DLL3 targeting chimeric antigen receptors and binding agents

Abstract

To provide methods and composition for the treatment of cancer and in particular malignancies involving aberrant expression of DLL3.SOLUTION: Provided herein are DLL3 binding agents and chimeric antigen receptors (CARs) comprising a DLL3 binding molecule that specifically binds to DLL3, as well as immune cells comprising these DLL3-specific CARs, e.g., CAR-T cells, and pharmaceutical compositions comprising the immune cells.SELECTED DRAWING: None

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 March 1, 2019, and U.S. Provisional Patent Application No. 62 / 969,976, filed February 4, 2020, the entire contents of which are incorporated herein by reference in their entireties.

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

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

[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). Survival rates for SCLC have remained low for decades, primarily due to the lack of new therapies to combat this form of lung cancer. Conventional curative treatments for the cancer include chemotherapy and radiation therapy. Patients typically respond well to current first-line therapies, including etoposide and cisplatin, but invariably relapse quickly with chemotherapy-resistant disease. Prognosis in the relapsed and refractory setting is very poor, with rapid disease progression and a short median survival of less than six months. Thus, 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 to treat cancer (see, e.g., 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 chimeric antigen receptors (CARs), fusion proteins composed of DLL3 antigen recognition moieties and T cell activation domains (see, e.g., 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)). CAR-containing immune cells, e.g., 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, and functions cell-autonomously to inhibit Notch signal transduction, thus blocking cell-cell interaction and the internalization of Notch in target cells. Delta-like ligand 3 (DLL3) is a 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 carcinoma, carcinoid, dispersed neuroendocrine tumors in pancreas, bladder, and prostate, testicular cancer, and lung adenocarcinoma with neuroendocrine features. There is a need for the treatment of cancer, particularly malignant tumors with aberrant expression of DLL3. Methods and compositions that address this need are provided herein. Summary of the Invention

[0007] Provided herein is a chimeric antigen receptor (CAR) that comprises a DLL3 antigen binding domain that specifically binds to DLL3, and immune cells, such as CAR-T cells, that comprise these DLL3-specific CARs.Also provided are methods for producing and using these DLL3-specific CARs, and immune cells that comprise DLL3-specific CARs.The DLL-3-targeting CAR T cells described herein show good transduction efficiency, in vitro phenotype, and strong in vitro and in vivo antitumor activity.

[0008] In one aspect, the 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 is selected from the group consisting of (a) a chimeric antigen receptor having a 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, 340, 350, 360, 370, 380, 382, ​​390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810 (b) a variable heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 352, 361, 370, 379, 388, 397, 406, 415, 424, 433, 442, 451, and 460; and (c) a variable heavy chain CDR1 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 CDR2 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, 40 (d) a variable heavy chain CDR3 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,(e) a variable light chain CDR1 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 (f) 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.

[0009] In another aspect, the 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 is selected from the group consisting of (a) a chimeric antigen receptor having a 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, 23 (b) a variable heavy chain CDR1 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, 170, 180, 190, 201, 210, 220, 230, 240, 250, 260, 270, 280, 298, 307, 316, 325, 334, 343, 352, 361, 370, 379, 388, 397, 406, 415, 424, 433, 442, 451, and 460; 3, 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 CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 142, 144, and a variable heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of: 4, 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 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 is selected from the group consisting of (a) 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, 24 7, 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 CDR1 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 CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 15, 24, 33, 42, 51, 60, 69, 78, 80, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, and a variable light chain CDR3 comprising an amino acid sequence selected from the group consisting of: 7, 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 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 is selected from the group consisting of (a) 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 heavy 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, wherein 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 is selected from the group consisting of (a) SEQ ID NOs: 7, 16, 25, 34, 43, 52, 61, 70, 79, 88, 97, 106, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 700, 710, 720, 730, 740, 750, 760, 770, 780, 800, 810, 820, 830, 840, 850, 860, 870, 880, 900, 910, 24, 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 (b) a variable heavy 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, 27 and a variable light chain comprising an amino acid sequence selected from the group consisting of: 8, 287, 296, 305, 314, 323, 332, 341, 350, 359, 368, 377, 386, 395, 404, 413, 422, 431, 440, 449, 458, and 467, wherein the variable heavy chain and the variable light chain are linked by at least one linker.

[0013] In one aspect, the 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 scFvs presented in Table 1d.

[0014] In another aspect, the disclosure provides a chimeric antigen receptor that specifically binds to DLL3, wherein 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 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, wherein 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 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 costimulatory domain.

[0017] In some embodiments, the costimulatory domain of the chimeric antigen receptor is selected from the group consisting of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function associated antigen-1 (LFA-1 (CD1 la / 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), activating NK cell receptor, BTLA, Toll ligand receptor Body, 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, 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, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The signal transduction domains are AM, 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, and ligands that specifically bind to CD83, or any combination thereof.

[0018] In some embodiments, the costimulatory domain comprises the signaling region of CD28.

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

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

[0021] In some embodiments, the 4-1BB / CD137 costimulatory 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, the CD3 comprises CD3 zeta.

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

[0026] In some embodiments, the chimeric antigen receptor is encoded by the polynucleotide sequence of any one 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 QR3, SR2, RSR, or R2S format.

[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, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0036] In another aspect, the present disclosure provides engineered immune cells that express a chimeric antigen receptor described herein.

[0037] In some aspects, the present disclosure provides engineered immune cells that express 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 TCR knocked out (e.g., TCRα, TCRβ).

[0042] In one aspect, the present disclosure provides a pharmaceutical composition comprising an engineered immune cell that expresses 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 an engineered immune cell or an engineered immune cell 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 disclosure provides articles of manufacture including engineered immune cells or pharmaceutical compositions comprising engineered immune cells expressing a chimeric antigen receptor described herein.

[0047] In some aspects, the disclosure provides an anti-DLL3 binding agent as 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, an scFv fragment, a dsFv fragment, or a dAb fragment.

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

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

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

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

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

[0054] In some aspects, the present disclosure provides a pharmaceutical composition comprising an anti-DLL3 binding agent disclosed herein and a pharma- ceutically acceptable excipient.

[0055] In some aspects, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an anti-DLL3 binding agent, or a pharmaceutical composition comprising 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] [Figure 1A-1B] 1 is a series of plots showing that purified anti-DLL3 antibodies described herein bind to three DLL3-expressing small cell lung cancer cell lines (SHP-77, DMS 273, and DMS 454). Solid and dashed lines represent staining with anti-DLL3 antibodies or mouse IgG2A isotype control antibody, respectively. [Figures 2A-2D] The results of epitope mapping experiments are shown. Figure 2A is a schematic diagram of full-length and truncated human DLL3 proteins expressed on CHO cells for epitope mapping, and all proteins were fused with an HA tag at the N-terminus for easy detection. Figures 2B and 2C show the amino acid sequences of full-length and truncated human DLL3 proteins shown in Figure 2A. Figure 2D is a series of plots showing the results of epitope mapping of anti-DLL3 antibodies, as well as examples of anti-DLL3 antibodies that recognize the DSL, EGF1, and EGF3 domains, respectively, where the x-axis shows the signal from the PE channel and the y-axis shows the counts. [Figure 3A-3D] A series of plots and tables showing the structure, transduction efficiency of cells from two different donors, and cytotoxic activity of anti-DLL3 CAR. Figure 3A is a schematic diagram of a construct encoding an anti-DLL3 CAR, which includes, from N-terminus to C-terminus, an anti-DLL3 scFv, a hinge and transmembrane region from human CD8α, a cytoplasmic region from human 41BB, and a cytoplasmic region from CD3ζ. Figure 3B shows experimental data showing that anti-DLL3 CAR can be expressed on the surface of primary T cells and recognize recombinant DLL3, with the plots gated on live CD3+ cells, and the numbers on the plots are the percentage of cells expressing each anti-DLL3 CAR. Figures 3C and 3D show the transduction efficiency of anti-DLL3 CARs that include the scFv sequences described herein. [Figure 4A-4C]A series of plots showing the killing data of several anti-DLL3 CARs. Figure 4A shows experimental data showing 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 (E:T) ratio. T cells that did not express anti-DLL3 CAR (labeled "empty vector") were used as a negative control. Figure 4B shows experimental data showing 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 ratio. Figure 4C shows experimental data showing 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 ratio. For all plots in Figures 4A-4C, target cell viability was assessed using the One-glo assay system (n=3). [Diagram 5] A series of bar graphs showing that anti-DLL3 CAR-T cells released cytokines after co-incubation with the DLL3-expressing SHP-77 cell line when CAR-T cells and SHP-77 cells were incubated for 24 hours at an effector:target ratio of 1:1 or 1:9. Supernatants were collected and IFN-γ, IL-2, and TNF-α levels were measured using the proinflammatory 9-plex kit from MSD (n=3). [Figure 6A-6C]Plots showing experimental data of serial killing assays after repeated exposure of anti-DLL3 CAR-T cells to DLL3+ cell lines. Figure 6A shows serial killing of anti-DLL3 CAR-T cells against DLL3+WM266.4 cells. Some of the clones remained active on day 12 of the assay. Figure 6B shows serial killing of anti-DLL3 CAR-T cells against DMS 454 and WM266.4 cells. Figure 6C shows serial killing of anti-DLL3 CAR-T against DMS 273 small cell lung cancer line. For all plots in Figures 6A-6C, target cell viability was assessed (n=3-5) using the one-glo assay or the CellTiter-glo system. [Figure 7A-7B] 1 is a plot showing that anti-DLL3 CAR-T cells dose-dependently eliminated established SHP-77 small cell lung cancer subcutaneous tumors in mice. [Figure 8] Plot showing that 10G1-K anti-DLL3 CAR-T cells dose-dependently inhibited the growth of established IV-injected SHP-77 small cell lung cancer tumors. Statistical analysis was performed using ANOVA with repeated measures (Dunnett's multiple comparisons), days 14-28, n=4-5. *, p<0.05. **, p<0.01. [Figure 9A-9C] Figure 9 shows the structure of primary T cells, transduction efficiency, and cytotoxic activity of anti-DLL3 CARs with safety switches. Figure 9A is a schematic diagram showing the structure of CAR designs with four different safety switches (QR3, SR2, RSR, and R2S). Figure 9B shows flow cytometry plots showing that anti-DLL3 CARs with safety switches shown in Figure 9A can be expressed on the surface of primary T cells and recognize recombinant DLL3. The plots are gated on live CD3+ cells, and the numbers on the plots indicate the percentage of cells expressing each anti-DLL3 CAR. Figure 9C shows experimental data showing that anti-DLL3 CARs with safety switches are active in sequential killing assays. [Figure 10A]Figure 1 shows plots demonstrating that two small cell lung cancer patient-derived xenograft (PDX) models express DLL3 on the cell surface. Solid and dashed lines represent staining with anti-DLL3 antibody or fluorescence minus one (FMO), respectively. [Figure 10B] Experimental data showing that anti-DLL3 CAR-T exhibits cytotoxic activity against the same two PDX models. [Figure 11A-11B] Figure 11A shows that the safety switch allows for the detection and depletion of DLL3 CAR-T cells by rituximab. Figure 11A shows that 8E11-SR2 and 26C8-R2S anti-DLL3 CAR-T cells were stained with recombinant DLL3 and PE-conjugated rituximab after 14 days of expansion and analyzed using flow cytometry. Numbers in the quadrants represent the percentage of total T cells. Figure 11B shows rituximab-mediated complement-dependent cytotoxicity (CDC) of 8E11-SR2 and 26C8-R2S anti-DLL3 CAR-T cells. CAR-T cells were incubated with 25% baby rabbit complement and rituximab for 3 hours, and cytotoxicity was evaluated using flow cytometry. [Figure 12A-12B] 12A shows plots showing that anti-DLL3 CAR-T cells with safety switches inhibited the growth of subcutaneously or IV-injected small cell lung cancer tumors. FIG. 12A shows that DLL3 CAR-T cells with safety switches eliminated established SHP-77 small cell lung cancer subcutaneous tumors in mice. FIG. 12B shows plots showing that anti-DLL3 CAR-T cells inhibited the growth of IV-injected DMS 273-DLL3 small cell lung cancer tumors. [Figure 13A] A representative image of mouse DLL3 RNA expression in the brain of NSG mice is shown, with circles indicating clusters of mouse DLL3 RNA. [Figure 13B] Anti-human CD3 staining of spleen, pituitary, and brain samples from animals administered untransduced T cells, 10G1-K DLL3 CAR-T cells, or 2G1 DLL3 CAR-T cells is shown. Arrows indicate CD3 positive cells in the spleen. [Fig. 14A-14F]The experimental design and results of a mouse safety study using a subcutaneous LN229-mDLL3 tumor model are shown. Figure 14A shows the study arms and experimental design. Figure 14B shows the timing of tissue harvest and tumor volume for animals receiving either non-transduced T cells or DLL3 CAR-T cells. Figure 14C is a table showing human CD3 staining scores for brain and pituitary samples. Figure 14D is a table showing histological analysis of harvested brain and pituitary samples. Figure 14E shows images of pituitary samples stained with anti-vasopressin antibodies. Figure 14F shows images of pituitary samples stained with anti-oxytocin antibodies. [Figure 15A-15D] Figure 15 shows the experimental design and results of a mouse safety study using intracranial LN229-mDLL3 tumor model. Figure 15A shows the study groups and experimental design. Figure 15B shows the timing of tissue collection and tumor volume of animals receiving non-transduced T cells, DLL3 CAR-T cells or EGFRvIII CAR-T cells. Figure 15C shows the human CD45 staining scores of brain, pituitary and spleen samples. Figure 15D shows the histological analysis of brain and pituitary samples. [Figures 16A-16C] Figure 16 shows experimental data of in vitro cytotoxicity of dissociated mouse pituitary cells. Figure 16A shows the cytotoxicity readout of target cells after 3 days of co-culture with DLL3 CAR-T cells, showing that DLL3 CART is not cytotoxic to mouse pituitary cells in vitro. Figure 16B shows flow cytometry analysis of surface staining for activation markers CD25 and 41BB of T cells co-cultured with targets, showing that mouse pituitary cells do not activate DLL3 CAR-T in vitro. Figure 16C shows cytokines secreted in cell culture medium, showing that no cytokines are secreted after 3 days of co-culture of DLL3 CAR-T cells with mouse pituitary cells in vitro, analyzed by MSD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Provided herein are DLL3-specific antibodies and chimeric antigen receptors (CARs).The DLL-3-specific CARs described herein comprise an extracellular domain, a transmembrane domain, and an extracellular domain, and the extracellular domain comprises a DLL3 antigen binding domain that specifically binds to DLL3, and a polynucleotide that encodes these CARs.Also provided are immune cells, such as CAR-T cells, that comprise these DLL3-specific CARs, and pharmaceutical compositions that comprise these immune cells.Methods of making and using these DLL3-specific CARs and immune cells that comprise these DLL3-specific CARs are also disclosed, for example, for the treatment of cancer.

