A variable heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 110, A variable heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 111, A variable light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 112, A variable light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 113, and A variable light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 114, An anti-DLL3 binding agent comprising, wherein the binding agent is an antibody, an antibody conjugate, or an antigen-binding fragment thereof, optionally F(ab') 2 fragment, Fab' fragment, Fab fragment, Fv fragment, scFv fragment, dsFv fragment, or dAb fragment, which may be an anti-DLL3 binding agent.

36. The anti-DLL3 binding agent according to claim 35, wherein the binding agent is a monoclonal antibody comprising an IgG constant region.

37. The anti-DLL3 binding agent according to any one of claims 35 to 36, comprising a VH sequence that is at least about 90% identical to the variable heavy chain (VH) sequence of SEQ ID NO:

115.

38. The anti-DLL3 binding agent according to any one of claims 35 to 37, comprising a VL sequence that is at least about 90% identical to the variable light chain (VL) sequence of SEQ ID NO:

116.

39. The anti-DLL3 binding agent according to any one of claims 35 to 38, wherein the binding agent comprises a sequence that is at least about 90% identical to the scFv sequence of SEQ ID NO:

117.

40. The anti-DLL3 binding agent according to any one of claims 35 to 39, wherein the binding agent comprises the scFv sequence of SEQ ID NO:

117.

41. The anti-DLL3 binding agent according to any one of claims 35 to 40, wherein the binding agent is a fusion protein comprising an scFv fragment fused to an Fc constant region.

42. A pharmaceutical composition comprising the anti-DLL3 binding agent according to any one of claims 35 to 41, and optionally comprising a pharmaceutically acceptable excipient.

43. The pharmaceutical composition according to claim 42, for treating a disease or disorder in a subject in need thereof.

44. The pharmaceutical composition according to claim 43, wherein the disease or disorder is cancer.

45. The pharmaceutical composition according to claim 43 or 44, wherein the disease or disorder is small cell lung cancer.

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