[0060] I. DLL-3 BINDING AGENTS 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 that comprises sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen (e.g., DLL-3). As known in the art, intact antibodies produced in nature are tetrameric agents of about 150 kD composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other in what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CHI, CH2, and carboxy-terminal CH3 (located at the base of the stem of the Y). A short region, known as a "switch", connects the heavy chain variable and constant regions. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in the intact antibody. Each light chain is composed of two domains, an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch". Those skilled in the art are familiar with antibody structure and sequence elements and will recognize the "variable" and "constant" regions in the provided sequences and will understand that there may be some flexibility in the definition of the "boundary" between such domains, and that different presentations of the same antibody chain sequence may exhibit such boundaries at positions that are, for example, 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 is typically based on 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. Pat. No. 7,709,226), contact numbering, or the AbM scheme (Antibody The numbering scheme follows that of the Oxford Molecular Modeling program.

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

[0063] An intact antibody tetramer is composed of two heavy-light chain dimers in which the heavy and light chains are linked together by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions together, so that the dimers are connected to each other and form a tetramer. Naturally produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., three-, four-, or five-stranded sheets) packed together in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complement determining regions" (CDR1, CDR2, and CDR3), and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR regions form a beta sheet that provides 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 where they are located at the tip of a 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 and / or other binding attributes of the Fc region for the Fc receptor can be modulated by glycosylation or other modifications. In some embodiments, the antibodies produced and / or utilized according to the present invention comprise a glycosylated Fc domain, including Fc domains with such glycosylation that have been modified or engineered.

[0064] For purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that contains sufficient immunoglobulin domain sequences as found in a natural antibody may be referred to and / or used as an "antibody," whether such a polypeptide is produced naturally (e.g., produced by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal, and in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences 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] Moreover, the term "antibody" as used herein, in appropriate embodiments (unless otherwise stated or clear from the context), can refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in some embodiments, antibodies utilized in accordance with the present invention may be intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; 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®); VHHs; Anticalins®; Nanobodies® minibodies; BiTEs®; ankyrin repeat proteins or The formats are selected from, but are not limited to, DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody may lack a covalent modification (e.g., glycan attachment) that it would have if it were produced naturally. In some embodiments, the antibody may contain a covalent modification (e.g., glycan attachment, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.).

[0066] Antibodies include antibody fragments. Antibodies may be polyclonal, monoclonal, chimeric dAb (domain antibody), single chain, F ab, F a , F (ab)2 Fragments, scFv, and F ab This 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 "light chains" (LC) and "heavy chains" (HC) (such light chain (LC) / heavy chain pairs are abbreviated herein as LC / HC). The light and heavy chains of such antibodies are polypeptides consisting of several domains. In a whole antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises heavy chain constant domains CHI, CH2, and CH3 (antibody classes IgA, IgD, and IgG), and optionally a heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain VL and a light chain constant domain CL. The variable domains VH and VL can be further subdivided into regions of hypervariability, 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 from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Janeway, CA, Jr, et al, (2001) Immunobiology., 5th ed., Garland Publishing, and Woof, J., Burton, D., Nat Rev Immunol 4 (2004) 89-99). 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, murine antibodies, human antibodies, chimeric antibodies, humanized antibodies, and genetically engineered antibodies (variant or mutant antibodies), so long as their characteristic properties are retained. In some embodiments, the antibody or binding agent is a humanized antibody, particularly as a recombinant human or humanized antibody.

[0068] In some embodiments, an antibody or binder may be "symmetric". By "symmetric", it is meant that the antibody or binder has the same type of Fv region (e.g., an antibody has two Fab regions). In some embodiments, an antibody or binder may be "asymmetric". By "asymmetric", it is meant that the antibody or binder has at least two different types of Fv region (e.g., an antibody has Fab and scFv regions, Fab and scFv2 regions, or Fab-VHH regions). A variety of 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 sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include 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 utilizing the structural and functional features of antibodies in alternative presentations. For example, antibody agents utilized in accordance with the present invention include intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; 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 (registered trademark); VHH; Anticalins (registered trademark); Nanobodies (registered trademark); BiTEs (registered trademark); Ankyrin repeat proteins or DARPINs (registered trademark); Avimers (registered trademark); DART; TCR-like antibodies; Adnectins (registered trademark); Affilins (registered trademark); Trans-bodies (registered trademark); Affibodies (registered trademark); TrimerX (registered trademark); MicroProteins; Fynomers (registered trademark), Centyrins (registered trademark); and KALBITOR (registered trademark).

[0070] In some embodiments, an antibody may lack a covalent modification (e.g., glycan attachment) that it would have if it were produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., glycan attachment, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.). In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs), and in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes 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, an antibody agent is or comprises a polypeptide whose amino acid sequence includes a structural element recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0071] The encoded antibody or antigen-binding molecule of the present invention can be single-chain or double-chain. In some embodiments, the antibody or antigen-binding molecule is single-chain. 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 greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (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 disclosure provides a composition comprising a DLL-3 binding agent (e.g., a DLL3-specific antibody) and a pharma- ceutically 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 antibody's two heavy chains, while the IgM and IgE Fc regions are composed of three heavy chain constant domains (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C31, C32, C43, C54, C65, C76, C87, C98, C99, C100, C111, C122, C131, C141, C152, C163, C174, C185, C196, C197, C198, C211, C222, C32, C43, C54, C65, C76, C87, C99, C101, C112, C123, C132, C144, C145, C151, C152, C164, C175, C185, C198, C199, C225, C231, C242, C32, C43, C54, C65, C76, C87, C99, C102, C113, C124, C131, C145, C153, C154, C165, C176, C187, C188, C199, C229, C231, C242, C32, H The Fc region of IgG contains a highly conserved N-glycosylation site (N297). Glycosylation of the Fc fragment may be essential for Fc receptor-mediated activity. The N-glycans attached to this site may be mainly core-fucosylated biantennary structures of complex type.

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

[0075] The disclosed anti-DLL-3 antibody agents can be antibodies 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] Provided herein is a DLL3 binding agent (e.g., antibody) that can bind to various regions or domains of DLL3 target.Epitope can be, for example, adjacent amino acids of DLL3 target (linear or adjacent epitope), or can be taken together from two or more non-adjacent regions of DLL3 target (conformational, non-linear, discontinuous, or non-adjacent epitope).The epitope that 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. Exemplary DLL3 antibodies described herein bind to the DLL3 domains provided in Table 1a. [Table 1]

[0078] In some embodiments, the DLL3 binding agent comprises a variable heavy chain (VH), and the amino acid sequence of the VH is selected from the VH sequences presented in Table lb. In some embodiments, the anti-DLL-3 binding agent 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 to an amino acid sequence presented in Table lb. [Table 2-1] [Table 2-2] [Table 2-3]

[0079] In some embodiments, the DLL3 binding agent 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 binding agent 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 an amino acid sequence presented in Table 1c. [Table 3-1] [Table 3-2] [Table 3-3]

[0080] Provided herein is a DLL3-binding agent (e.g., an antibody), wherein the DLL3 antigen-binding domain comprises a variable heavy chain (VH) and a variable light chain, wherein 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 binding agent comprises a 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 binding agent comprises a 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 containing the sequences shown in Tables 1b-1e, including functionally equivalent DLL3 antibody agents with modifications that do not significantly affect their properties, as well as variants with enhanced or decreased activity and / or affinity. For example, the amino acid sequence may be mutated to obtain a DLL3 antigen binding agent with a desired binding affinity to DLL3. Modification of polypeptides is a routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, with one or more deletions or additions of amino acids that do not significantly deleteriously alter the functional activity or mature (enhance) the affinity of the polypeptide for its ligand, or with the use of chemical analogs.

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

[0085] Substitution variants have at least one amino acid residue in the antigen-binding domain removed and a different residue inserted in its place. In some embodiments, the sites of interest for substitution mutagenesis include hypervariable regions / CDRs, although FR alterations are also contemplated. Conservative substitutions are shown in Table 2 under the heading of "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 fragment In one aspect, the anti-DLL-3 antibody agent according to any of the above embodiments may be an antibody fragment. An antibody fragment comprises 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, diabodies, linear antibodies, multispecifics formed from antibody fragments and scFv fragments, and other fragments described below. In some embodiments, the antibody is a full-length antibody, such as an intact IgG1 antibody, or other antibody class or isotype as described herein. (See, e.g., 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); WO 93 / 01161, and U.S. Patent Nos. 5,571,894, 5,869,046, 6,248,516, and 5,587,458.) A full-length, intact, or whole antibody is an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein. Antibody fragments can be produced by a variety of techniques, as known in the art, including, but not limited to, proteolytic digestion of an intact antibody and production by recombinant host cells (e.g., E. coli or phages).

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

[0088] Diabody is V H Domain and V L Small antibody fragments prepared by constructing sFv fragments with short linkers (e.g., about 5-10 residues) between the V domains, such that interchain rather than intrachain pairing of the V domains is achieved, resulting in bivalent fragments. Bispecific diabodies are heterodimers of two crossover sFv fragments, where the V domains of two antibodies are linked together. H Domains and V L The domains may be present on different polypeptide chains (see, e.g., EP 404,097, WO 93 / 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, ranging from about 11 kDa to about 15 kDa. DAbs are composed of the robust variable regions of the immunoglobulin heavy and light chains (V and V, respectively). H and V L). They are highly expressed in microbial cell culture, exhibit favorable biophysical properties including, but not limited to, solubility and temperature stability, and are well suited for selection and affinity maturation by in vitro selection systems such as, for example, phage display. dAbs are biologically active as monomers, and because of their small size and inherent stability, can be formatted into larger molecules to generate agents with extended serum half-lives or other pharmacological activity. (See, e.g., WO9425591 and US2003 / 0130496).

[0090] Fv and scFv are species with intact binding sites that do not contain constant regions. Thus, they may be suitable for reduced non-specific binding during in vivo use. Single-chain Fv (sFv or scFv) consists of VFs connected to a single polypeptide chain. H and V L An sFv polypeptide is an antibody fragment that contains an antibody domain. The sFv polypeptide has a V domain that enables the sFv to form the desired structure for antigen binding. H Domain and V L A polypeptide linker between the domains may further be included (see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra). scFv fusion proteins can be constructed to provide fusion of an effector protein at either the amino or carboxy terminus of the sFv. An antibody fragment may also be a "linear antibody" (see, e.g., U.S. Patent No. 5,641,870). Such linear antibody fragments may be monospecific or bispecific. Exemplary DLL3-specific scFvs are 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 an scFv comprising the light chain variable (VL) region and the heavy chain variable (VH) region of a DLL3-specific monoclonal antibody linked by a flexible linker. Single chain variable region fragments can be generated by linking the light and / or heavy chain variable regions by using a linking peptide. An example of a linking peptide has the amino acid sequence (GGGGS): x where x is 1, 2, 3, 4, or 5 (SEQ ID NO: 470). In some embodiments, x is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or any integer less than about 20. In some embodiments, the linker is (GGGGS)4 (SEQ ID NO: 478). In general, the linker can be a short, flexible polypeptide, in some embodiments, composed of about 20 or less amino acid residues. The linker can then be modified for additional functions, such as attachment of drugs or attachment to solid supports. Single-chain variants can be produced either recombinantly or synthetically. For synthetic production of scFvs, automated synthesizers can be used. For recombinant production of ScFvs, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. A polynucleotide encoding the scFv of interest can be produced 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 an exemplary embodiment, provided herein is a DLL3 antigen binding domain comprising a VH region comprising the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in Table 1b, and / or a VL region comprising the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in Table 1c. In some embodiments, the 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 GGGGSGGGSGGGGS (SEQ ID NO: 534). In some embodiments, the linker is a scFv Whitlow linker, which may comprise the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 535). The scFv Whitlow linker may be encoded by a DNA sequence comprising GGGTCTACATCCGGCTCCGGGAAGCCCGGAAGTGGCGAAGGTAGTACAAAGGGG (SEQ ID NO: 566). In some embodiments, the VH and VL sequences of the disclosed scFvs may be oriented with the VH sequence located at the N-terminus of the ScFv, followed by the linker, then the VL sequence, and in other embodiments, the scFvs may be oriented with the VL sequence at the N-terminus, followed by the linker, then the VH sequence.

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

[0094] In some embodiments, the anti-DLL-3 antibody agents provided herein can be chimeric antibodies (see, e.g., U.S. Pat. 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-switched" antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0095] In some embodiments, a chimeric antibody can be a humanized antibody (see, e.g., 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. Pat. 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., (see Osbourn 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 a non-human hypervariable region and amino acid residues from a human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, and 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. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody.

[0096] A non-human antibody can be humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. A humanized antibody can comprise one or more variable domains comprising one or more CDRs, or portions thereof, derived from a non-human antibody. A humanized antibody can comprise one or more variable domains comprising one or more FRs, or portions thereof, derived from a human antibody sequence. A humanized antibody can optionally comprise at least a portion of a human constant region. In some embodiments, one or more FR residues in a humanized antibody are replaced with the corresponding residues from a 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 the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions, human mature (somatically 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. Immunol, 151:2623 (1993)). al., J. Biol. Chem., 271:22611-22618 (1996).

[0098] iii. Human antibodies In some embodiments, the anti-DLL-3 antibody agent provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art (see, for example, van Dijk and van de Winkel, Curr.Opin.Pharmacol,5:368-74(2001), and Lonberg, Curr.Opin.Immunol,20:450-459(2008)). Human antibodies can be produced by humans or human cells, or have amino acid sequences corresponding to those of antibodies derived from non-human sources that utilize human antibody repertoires or other human antibody-encoding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be prepared by administering an immunogen (e.g., a DLL-3 protein) to intact human antibodies or to transgenic animals that have been modified to produce intact antibodies with human variable regions in response to 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 with a different human constant region.

[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, as well as 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. Pat. 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 the desired human constant regions.

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

[0101] iv. Antibody derivative In some embodiments, the antibody agents provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies may include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers may include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, 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-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.

[0102] The polymers 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, a conjugate of antibody and non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation.In some embodiments, the non-proteinaceous moiety can be a carbon nanotube (see, for example, Kam et al., Proc.Natl.Acad.Sci.USA,102:11600-11605(2005)).The radiation can be of any wavelength, including but not limited to wavelengths that do not harm normal cells but heat the non-proteinaceous moiety to a temperature that kills cells in the vicinity of the antibody-non-proteinaceous moiety.

[0104] A DLL3 binding agent (e.g., a molecule that contains an antigen-binding domain) is said to "specifically bind" to its target antigen (e.g., human, cynomolgus monkey, or mouse DLL3) if the dissociation constant (Kd) is about 1 nM. An antigen-binding domain specifically binds to an antigen with "high affinity" if the Kd is between 1-5 nM and "very high affinity" if the Kd is between 0.1-0.5 nM. 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 is administered to human DLL3 at a concentration of about 1×10 -7 M~1×10 -12 In yet another embodiment, the antigen-binding domain binds with a Kd of about 1x10 -5 M~1x10 -12 It will bind with a Kd of M.

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

[0106] In some embodiments, the DLL3 antigen binding domain is administered to a mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3, or mouse DLL3) at 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 equal to 1 × 10 -7 M -1 s- 1 Association rate (k on In certain embodiments, k on is determined using a monovalent antibody, such as a Fab fragment, as measured, for example, by BlAcore® surface plasmon resonance technology. on is determined using a bivalent antibody as measured, for example, by BlAcore® surface plasmon resonance technology.

[0107] In some embodiments, the DLL3 antigen binding domain is administered to a 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 Dissociation rate (k off In certain embodiments, k off is determined using a monovalent antibody, such as a Fab fragment, as measured, for example, by BlAcore® surface plasmon resonance technology. off is determined using a bivalent antibody as measured, for example, by BlAcore® surface plasmon resonance technology.

[0108] II. Chimeric Antigen Receptor As used herein, 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 a target antigen, CAR can activate immune cells to attack and destroy the cells (e.g., cancer cells) that carry that antigen. CAR can also incorporate a co-stimulatory domain or a signal transduction domain to enhance their efficacy. 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 Nos. 7,741,465 and 6,319,494.

[0109] The chimeric antigen receptor described herein comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain comprises a DLL3 antigen binding domain that specifically binds to DLL3. In some embodiments, the DLL-3 specific CAR comprises 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 comprises the following elements from 5' to 3': a CD8α signal sequence, a DLL3 scFv comprising a DLL3 variable heavy chain and / or a 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 comprises a safety switch and / or a monoclonal antibody specific epitope.

[0111] A.Antigen-binding domain As discussed above, the DLL3 CAR described herein comprises an antigen-binding domain. As used herein, "antigen-binding domain" refers to any polypeptide that binds to a specific target antigen, for example, the specific target antigen can be DLL3 (DLL-3) protein or a fragment thereof (interchangeably 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 hyperproliferative disease.

[0112] In some embodiments, the antigen-binding domain comprises 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) comprising a light chain CDR, CDR1, CDR2, and CDR3, and a heavy chain CDR, CDR1, CDR2, and CDR3.

[0113] In some embodiments, the DLL-3 specific CAR comprises a VH as shown in Table lb. In some embodiments, the DLL-3 specific CAR comprises a VL as shown in Table lc. In some embodiments, the DLL-3 specific CAR comprises a heavy chain CDR1, CDR2, CDR3 as shown in Table le. In some embodiments, the DLL-3 specific CAR comprises a light chain CDR1, CDR2, CDR3 as shown in Table lf.

[0114] Variants of antigen-binding domains (e.g., variants of CDRs, VHs, and / or VLs) are also within the scope of the present disclosure, e.g., variable light chains and / or variable heavy chains each having at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or greater than 99% identity to the amino acid sequence of the antigen-binding domain sequences described herein. In some examples, such molecules include at least one heavy chain and one light chain, while in other examples, the variant forms contain two variable light chains and two variable heavy chains (or subparts thereof). Those of skill in the art will be able to determine suitable variants of the antigen-binding domains described herein using well-known techniques. In certain embodiments, those of skill in the art will be able to identify suitable regions of the molecule that can be altered without destroying activity by targeting regions not believed to be important for activity.

[0115] In certain embodiments, the polypeptide structure of the antigen-binding domain is based on an antibody, including, but not limited to, a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, a synthetic antibody (sometimes referred to herein as an "antibody mimetic"), a chimeric antibody, a humanized antibody, a human antibody, an antibody fusion (sometimes referred to herein as an "antibody conjugate"), and fragments thereof. In some embodiments, the antigen-binding domain comprises or consists of an avimer.

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

[0117] In some embodiments, the DLL3 antigen binding domain is an scFv. In some embodiments, the DLL3-specific CAR comprises an 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 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 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 present disclosure relates to an isolated polynucleotide encoding any one of the DLL3 antigen binding domains described herein.In some embodiments, the present disclosure relates to an isolated polynucleotide encoding a DLL3 CAR described in Table 10.Also provided herein are vectors comprising the polynucleotides and methods for producing the same. [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

[0120] b. Safety Switch and Monoclonal Antibody Specific Epitope Safety switch It will be understood that adverse events can be minimized by transducing the immune cells (containing one or more CARs) with suicide genes. It may also be desirable to incorporate an inducible "on" or "facilitator" switch into the immune cells. Suitable techniques include the use of inducible caspase-9 (US Patent Application No. 2011 / 0286980) or thymidine kinase before, after, or at the same time as the cells are transduced with the CAR constructs of the present disclosure. Additional methods for introducing suicide genes and / or "on" switches include TALENS, zinc finger, 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 utilizes the FKBP / rapalog dimerization system in certain cells, the contents of which are incorporated herein by reference in their 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 contents of which are also incorporated herein by reference in their entirety. Additional dimerization pairs may include cyclosporine-A / cyclophilin, receptor, estrogen / estrogen receptor (optionally with 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 contents of which are further incorporated herein by reference in their entireties.

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

[0123] The suicide polypeptide may also include a signal peptide at the amino terminus, such as MGTSLLCWMALCLLGADHADA (SEQ ID NO: 553). In some embodiments, the suicide polypeptide includes the amino acid sequence shown in SEQ ID NO: 554, which is SEQ ID NO: 553: It contains a signal sequence of MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (sequence number 554).

[0124] When the suicide polypeptide is expressed on the surface of a CAR immune cell (CAR-T cell), the binding of rituximab to the R epitope of the polypeptide causes the lysis of the cell. 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 deletion of DLL3-specific CAR immune cells (e.g., CAR-T cells) may occur in vivo, for example, by administering rituximab to the patient. The decision to delete the transferred cells may result from undesirable effects detected in the patient due to the transferred cells, for example, when an unacceptable level of toxicity is detected.

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

[0126] In some embodiments, the extracellular domain of any one of the DLL3-specific CARs disclosed herein may comprise one or more epitopes specific to (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 incorporated herein in its entirety. In these embodiments, the extracellular domain of the CAR comprises an antigen-binding domain that specifically binds to DLL3 and one or more epitopes that bind to one or more monoclonal antibodies (mAbs). CARs that comprise mAb-specific epitopes may be single-chain or multi-chain.

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

[0128] Thus, in some embodiments, the present disclosure relates to methods for selecting and / or depleting engineered immune cells that have been equipped with CARs that contain mAb-specific epitopes, as well as methods for promoting the recovery of endogenous DLL3-expressing cells.

[0129] Several epitope-monoclonal antibody couples can be used to generate CARs that contain monoclonal antibody specific epitopes, particularly those already approved for medical use, such as CD20 epitope / rituximab as a non-limiting example.

[0130] The present disclosure also encompasses methods for selecting engineered immune cells loaded with DLL3-specific CARs that express mAb-specific epitopes, and therapeutic methods in which activation of engineered immune cells loaded with these CARs is modulated by depleting the cells using an antibody that targets the external 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 sequence: -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 Including, -In the formula, -V1 is V L And V2 is V H or V1 is V H And V2 is V L and -L1 is V H Chain V L is a linker suitable for linking to a chain, epitope 1, epitope 2, epitope 3, and epitope 4 are mAb-specific epitopes and are identical, or

[0132] C hinge domain The extracellular domain of the CAR of the present disclosure may comprise a "hinge" domain (or hinge region). The term generally refers to any polypeptide that functions to link the transmembrane domain in the 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 comprise up to 300 amino acids, in some embodiments 10-100 amino acids, or in some embodiments 25-50 amino acids. The hinge domain may be derived from all or a portion of a naturally occurring molecule, such as CD8, CD4, CD28, 4-1BB, or all or a portion of the extracellular region of IgG (particularly the hinge region of IgG, it will be understood that the hinge region may contain a portion 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 a portion of an antibody heavy chain constant region. Alternatively, the A domain may be a synthetic sequence that corresponds to a naturally occurring A sequence, or may be a completely synthetic A sequence. In some embodiments, the A domain is a portion of the human CD8 alpha chain (e.g., NP_001139345.1). In another particular embodiment, the hinge and transmembrane domains comprise a portion of the human CD8 alpha chain. In some embodiments, the hinge domain of the CAR described herein comprises a subsequence 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 comprises a human CD8α hinge, a human IgG1 hinge, a human IgG4, a human PD-1, or a human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein comprises an scFv, a CD8α human hinge and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. Table 5 provides the amino acid sequences of exemplary 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 an extracellular domain amino acid sequence set forth in Table 5 herein.

[0135] D transmembrane domain The CAR of the present disclosure is designed with a transmembrane domain fused to the extracellular domain of the CAR.Similarly, it 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 avoid such domain binding to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex.In some embodiments, a short linker can form a bond between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0136] Suitable transmembrane domains for the CARs disclosed herein include (a) transmembrane domains that are specific for, for example and without limitation, T helper (T h ) cells, cytotoxic T(T c ) cells, T regulatory (T reg ) cells, or lymphocyte cells, such as natural killer (NK) cells, and / or (b) the ability to interact with an extracellular antigen-binding domain and an intracellular signaling domain to induce a cellular response of an immune cell against a target cell.

[0137] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0138] Transmembrane regions of particular use in the present disclosure include those derived from CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (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, IgA, IgAg, IgAb ... Integrin, signaling lymphocyte activation molecule (SLAM protein), activating 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 The present invention may be derived from (including or corresponding to) a ligand that specifically binds to 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 non-limiting examples, the transmembrane region may be derived from or part of a T cell receptor, such as the α, β, γ, or δ polypeptides that make up the CD3 complex, the IL-2 receptor p55 (chain), p75 (β chain) or γ chain, a subunit chain of an Fc receptor, in particular the Fcγ receptor III or CD protein. Alternatively, the transmembrane domain may be synthetic and may contain primarily hydrophobic residues such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8 α chain (e.g., NP_001139345.1).

[0140] In some embodiments, the transmembrane domain in a CAR of the present disclosure is a CD8α transmembrane domain. In some embodiments, the transmembrane domain in a 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 a 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 a 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 a CAR of the present disclosure can provide activation of at least one of the normal effector functions of an immune cell that contains a CAR, e.g., signal 1 / activation and / or signal 2 / costimulation. For example, effector function of a T cell can refer to cytolytic activity or helper activity, including secretion of cytokines. In some embodiments, activating intracellular signaling domains for use in CARs can be, for example, without limitation, the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction following ligation of an antigen receptor, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0143] Suitable (e.g., activation) 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 costimulatory factor (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, sarcolemmal cell line ... Integrin, signaling lymphocyte activation molecule (SLAM protein), activating 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 It will be understood that the present invention includes signaling domains derived from (or corresponding to) a ligand that specifically binds to 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.

[0144] In addition to the activation domains described above, the intracellular domains of the CARs of the present disclosure may incorporate costimulatory signaling domains (interchangeably referred to herein as costimulatory molecules) to enhance their efficacy. The costimulatory domains can provide signals in addition to the primary signals provided by the activation molecules described herein.

[0145] Suitable costimulatory domains within the scope of the present disclosure include, 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 (CD1 1a / 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, signaling 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, CD 8beta, IL-2Rbeta, IL-2Rgamma, IL-7Ralpha, 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 The polypeptide may be derived from (or correspond to) 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 ligand, or a fragment or combination thereof.It will be understood that additional costimulatory molecules, or fragments thereof, not listed above, are within the scope of the present disclosure.

[0146] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include a 41BB / CD137 domain, either by itself or in combination with any other desired intracellular domain useful in the context of the CAR 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 a CD28 domain, either by itself or in combination with any other desired intracellular domain useful in the context of the CAR of the present disclosure. The complete native amino acid sequence of CD28 is described in NCBI Reference Sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI Reference Sequence: NM_006139.1.

[0148] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include a CD3 zeta domain, either by itself or in combination with any other desired intracellular domain useful in the context of the CAR 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 set forth in SEQ ID NO: 481 in Table 7. For example, the intracellular domain of the CAR can include a CD3 zeta chain portion and a portion of a costimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR 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 costimulatory / signaling domain of 4-1BB.

[0149] In some embodiments, 4-1BB (the 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: AAGCGCGGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTATGAGGCCCGTACAGACAACACAGGAGGAAGATGGCTGTAGCTGCAGATTTCCCGAGGAGGAGGAAGGTGGGTGCGAGCTG (SEQ ID NO: 568).

[0150] In some embodiments, the intracellular domain in the CAR is designed to comprise a portion of CD28 and CD3 zeta, wherein the intracellular CD28 comprises the nucleic acid sequence set forth in SEQ ID NO:567. AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCC ACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC (SEQ ID NO: 567).

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

[0152] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises 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 a CAR of the present disclosure comprises an amino acid sequence that comprises at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 480 and SEQ ID NO: 551. In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises an amino acid sequence that comprises at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 480, and / or at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 551.

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

[0154] III. Immune cells containing CAR a. Immune cells Provided herein are engineered immune cells (e.g., CAR-T cells) that express the CARs of the disclosure.

[0155] In some embodiments, the engineered immune cells comprise a population of CARs, each of which comprises a different extracellular antigen binding domain. In some embodiments, the immune cells comprise a population of CARs, each of which comprises 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 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, which may 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, cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, particle bombardment (e.g., gene gun), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV), or polyplexes.

[0161] In some embodiments, engineered immune cells expressing a DLL3-specific CAR of the present disclosure on their cell surface membrane comprise a percentage of stem cell memory and central memory cells that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, engineered immune cells expressing a DLL3-specific CAR of the present disclosure on their cell surface membrane comprise a percentage of stem cell memory and central memory cells that is greater than about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 15% to about 100%, about 15% to about 90%, or more than about 10% to about 10%. %, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 100%, about 30% to about 90 %, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60 %, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 90%, about 70% to about 80%, about 80% to about 100%, about 80% to about 90%, about 90% to about 100%, about 25% to about 50%, about 75% to about 100%, or 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 expressing any one of the CARs described herein.In some embodiments, the immune cell is a cytotoxic T lymphocyte expressing any one of the CARs described herein.In some embodiments, the immune cell is a regulatory T lymphocyte expressing any one of the CARs described herein.In some embodiments, the immune cell is a helper T lymphocyte expressing any one of the CARs described herein.

[0163] Prior to expansion and genetic modification, the source of cells can be obtained from a subject through various non-limiting methods. Cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumor. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, cells can be derived from healthy donors, patients diagnosed with cancer, or patients diagnosed with infectious diseases. In some embodiments, cells can be part of a mixed population of cells that exhibit different phenotypic characteristics.

[0164] Also provided herein is a cell line obtained from an immune cell (e.g., T cell) transformed according to any of the above methods. Also provided herein is a modified cell that is resistant to immunosuppressive treatment. In some embodiments, the isolated cell according to the present disclosure comprises a polynucleotide encoding a CAR.

[0165] The immune cells of the present disclosure can be activated and expanded either before or after genetic modification of the immune cells using commonly known methods.Generally, the engineered immune cells of the present disclosure can be expanded, for example, by contacting with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of T cells to create an activation signal for 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 T cells.

[0166] In some embodiments, a population of T cells can be stimulated in vitro, for example, by contact with an anti-CD3 antibody, such as an 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 costimulation of accessory molecules on the surface of T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody (e.g., an OKT3 antibody) and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. The anti-CD3 antibody and the anti-CD28 antibody can be placed on beads, such as plastic or magnetic beads, or on a plate or other substrate. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimal Essential Media or RPMI Media 1640, or X-vivo 15 (Lonza)) that may contain 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 of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones, and / or cytokines (e.g., IL-7 and / or IL-15) in sufficient amounts for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells that are infused into subjects.The target cells are maintained under the necessary conditions to support growth, for example, appropriate temperature (e.g., 37°C) and atmosphere (e.g., air and 5% CO2). T cells 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 contain one or more disrupted or inactivated genes. In some embodiments, the engineered immune cells according to the present disclosure contain 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 contain a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, the isolated cells according to the disclosure are 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 The cell comprises two disrupted or inactivated genes selected from the group consisting of 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 expresses 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 the gene 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 is rendered non-functional in the cell according to the present disclosure by disrupting or inactivating the TCR alpha and / or TCR beta genes. In some embodiments, a method is provided for obtaining modified cells from an individual, the cells being capable of growing independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells capable of growing independently of the MHC signaling pathway and amenable to obtain by this method are 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, within the scope of the present disclosure is a method for treating a patient in need of treatment for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising administering to the patient an effective amount of modified cells comprising disrupted or inactivated TCR alpha and / or TCR beta genes.

[0169] In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapeutic drugs. The chemotherapeutic drugs can be, for example, purine nucleotide analogs (PNAs), thus making the immune cells suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) to monophosphate, diphosphate, and triphosphate PNAs. Their triphosphate forms compete with ATP for DNA synthesis, act as proapoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production. Provided herein are DLL3-specific CAR-T cells that contain a disrupted or inactivated dCK gene. In some embodiments, dCK knockout cells are generated by transfection of T cells with a polynucleotide encoding a specific TAL nuclease directed to the dCK gene, for example, by electroporation of mRNA. dCK knockout DLL3-specific CAR-T cells are resistant to PNAs, including, for example, chlorofarabine and / or fludarabine, and maintain the cytotoxic activity of T cells against DLL3-expressing cells.

[0170] In some embodiments, the isolated cells or cell lines of the present disclosure may comprise pTα or a functional variant thereof. In some embodiments, the isolated cells or cell lines may 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 the immune cells as a transgene via a plasmid vector. In some embodiments, the plasmid vector can also include a selection marker, for example, to provide for identification and / or selection of cells that have received the vector.

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

[0173] In some embodiments, an isolated nucleic acid is provided that comprises a promoter operably linked to a first polynucleotide encoding a DLL3 antigen binding domain, at least one costimulatory molecule, and an activation domain. In some embodiments, the nucleic acid construct is comprised 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, a SFG vector, an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, and a vaccinia virus vector. In some embodiments, the nucleic acid is comprised within a plasmid.

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

[0175] A variety of known techniques can be used in producing polynucleotides, polypeptides, vectors, antigen-binding domains, immune cells, compositions, and the like, according to the present disclosure.

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

[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, umbilical cord blood, thymus tissue, tissue from an infection 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 skilled 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 plasma fraction and placed in a suitable 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 through a PERCOLL™ gradient. Specific subpopulations of T cells (e.g., CD28+, CD4+, CDS+, CD45RA-, CD45RO+, CDS+, CD62-, CD95-, CD95+, IL2Rβ+, IL2Rβ-, CCR7+, CCR7-, CDL-, CD62L+, and combinations thereof) can be further isolated by positive or negative selection techniques known in the art. In one example, the subpopulation of T cells is CD45RA+, CD95-, IL-2Rβ-, CCR7+, CD62L+. In one example, the subpopulation of T cells is CD45RA+, CD95+, IL-2Rβ+, CCR7+, CD62L+. In one example, the subpopulation of T cells is CD45RO+, CD95+, IL-2Rβ+, CCR7+, CD62L+. In one example, the subpopulation of T cells is CD45RO+, CD95+, IL-2Rβ+, CCR7-, CD62L-. In one example, the subpopulation of T cells is CD45RA+, CD95+, IL-2Rβ+, CCR7-, CD62L-. For example, enrichment of T cell population by negative selection can be achieved with a combination of antibodies directed against surface markers unique to 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 against cell surface markers present on negatively selected cells. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

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

[0181] In some embodiments, the 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 expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is 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 positive for granzyme B and perforin.

[0182] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with distinct cell surface antigens.

[0183] iii. Stem cell-derived immune cells In some embodiments, 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 directly isolated from patients. Various methods for isolating, developing, and / or culturing stem cells are known in the art and can be used to implement 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 peripheral blood mononuclear cell isolates, 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 before being genetically modified. In some embodiments, the isolated immune cells are genetically modified to reduce or eliminate 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 expression of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, the immune cells such as T cells are optionally further genetically modified with a chimeric antigen receptor 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 incorporated herein by reference in their entirety. In general, such methods involve contacting PBMCs or isolated T cells with stimulatory and costimulatory molecules, such as anti-CD3 and anti-CD28 antibodies, typically 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, which is 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. Pat. No. 6,040,177, U.S. Pat. No. 5,827,642, and WO2012 / 129514, the contents of which are incorporated by reference in their entireties.

[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 incorporated herein 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. An expression vector carrying a coding sequence for a chimeric receptor as disclosed herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. Successfully transduced T cells carrying the expression vector can be sorted using flow cytometry to isolate CD3 positive T cells, which can then be further expanded to increase the number of these CAR-expressing T cells in addition to cell activation using anti-CD3 antibodies and IL-2 or other methods known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of CAR-expressing T cells 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 non-human animal derived products such as 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 polynucleotides, vectors are introduced into host cells (isolated host cells) to allow the vector to replicate itself, thereby amplifying copies of the polynucleotides contained therein. Cloning vectors generally contain sequence components, including, but not limited to, replication origins, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements can be appropriately selected by those skilled in the art. For example, replication origins can be selected to promote the autonomous replication of vectors in host cells.

[0190] In certain embodiments, the present disclosure provides an isolated host cell that contains the vector provided herein.The host cell that contains the vector can be useful for expressing or cloning 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, 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, polylysine, histone, chitosan, and peptide mediated delivery. Standard methods for viral transfection and transformation of cells for expression of a vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used in genetically modifying a donor population of immune effector cells, each vector encoding a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, with a percentage of the engineered cells expressing two or more different CARs.

[0192] In one embodiment, the present disclosure provides a method of preserving engineered cells expressing a CAR targeting the DLL3 protein. In one embodiment, this involves cryopreserving the immune cells so that the cells remain viable upon thawing. In one embodiment, cryopreservation can include freezing in a suitable medium, such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions). A fraction of the 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 suffering from malignant tumors. If necessary, the cryopreserved transformed immune cells can be thawed, grown, and expanded for more such cells.

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

[0194] iv. Allogeneic CAR T cells Briefly, the process for manufacturing allogeneic CAR T therapy, or AlloCAR™, involves harvesting healthy, selected, screened, and tested T cells from healthy donors. The 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 make the CAR T product resistant to anti-CD52 antibody treatment. Thus, anti-CD52 antibody treatment can be used to lymphodeplete the host immune system, leaving the CAR T cells engrafted to achieve full therapeutic effect. Next, the T cells are engineered to express a CAR, which recognizes a specific cell surface protein (e.g., DLL-3) expressed on hematological or solid tumors. The engineered T cells then undergo a purification step and are eventually cryopreserved in vials for delivery to the 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) and 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 kills 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. Treatment Methods The present disclosure includes a method for treating or preventing a condition associated with undesirable 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 immune cell comprising a CAR disclosed herein.

[0197] Methods for treating diseases or disorders, 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 an engineered immune cell of the present application. In some embodiments, the T cell-mediated immune response is directed against a target cell. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the disclosure includes a method for treating or preventing a malignant tumor, 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 a method for treating or preventing a malignant tumor, 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. A CAR containing the immune cell of the disclosure can be used to treat a malignant tumor associated with aberrant expression of DLL3. In some embodiments, CARs containing the immune cells of the present disclosure can be used to treat malignancies such as small cell lung cancer, melanoma, low-grade glioma, glioblastoma, medullary thyroid carcinoma, carcinoid, dispersed neuroendocrine tumors in the pancreas, bladder, and prostate, testicular cancer, and lung adenocarcinoma with neuroendocrine features. In an exemplary embodiment, CAR-containing immune cells, such as anti-DLL3 CAR-T cells of the present 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 an engineered cell of the present disclosure, wherein the cell comprises a chimeric antigen receptor comprising a DLL3 antigen binding domain and binds to a DLL3 antigen on the tumor.

[0199] In some embodiments, the subject has a solid tumor or a hematological malignancy such as lymphoma or leukemia. In some embodiments, the engineered cells are delivered to a tumor bed, such as that found in small cell lung cancer. In some embodiments, the cancer is present in the subject's bone marrow. In some embodiments, the engineered cells are autologous immune cells, e.g., autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, e.g., allogeneic T cells. In some embodiments, the engineered cells are xenogeneic immune cells, e.g., xenogeneic T cells. In some embodiments, the engineered cells are transfected or transduced ex vivo. As used herein, the term "in vitro cells" refers to any cells that are cultured ex vivo.

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

[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 medical treatment, subjects at risk for developing a disorder, and the like.

[0202] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and applications in which a subject reduces the risk of developing a disorder or other risk factor. Treatment does not require a complete cure of the disorder, but encompasses embodiments in which symptoms or underlying risk factors are reduced. The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it indicates a reduced likelihood of the occurrence of an event in the presence of a compound or method.

[0203] The desired total therapeutic amount of cells in the composition will include at least 2 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, 10 2 More than 10 cells, and up to 10 6 pieces, maximum 10 8 or 10 9 cells, 10 10 or 10 12 The number of cells will depend on the desired use for which the composition is intended, and the type of cells contained therein. Desired cell densities are typically between 10 and 20. 6 cells / ml, typically 10 7 cells / ml, typically 10 8 ≥ 10 cells / ml. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 The total number of cells injected can be distributed over multiple infusions that cumulatively equal or exceed 10 cells. In some embodiments of the present disclosure, particularly since all infused cells are redirected to a specific target antigen (e.g., DLL3), the total number of cells injected can be as high as 10. 6 / kilogram (10 per patient) 6 ~10 11) may be administered. CAR therapy may be administered multiple times at dosages within these ranges. Cells may be autologous, allogeneic, or xenogeneic to the patient undergoing therapy.

[0204] In some embodiments, the therapeutically effective amount of CAR T cells is about 1×10 5 cells / kg, approx. 2X10 5 cells / kg, approx. 3X10 5 cells / kg, approx. 4X10 5 cells / kg, approx. 5X10 5 cells / kg, approximately 6X10 5 cells / kg, approximately 7X10 5 cells / kg, about 8X10 5 cells / kg, approx. 9X10 5 cells / kg, 2X10 6 cells / kg, approx. 3X10 6 cells / kg, approx. 4X10 6 cells / kg, approx. 5X10 6 cells / kg, approximately 6X10 6 cells / kg, approximately 7X10 6 cells / kg, about 8X10 6 cells / kg, approx. 9X10 6 cells / kg, approximately 1X10 7 cells / kg, approx. 2X10 7 cells / kg, approx. 3X10 7 cells / kg, approx. 4X10 7 cells / kg, approx. 5X10 7 cells / kg, approximately 6X10 7 cells / kg, approximately 7X10 7 cells / kg, about 8X10 7 cells / kg, or approximately 9X10 7 cells / kg.

[0205] In some embodiments, the target dose of CAR+ / CAR-T+ cells is about 1×10 6 ~Approx. 1×10 10 In the range of cells / kg, e.g., about 1 x 10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 1×10 8 cells / kg, approximately 1×10 9cells / kg, or approximately 1 x 10 10 The dose is 100 mg / kg of cells. It will be understood that doses above and below this range may be appropriate for a particular subject, and appropriate dose levels may be determined by a medical provider as necessary. Additionally, multiple doses of cells may be provided in accordance with the present disclosure.

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

[0207] The CAR-expressing cell population of the present disclosure may be administered as a pharmaceutical composition alone or in combination with other components such as diluents and / or IL-2 or other cytokines or cell populations. The pharmaceutical composition of the present disclosure may include a CAR-expressing cell population, such as T cells, described herein, in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may 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 (such as solutions, suspensions, etc.) may contain one or more of the following: sterile diluents, such as water for injection, saline solution, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol or other solvents that can serve as solvents or suspension media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and 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 expressing 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 DLL3-expressing endogenous cells or cells of a 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 DLL3-expressing endogenous cells or cells of a cell line expressing DLL3 by engineered immune cells expressing any one of the DLL3-specific CARs described herein is about 5% to about 95%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75%, or about 25% to about 60%. In one embodiment, the endogenous DLL3-expressing cells are endogenous DLL3-expressing bone marrow cells.

[0210] In one embodiment, the percent reduction or lysis of target cells, e.g., cell lines 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 include administering one or more chemotherapeutic agents to the patient 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 can include administering to the patient a specific beneficial dose of cyclophosphamide (200 mg / m 2 / day~2000mg / m 2 / day, about 100mg / m2 / day~about 2000mg / m 2 / day, e.g., about 100 mg / m 2 / day, about 200mg / m 2 / day, about 300mg / m 2 / day, about 400mg / m 2 / day, about 500mg / m 2 / day, about 600mg / m 2 / day, about 700mg / m 2 / day, about 800mg / m 2 / day, approximately 900mg / m 2 / day, about 1000mg / m 2 / day, about 1500mg / m 2 / day, or approximately 2000 mg / m 2 / day), and specific doses of fludarabine (20 mg / m 2 / day~900mg / m 2 / day, about 10mg / m 2 / day~about 900mg / m 2 / day, e.g., about 10 mg / m 2 / day, about 20mg / m 2 / day, about 30mg / m 2 / day, about 40mg / m 2 / day, about 40mg / m 2 / day, about 50mg / m 2 / day, about 60mg / m 2 / day, about 70mg / m 2 / day, about 80mg / m 2 / day, approximately 90mg / m 2 / day, about 100mg / m 2 / day, about 500mg / m 2 / day, or approximately 900 mg / m 2 An exemplary dosing regimen includes administering about 30 mg / m2 of engineered T cells to a patient for three days prior to administration of a therapeutically effective amount of engineered T cells to the patient. 2 about 300 mg / m 2 The method includes administering daily to the patient 100 mg / day of cyclophosphamide.

[0212] In some embodiments, particularly where the engineered cells provided herein have been genetically edited to eliminate 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 1-20 mg / day IV, e.g., about 13 mg / day IV, for 1, 2, 3 or more days. The antibody may be administered in combination with, prior to, or following administration of other components 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 the subject.

[0214] In certain embodiments, compositions comprising the CAR-expressing immune effector cells disclosed herein may be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylameramines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaolamide, and trimethylololamine resembling; chlorambucil, ... Nitrogen mustards such as lornafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosuureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, and caliceramide. Amycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimec antibiotics such as azoxymethane, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU;Androgens such as calcineurin, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopida Mol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark); Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitrol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, such as paclitaxel (TAXOL (trademark), Bristol-Myers Squibb), and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues 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 inhibitors RF S2000; difluoromethylfolate (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™, (alitretinoin); ONTAK™ (denileukin diftitox); esperamicin; capecitabine;and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapristone, and toremifene (Farestone); and antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Optionally, a combination of chemotherapy agents is also administered, including, but not limited to, CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.;

[0215] In some embodiments, the chemotherapeutic agent is administered simultaneously with or within one week after administration of the engineered cells, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered for 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 administration of the engineered cells, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least one month prior to administration of the cells, polypeptide, or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0216] Various additional therapeutic agents may 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 ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), imatinib (G leevec®), 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, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, and pak These include, but are not limited to, mTOR inhibitors such as ritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as the CDK inhibitor palbociclib.

[0218] In some embodiments, the composition comprising the CAR-containing immune cells can be administered with a therapeutic regimen to prevent or reduce cytokine release syndrome (CRS) or neurotoxicity. The therapeutic regimen to prevent cytokine release syndrome (CRS) or neurotoxicity can include lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (e.g., L-NIL or 1400W). In additional embodiments, the composition comprising the 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, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialic acid salts. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporoxifene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as 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 cytokines. 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 inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-beta; platelet growth factor; transforming growth factor, such as TGF-alpha and TGF-beta. These include transforming growth factor (TGF); insulin-like growth factor-I and -II; erythropoietin (EPO); bone morphogenetic factor; interferons such as interferon-alpha, beta, and gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) 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 the native sequence cytokines.

[0220] V. Selection and Depletion Methods In some embodiments, a method is provided for in vitro sorting of a population of immune cells, a subset of the population of immune cells comprising engineered immune cells expressing any one of the DLL3-specific CARs comprising an epitope (e.g., an exemplary mimotope sequence) specific for a monoclonal antibody. The method comprises contacting the 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 for engineered immune cells expressing the DLL3-specific CAR.

[0221] In some embodiments, the monoclonal antibody specific for the epitope is optionally conjugated to a fluorophore, in which embodiment, selecting cells that bind 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 a magnetic particle, in which 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 a CAR is alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, daclizumab, In some embodiments, the mAb is selected from the group consisting of rituximab, 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 other embodiments, the mAb is QBEND-10.

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

[0225] In some embodiments, the population of CAR-expressing immune cells obtained when using the methods for in vitro selection of CAR-expressing immune cells described above exhibits increased cytotoxic activity in vitro compared to the initial (unselected) cell population. In some embodiments, the cytotoxic activity in vitro is increased 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 may 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 selecting cells that express an antigen, such as the CD34 antigen, using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting.

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

[0229] Flow cytometry analysis involves two major steps: 1) labeling selected cell types with one or more labeling markers, and T) determining the number of labeled cells relative to the total number of cells in a population. In some embodiments, the method of labeling a cell type involves binding a labeled antibody to a marker expressed by a particular 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 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 pure cell populations. Cells in single cell suspension can be magnetically labeled with microbeads. The sample is applied to a column composed of ferromagnetic spheres, covered with a cell-friendly coating that allows for quick and gentle separation of cells. Unlabeled cells pass through while magnetically labeled cells are retained in the column. 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 present disclosure provides a method for depleting DLL3-specific CAR-expressing immune cells by in vivo depletion. In vivo depletion can include administering a treatment (e.g., a molecule that binds to an epitope on the CAR) to a mammalian organism with the aim of stopping 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 a mAb-specific epitope, 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 a 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 immune engineered cells is performed on 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 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 expressing a CAR containing a mAb-specific epitope (CAR-expressing immune cells), comprising contacting said CAR-expressing immune cells with at least one epitope-specific mAb.

[0235] In some embodiments, contacting the engineered immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb comprises injecting the patient with the epitope-specific mAb (e.g., rituximab). In some embodiments, the amount of 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, contacting the engineered immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb provides the patient with about 375 mg / m 2 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 a 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 is reduced. In some embodiments, the amount of viable CAR-expressing immune cells is reduced 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, the in vivo depletion of CAR-engineered immune cells is performed by injecting bispecific antibodies. By definition, bispecific monoclonal antibodies (BsAbs) are artificial proteins that are composed of fragments of two different monoclonal antibodies and, as a result, bind to two different types of antigens. These BsAbs and their use in immunotherapy are reviewed in Muller D and Kontermann RE (2010) Bispecific Antibodies for Cancer Immunotherapy, BioDrugs 24(2):89-98.

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

[0240] In some embodiments, cytotoxic drugs are conjugated to epitope-specific mAbs that can be used to deplete CAR-expressing immune cells.By combining the targeting ability of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, antibody-drug conjugates (ADCs) allow for sensitive discrimination between healthy and diseased tissues when compared to the use of drugs alone.Some ADCs have received market approval, and the technology for making them, especially on linkers, has been developed (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 epitope-specific mAb injected is pre-conjugated with a molecule that can promote complement-dependent cytotoxicity (CDC). Thus, the complement system supports or complements the ability of antibodies to remove pathogens from the organism. When stimulated, an activation cascade is triggered as a massive amplification of the response and activation of cell-killing membrane attack complex. Different molecules such as glycans can be used to conjugate mAbs (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 present disclosure comprise allogeneic DLL3 CAR-containing T cells and a CD52 antibody for administering a lymphodepletion regimen and a CAR-T regimen to a subject.

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

[0245] Example 1: Generation and testing of DLL3-targeting antibodies Monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or may be made 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 DLL3-specific antibodies can recognize cells expressing endogenous DLL3, DMS 273 (Sigma, Cat. No. 95062830), DMS 454 (Sigma, Cat. No. 95062832), and SHP-77 (ATCC, Cat. No. CRL-2195) cells were stained with 2ug / ml purified DLL3 antibody with mouse IgG2A backbone (mIgG2a) or control mIgG2a antibody in PBS supplemented with 1% BSA. Bound DLL3 antibody was detected with PE-labeled anti-mouse IgG antibody (Biolegend, Cat. No. 405307). Samples were analyzed by flow cytometry. Representative images showing binding of DLL3 antibody to DMS 273, DMS 454, and SHP-77 cells are included in Figure 1.

[0247] Example 2: Determination of Kinetics and Affinity of Anti-DLL3 Antibodies to 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 monkey (cyno), and mouse DLL3. For the scFvs, the variable regions of anti-DLL3 antibodies 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 part of Fc from a human IgG2 sequence, resulting in scFv-Fc fusions and expressed using Expi293. The extracellular domains (ECDs) from human, cynomolgus monkey, and mouse DLL3 were fused with 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] Antibody binding kinetics were determined by surface plasmon resonance (Biacore™ surface plasmon resonance (SPR) system, GE Healthcare Bio-Sciences, Pittsburg PA). Antibodies diluted in HBS-T+ running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 0.05% vol / vol Tween 20, 1mg / 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 in HBS-T+ and injected for 2 minutes at 30uL / min, after a 10 minute dissociation time, the surface was regenerated with either 10mM glycine-HCl pH 1.7 or phosphoric acid between injections. The kinetic association rate (kon) and dissociation rate (koff) are 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 k off / k on It is calculated as:

[0249] The kinetics and affinity parameters of the anti-DLL3 antibodies tested are shown in Table 8. Specifically, Table 8 shows the affinity of the 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 of the anti-DLL3 antibodies recognizes. [Table 11]

[0250] Example 3: Generation of CHO cells expressing full-length and truncated DLL3 A panel of CHO cells expressing full-length and various truncated human DLL3 was used to determine which domains 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 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 truncated DLL3 proteins that were generated (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 (sequence number 556, GeneBank record NM_016941) and 7 HA-tagged truncated human DLL3 (sequence numbers 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, psPAX2, and pMD2G vectors. Two days after transfection, the supernatant containing the viral particles was collected and used to transduce CHO cells together with 5ug / ml polybrene.

[0253] The expression of full-length and truncated DLL3 was verified by FACS assay using PE-conjugated anti-HA antibody (Biolegend, Cat. No. 901518). As a negative control, cells were incubated with an isotype-matched, PE-labeled antibody (Biolegend, Cat. No. 400111) instead of anti-HA antibody. The lower panel of 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 supernatant or purified DLL3 antibody in PBS+1% BSA. Bound DLL3 antibody was detected with PE-labeled anti-mouse IgG antibody (Biolegend, Cat. No. 405307). Samples were analyzed by flow cytometry. The binding domain of each clone was determined using a panel of CHO expressing full-length or truncated DLL3 as described in Example 2. Flow cytometry analysis showed that such clones recognize EGF3, for example, if the clone binds to all truncated proteins including EGF3, but not to any truncated proteins that do not contain EGF3. As shown in the representative images in Figure 2D, anti-DLL3 antibodies recognize the DSL, EGF1, and EGF3 domains, respectively. Signal from the PE channel is shown on the x-axis and counts are 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. The amino acid sequences of the heavy and light chain variable regions of these antibodies (Tables 1b and 1c) were used to design single chain variable fragments (scFvs) (Table 1d) with the following general structure: heavy chain variable region--linker--light chain variable region. 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': the CD8α signal sequence (SEQ ID NO:477), the anti-DLL3 scFv, the hinge and transmembrane region of the human CD8α molecule (SEQ ID NO:479), the cytoplasmic portion of the 41BB molecule (SEQ ID NO:291), and the 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

[0258] A schematic of the CAR structure is shown in Figure 3A. Representative CAR sequences reformatted from anti-DLL3 clones are included in SEQ ID NOs: 482-533. A 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, Cat. No. 130-096-535). Alternatively, primary human T cells can be directly purified from LeukoPak (StemCell Technologies).

[0260] To generate lentivirus encoding DLL3 CAR, HEK-293T cells were seeded 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 0. On day 1, lentivirus was prepared by mixing together lentiviral packaging vectors 1.5ug psPAX2, 0.5ug pMD2G, and 0.5ug of the appropriate transfer CAR vector in 250uL of Opti-MEM (Gibco) per well of a 6-well plate ("DNA mix"). 10uL of Lipofectamine 2000 (Invitrogen) in 250uL 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 a total volume of 500uL was slowly added to the side of the well containing the HEK-293T. Purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, Cat. No. 130-111-160, 1:100 dilution). On day 2, medium from each well of a 6-well plate was replaced with 2 mL per well of T cell transduction medium, i.e., X-Vivo-15 supplemented with 10% FBS. On day 3, 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, Cat. No. 80192M). Lentiviral supernatant from HEK293T cells was harvested and passed through a 0.45 micron filter (EMD Millipore) to remove cell debris and then added to the T cells along with 100 IU / mL 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.Transduction efficiency was determined on days 9 and 13 by detecting the percentage of T cells that recognize recombinant DLL3 (Adipogen) using flow cytometry. Cells were expanded in larger flasks or G-Rex vessels (Wilson Wolf) as needed using T cell expansion medium. 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 1ug / ml Flag-tagged recombinant DLL3 (Adipogen) in PBS+1% BSA for 20 minutes at 4° C. Cells were then washed with PBS+1% BSA, stained with PE-labeled anti-Flag antibody (Biolegend, Cat. No. 637310), and analyzed using flow cytometry.

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

[0263] Example 6: In vitro characterization This example describes experiments used to determine the specificity and in vitro activity of 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 lentivirus encoding full-length human DLL3.

[0265] To test DLL3-specific killing, firefly luciferase expressing HEK-293T cells with or without human DLL3 expression were then seeded in 96-well assay plates (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 effector:target (E:T) ratios ranging from 1:9 to 9:1 in T cell growth medium, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio). Cell survival was measured after 72 hours using the one-glo assay kit (Promega). Representative DLL3 CAR-T cells showed potent killing against HEK-293T-DLL3 cells but no detectable activity in 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 effector:target (E:T) ratios ranging from 1:9 to 9:1 in T cell growth medium, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio). Cell viability was measured after 72 h using the one-glo assay kit (Promega). Each condition was assayed in three replicates. The mean percentage of viable cells and standard deviation were plated (Figure 4B and Figure 4C).

[0267] Figure 4A shows experimental data demonstrating 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 that did not express the anti-DLL3 CAR (labeled empty vector) were used as a negative control.

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

[0269] Figure 4C shows experimental data showing 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 assessed using the One-glo assay system (n=3).

[0270] To measure cytokines secreted by 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, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio). After 24 h, tissue culture supernatants were collected and the levels of three cytokines [interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-2] in the supernatants were measured using a human proinflammatory tissue culture 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 h (n=3).

[0271] Example 7: Sequential killing assay The sequential killing assay involves repeated exposure of CAR-T cells to their targets, which expands and, in certain cases, 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 a 96-well plate with white walls and a flat clear bottom in 100ul of X-Vivo-15 medium with 5% human serum. After target cells were allowed to attach to the bottom of the plate, 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 with 5% human serum. Every 2 days thereafter, 100μl of medium containing DLL3 CAR-T cells was transferred to freshly seeded target cells, and the percentage of lysis of 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 of lysis and standard deviation were plated (Figure 6A-6C). The optimal clone was the one with the highest target cell lysis throughout the assay at day 12. These data show experimental data from a serial killing assay, demonstrating that some of the clones remained active after repeated exposure of anti-DLL3 CAR-T cells to DLL3+WM266.4 cells. Target cell survival was assessed using the One-glo assay system or CellTiter-glo at each time point indicated (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 frozen stock vials, thawed, and counted according to standard procedures. Cells were cultured at 50 × 10 in complete growth medium (RPMI + 10% FBS). 6 The cells were diluted to 5x10 viable cells / mL. The cell suspension was kept on ice until transplantation. Immediately after transplantation, the cells were mixed 1:1 with BD Matrigel Matrix (Cat. No. 354234) and diluted at 5x10 per mouse. 6200 μL of cell / matrigel suspension containing 100 SHP-77 cells were injected subcutaneously. Tumor growth was monitored by caliper measurements using digital calipers starting 5 days after implantation. Tumor size was calculated using the formula: Tumor volume = (width^2 x length / 2). Mice were randomized into groups of 5 based on tumor volume approximately 2 weeks after implantation. The mean tumor volume per group was 314 mm. 3 The results were as follows. One day after randomizing the mice, non-transduced T cells and DLL3 CAR-T cells were thawed and counted according to standard procedures. Cells were resuspended in RPMI+10% FBS and injected by tail vein IV injection at 2 million or 5 million CAR+ cells per mouse in a volume of 200 uL per mouse. Tumors continued to be 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 showing that anti-DLL3 CAR-T cells can eliminate established small cell lung cancer tumors.

[0274] To test the antitumor activity of DLL3 CAR-T cells in a model that exhibits metastasis 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 cultured at 40x10 in complete growth medium (RPMI + 10% FBS). 6The cells were diluted to 10000 viable cells / mL. The cell suspension was kept on ice until implantation and 200 μL of cell suspension per mouse was injected by tail vein IV. On day 7 after implantation, 200 uL of luciferin (15 mg / mL) was injected and tumor growth was monitored by IVIS imaging. Mice were randomized into groups of 5 based on total light flux on day 11 after implantation. On day 12 after implantation, CAR-T were thawed and counted according to standard procedures. Cells were resuspended in RPMI + 10% FBS and injected by tail vein IV injection in a volume of 200 uL per mouse at 2 million or 7 million CAR+ cells per mouse. Tumors continued to be monitored every 3-4 days until the end of the study. As shown in Figure 8, 10G1-K anti-DLL3 CAR-T cells can inhibit established small cell lung cancer tumors in mice in a dose-dependent manner.

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

[0276] Protein sequences encoding anti-DLL3 CAR constructs containing a safety switch are shown in Table 9. An exemplary safety switch construct may include a CD8α signal sequence (SEQ ID NO:477), an anti-DLL3 scFv described herein, a CD20 mimotope (SEQ ID NO:536), a QBEND-10 epitope (SEQ ID NO:544), a hinge and transmembrane region of a human CD8α molecule (SEQ ID NO:479), a cytoplasmic portion of a 4-1BB molecule (SEQ ID NO:291), and a cytoplasmic portion of a 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 activity was investigated using the method described in Example 7. Figure 9A is a plot showing the structure of four different safety switches. Figure 9B shows experimental flow cytometry data showing that anti-DLL3 CARs 2G1, 4H8, and 10G1-K with safety switches can be expressed on the surface of primary T cells and recognize recombinant DLL3. The plots are gated on live CD3+ cells, and the numbers on the plots are the percentage of cells expressing each anti-DLL3 CAR. Figure 9C shows experimental data showing that anti-DLL3 CARs with safety switches are active in a serial killing assay of 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 cell surface DLL3 expression, frozen vials of PDX models were thawed and 200,000 cells were used for each staining sample. DLL3 expression was verified by FACS assay using 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 showing that DLL3 is expressed on the surface of two small cell lung cancer PDX models. Figure 10B shows experimental data showing 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 ratio. T cells that did not express anti-DLL3 CAR (labeled empty vector) were used as 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 insertion of a rituximab mimotope at various positions in the extracellular domain of the CAR as 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, frozen CAR-T cells were thawed and 1x10 5 Cells were incubated in RPMI1640 medium supplemented with 10% FBS in 96-well plates. Cells were incubated for 3 hours in the absence or presence of 25% baby rabbit 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 showing that anti-DLL3 CAR-T cells can be detected by both recombinant DLL3 and rituximab staining. Figure 11B shows experimental data showing 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 antitumor activity of DLL3 CAR-T cells with a safety switch, SHP-77 tumor-bearing NSG mice were used. SHP-77 cells were thawed from a frozen vial, counted, and diluted to 50 × 10 in RPMI medium / Matrigel suspension per mouse. 6 Viable cells / mL were injected subcutaneously. Tumor growth was monitored by caliper measurements using digital calipers starting 5 days after implantation. Tumor size was calculated using the formula: Tumor volume = (width^2 x length / 2). Mice were randomized into groups of 8 based on tumor volume approximately 14 days after implantation. The mean tumor volume per group was 178 mm 3 On the same day that mice were randomized, non-transduced T cells and DLL3 CAR-T cells were thawed and counted according to standard procedures. Cells were counted at 5 × 10 per mouse. 6 10 CAR+ cells were resuspended in RPMI via tail vein IV injection in a volume of 200uL per mouse. Tumors continued to be monitored every 3-4 days until the end of the study. All groups of DLL3 CAR-T cells with safety switch induced significant tumor inhibition and complete or near complete elimination of detectable tumors by day 50 (Figure 12A).

[0282] To test the antitumor activity of DLL3 CAR-T cells in a model that exhibits metastasis like the 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 cultured at 5 × 10 per mL in RPMI medium. 5 The cells were diluted to viable cells. 200 μL of cell suspension per mouse was injected via tail vein IV. On day 3 after implantation, mice were randomized into groups of 9. On the same day, DLL3 CAR-T were thawed, counted, and 5x10 per mouse were injected. 610 CAR+ cells were resuspended in RPMI media by tail vein IV injection in a volume of 200uL per mouse. Tumors continued to be 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 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 mouse brain, brains from three NSG mice were fixed in 10% neutral buffered formalin (NBF), embedded, serially sectioned at 4-6 microns, and analyzed with the RNAscope® LS Red ISH assay (ACDBio). DLL3 RNA was detected at low levels in NSG mouse brain samples. Figure 13A shows a representative image of mouse DLL3 RNA staining observed with this assay.

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

[0285] Example 14: Mouse safety study using animals bearing subcutaneous tumors To further evaluate the liability of potential brain and pituitary toxicity, 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 may potentially result in increased sensitivity and activity against normal tissues expressing DLL3. The experimental design is shown in Figure 14A. Three days prior to tumor implantation (day -3), adeno-associated virus (AAV) (Vigene Biosciences) encoding IL-7 and IL-15 was injected through the tail vein to support the proliferation and persistence of CAR-T cells. LN229-mDLL3 cells were then thawed from frozen vials and 4.25 × 10 7 The cells were diluted to 4.25x10 cells / mL. The cell suspension was kept on ice until transplantation. Just prior to transplantation, the cells were mixed 1:1 with BD Matrigel Matrix (Cat. No. 354234) and diluted to 4.25x10 cells / mL per mouse. 6 200 μL of cell / matrigel suspension containing LN229-mDLL3 cells were injected subcutaneously. Tumor growth was monitored by caliper measurements using digital calipers starting from day 8 post-implantation. Tumor size was calculated using the formula: tumor volume = (width^2 x length / 2). On day 22 post-implantation, mice were randomized into groups of 5 based on tumor volume and serum concentrations of IL-7 and IL-15. On the same day (day 22), non-transduced T cells and mouse cross-reactive 10G1-K DLL3 CAR-T cells were thawed, resuspended in RPMI + 10% FBS, and injected at 1x10 per mouse. 7 CAR+ cells were injected via tail vein IV injection in a volume of 200 uL per mouse. Tumors continued to be 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] At day 49, if animals that received DLL3 CAR-T cells were tumor-free, brain tissue from the animals was fixed in 10% NBF and embedded to reveal the ventricular system, including the third and fourth lateral ventricles, and three sections were placed into a single block that was serially sectioned at 4-6 microns and stained with hematoxylin and eosin (H&E) or stained to detect human-specific CD3 (hCD3) by immunohistochemistry. Pituitaries were fixed in 10% NBF, processed, and stained with H&E or immunohistochemically stained to show hCD3. H&E slides were examined microscopically, and 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, and relatively few T cells in the anterior lobe (Figure 14C and data not shown). Sparse-moderately low or moderately-moderately high hCD3 staining of T cells was present in the brain neuropil and vasculature (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 anti-vasopressin antibody (ImmunoStar, 20069) at 1 / 7,000 dilution for 1 h at room temperature, then with Rabbit-on-Rodent HRP-Polymer (Biocare Medical) for 30 min at room temperature. For oxytocin detection, samples were stained with anti-oxytocin antibody (ImmunoStar, 20068) at 1 / 10,000 dilution for 15 min at room temperature, then with Rabbit-on-Rodent HRP-Polymer (Biocare Medical) for 30 min at room temperature. Both hormones could be detected in the pituitary neural lobe of animals that received non-transduced T cells or DLL3 CAR-T cells, indicating that 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 bearing 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 a frozen vial and diluted to 1 x 10 in RPMI. 7 The cells were then diluted to 3 x 10 viable cells / mL in 3 µL per mouse. 4 Cell suspensions containing LN229-mDLL3 cells were injected intracranially. Tumor growth was monitored by an IVIS imaging system. On day 17 after implantation, mice were randomized into groups of 10 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 at 1x10 7 10 CAR+ cells / mouse were injected via tail vein IV injection in a volume of 200uL / mouse. EGFRvIII CAR-T cells were included as a control to evaluate potential inflammation caused by tumor lysis in the brain. To support CAR-T cell proliferation and persistence, 0.5ug of IL-15 (Peprotech AF-200-15) and 3ug of IL-15Ra Fc fusion protein (R&D Systems 7194-IR) were administered to each animal twice weekly starting on day 17 until the end of the study. Tumors continued to be 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 tissue from all animals was trimmed, processed, embedded, and the ventricular system, including the third and fourth lateral ventricles, was revealed, and three sections were placed into a single block, which was serially sectioned 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, intermediate, and anterior lobes, and stained with H&E or stained immunohistochemically to detect hCD45 as a marker for human T cells.

[0288] At day 22, animals receiving non-transduced T cells or 10G1-K DLL3 CAR-T cells had rare / sparse hCD45 staining T cells in the brain or pituitary (data not shown). Meanwhile, for animals treated with EGFRvIII CAR-T cells, hCD45+ staining ranged from rare / sparse to moderately low or moderate to moderately high in areas of infiltration / gliosis or glioma, consistent with antitumor activity in this group (data not shown). At day 38, animals receiving non-transduced T cells or EGFRvIII CAR-T cells had rare / sparse hCD45+ staining in the brain and pituitary. Animals receiving 10G1-K DLL3 CAR-T cells had minimal or mild mononuclear cell infiltration in the pituitary, mainly in the intermediate and neural lobes (Figure 15C-D). These animals also had slightly more (moderately less) hCD45+ staining associated with small foci of glioma compared to rare / sparse staining in other regions of the brain (cerebral vasculature, choroid plexus, and meninges), consistent with the antitumor 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 on the pituitary gland, mouse pituitaries from NSG mice were harvested under sterile conditions for in vitro analysis. Tissues were dissociated by three rounds of incubation at 37°C in 1 mL of dissociation mix [5 mL DMEM, high glucose, GlutaMax (Gibco, Cat. No. 10564), 50 uL Enzyme H, 5 uL Enzyme R, 6.25 uL Enzyme A (Miltenyi Tumor Dissociation Kit No. 130-095-929)], followed by mechanical dissociation using trituration. 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. Cells were pelleted and treated with ACK lysis buffer for 3 min at RT, followed by neutralization in complete medium. The cell suspension was filtered through a 70u filter and centrifuged to remove the buffer. Cells were counted and plated in a 96-well plate at 5x10 per well in complete medium. 4 Cells were seeded and allowed to recover for 3 days before CAR-T cells were added, at which point the expected target density was 1 × 10 per well. 4 As a 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 targets for 3 days. At the end of the 3-day co-culture, media was separated from the wells and centrifuged to pellet the T cells. Target cells were treated with 50uL / well Cell Titer Glo (Promega, G7570) for 10 minutes and analyzed for cytotoxicity readings on a SpectraMax plate reader.

[0290] Figure 16A shows experimental data showing that DLL3 CAR-T cells are active against 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 analysis by flow cytometry. 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 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 DLL3+DMS 273 cell lines, but there is no cytokine secretion after co-culture of 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 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, 454, 463, and 696; (e) 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 (f) a chimeric antigen receptor comprising at least one of the 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.

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) 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; and (c) a chimeric antigen receptor comprising 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.

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 (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 chimeric antigen receptor comprising 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.

4. 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) at least one of 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; The chimeric antigen receptor, wherein the variable heavy chain and the variable light chain are linked by at least one linker.

5. 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; The chimeric antigen receptor, wherein the variable heavy chain and the variable light chain are linked by at least one linker.

6. 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 comprises a sequence selected from the group consisting of a scFv presented in Table 1d.

7. 1. A chimeric antigen receptor that specifically binds to DLL3, wherein 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 632-683.

8. The chimeric antigen receptor of any one of claims 1 to 7, wherein the intracellular domain comprises at least one costimulatory domain.

9. The costimulatory domain may be any of the following: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function associated antigen-1 (LFA-1 (CD1 la / 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), activating NK cell receptor, BTLA, Toll ligand receptor Body, 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, 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 , DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT The chimeric antigen receptor according to claim 8, which is a signal transduction region of a ligand that specifically binds to AM, 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 any combination thereof.

10. The chimeric antigen receptor of claim 9, wherein the costimulatory domain comprises the signal transduction region of 4-1BB / CD137.

11. The chimeric antigen receptor of claim 10, wherein the 4-1BB / CD137 costimulatory domain comprises SEQ ID NO: 480 or a fragment thereof.

12. The chimeric antigen receptor of any one of claims 1 to 11, wherein the intracellular domain comprises at least one activation domain.

13. The chimeric antigen receptor of claim 12, wherein the activation domain comprises CD3.

14. The chimeric antigen receptor of claim 13, wherein the CD3 comprises CD3 zeta.

15. The chimeric antigen receptor of claim 14, wherein the CD3 zeta comprises SEQ ID NO: 481 or a fragment thereof.

16. The chimeric antigen receptor of any one of claims 1 to 15, wherein the chimeric antigen receptor is encoded by a polynucleotide sequence of any one of SEQ ID NOs: 570-621 and 631.

17. The chimeric antigen receptor of any one of claims 1 to 16, further comprising a safety switch.

18. The chimeric antigen receptor of claim 17, wherein the safety switch comprises a CD20 mimotope or a QBEND-10 epitope.

19. The chimeric antigen receptor of claim 18, wherein the safety switch comprises one or more CD20 mimotopes or one or more QBEND-10 epitopes, or a combination thereof.

20. The chimeric antigen receptor of any one of claims 17 to 19, wherein the chimeric antigen receptor comprises one or more safety switches in the format of QR3, SR2, RSR, or R2S.

21. The chimeric antigen receptor of any one of claims 17-20, wherein 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.

22. An isolated polynucleotide encoding any one of the chimeric antigen receptors of claims 1 to 21.

23. A vector comprising the polynucleotide of claim 22.

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

25. An engineered immune cell expressing any one of the chimeric antigen receptors of claims 1 to 21.

26. 25. An engineered immune cell expressing the polynucleotide of claim 22 or the vector of claim 23 or 24.

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

29. 28. The engineered immune cell of claim 27, wherein the cell is an allogeneic T cell.

30. A pharmaceutical composition comprising an engineered immune cell according to any one of claims 25 to 29.

31. 31. A method of treating a disease or disorder in a subject in need thereof, comprising administering to said subject an engineered immune cell according to any one of claims 25 to 29, or a pharmaceutical composition according to claim 30.

32. 32. The method of claim 31 , wherein the disease or disorder is cancer.

33. 33. The method of claim 31 or 32, wherein the disease or disorder is small cell lung cancer.

34. 32. An article of manufacture comprising an engineered immune cell according to any one of claims 26 to 325 to 29, or a pharmaceutical composition according to claim 30.

35. (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, 454, 463, and 696; (e) 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 (f) an anti-DLL3 binding agent comprising 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.

36. The binding agent is an antibody, an antibody conjugate, or an antigen-binding fragment thereof, optionally an F(ab') 2 36. The DLL3-binding agent of claim 35, which is a Fab' fragment, a Fab fragment, an Fv fragment, a scFv fragment, a dsFv fragment, or a dAb fragment.

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

38. 38. The anti-DLL3 binding agent of any one of claims 35-37, comprising a variable heavy (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to a VH sequence provided in Table lb.

39. 39. The anti-DLL3 binding agent of any one of claims 35-38, comprising a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to a VL sequence provided in Table 1c.

40. 40. The anti-DLL3 binding agent of any one of claims 35-39, wherein the binding agent comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to a scFv sequence presented in Table 1d.

41. 41. The anti-DLL3 binding agent of 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. 42. A pharmaceutical composition comprising an anti-DLL3 binding agent according to any one of claims 35 to 41 and a pharma- ceutically acceptable excipient.

43. A method of treating a disease or disorder in a subject in need thereof comprising administering to the subject an anti-DLL3 binding agent according to any one of claims 35 to 41, or a pharmaceutical composition according to claim 42.

44. 44. The method of claim 43, wherein the disease or disorder is cancer.

45. 45. The method of claim 43 or 44, wherein the disease or disorder is small cell lung cancer.

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