DLL3-targeted chimeric antigen receptor and conjugate

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 of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 812,585, filed on 1 March 2019, and U.S. Provisional Patent Application No. 62 / 969,976, filed on 4 February 2020, all of which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to a chimeric antigen receptor (CAR) comprising a DLL3 conjugate and an antigen-binding molecule that binds to DLL3, a polynucleotide encoding the same, and a method for treating cancer in a patient using the same.

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

[0004] Small cell lung cancer (SCLC) is an invasive form of lung cancer with a poor prognosis and limited treatment options. SCLC accounts for 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 therapeutic treatments for cancer include chemotherapy and radiotherapy. Patients typically respond well to current first-line therapies, including etoposide and cisplatin, but always relapse quickly with chemotherapy-resistant disease. The prognosis in relapsed, refractory situations is very poor, with rapid disease progression and a short median survival of less than 6 months. Therefore, there remains a great need to develop more targeted and potent therapies for proliferative disorders.

[0005] Adoptive transfer of immune cells genetically modified to recognize malignant tumor-associated antigens has shown promise as a novel approach to treating 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 consisting 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)). Immune cells containing CARs, such as CAR-T cells (CAR-Ts), are engineered to confer antigen specificity to them while retaining or enhancing their ability to recognize and kill target cells.

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

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

[0008] In one embodiment, the present disclosure provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises (a) 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, 3 (b) Variable heavy chain CDR1 containing an amino acid sequence selected from the group consisting of 43, 352, 361, 370, 379, 388, 397, 406, 415, 424, 433, 442, 451, and 460, (b) 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 Variable heavy chain CDR2 containing an amino acid sequence selected from the group consisting of 389, 398, 407, 416, 425, 434, 443, 452, 461, and 695, (c) 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 Variable heavy chain CDR3 containing an amino acid sequence selected from the group consisting of 8, 417, 426, 435, 444, 453, and 462, (d) 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,Variable light chain CDR1 containing an amino acid sequence selected from the group consisting of 454, 463, and 696, (e) 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 (f) A variable light chain CDR2 containing a column, and at least one variable light chain CDR3 containing 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 comprises (a) 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 Variable heavy chain CDR1 containing an amino acid sequence selected from the group consisting of 5, 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) SEQ ID NOs: 2, 11, 20, 38, 47, 56, 65, 74, 83, 92, 101, 110, 119, 128, 137, 146, 155, 164, 17 Variable heavy chain CDR2 containing amino acid sequences selected from the group consisting of 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, as well as (c) SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57, 66, 75, 8 It contains a variable heavy chain CDR3 containing 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 embodiment, the present disclosure provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen-binding domain that specifically binds to DLL3, and the antigen-binding domain comprises (a) 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 (b) Variable light chain CDR1 containing an amino acid sequence selected from the group consisting of 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) SEQ ID NOs: 5, 14, 23, 32, 41, 50, 59, 68, 77, 86, 95, 104, 113, 122, 131, 140, 149, 158, 167 Variable light chain CDR2 containing an amino acid sequence selected from the group consisting of 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, as well as (c) SEQ ID NOs: 6, 15, 24, 33, 42, 51, 60, 69, 78, 8 It contains a variable light chain CDR3 having 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 comprises (a) SEQ ID NOs: 7, 16, 25, 34, 43, 52, 61, 70, 79, 88, 97, 106, 115, 124, From 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 Variable heavy chains containing amino acid sequences selected from the group, and (b) 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 It comprises at least one variable light chain containing an amino acid sequence selected from the group consisting of 305, 314, 323, 332, 341, 350, 359, 368, 377, 386, 395, 404, 413, 422, 431, 440, 449, 458, and 467, and the variable heavy chain and variable light chain are linked by at least one linker.

[0012] In a further embodiment, 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) SEQ ID NOs: 7, 16, 25, 34, 43, 52, 61, 70, 79, 88, 97, 106, 115, 1 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, o (b) Variable heavy chains containing amino acid sequences selected from the group consisting of 466, and (b) 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 The variable light chain comprises 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, and the variable heavy chain and variable light chain are linked by at least one linker.

[0013] In one embodiment, 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 scFv presented in Table 1d.

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

[0015] In some embodiments, the Disclosure provides a chimeric antigen receptor that specifically binds to DLL3, the chimeric antigen receptor comprising 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.

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

[0017] In some embodiments, the co-stimulatory domain of the chimeric antigen receptor may include CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (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 I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activator molecule (SLAM protein), activated NK cell receptor, BTLA, and 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 These are signaling regions of ligands that specifically bind 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, and CD83, or any combination thereof.

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

[0019] In some embodiments, the CD28 co-stimulatory domain includes sequence number 550.

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

[0021] In some embodiments, the 4-1BB / CD137 co-stimulatory domain includes sequence number 480.

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

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

[0024] In some embodiments, CD3 includes a CD3 zeta.

[0025] In some embodiments, the CD3 zeta includes sequence number 481.

[0026] In some embodiments, the chimeric antigen receptor is encoded by one of the polynucleotide sequences of sequence numbers 571-621 and 631.

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

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

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

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

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

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

[0033] In some embodiments, the disclosure provides isolated polynucleotides 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 adenovirus vector, an adenovirus-related vector, a lentiviral vector, or any combination thereof.

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

[0037] In some embodiments, the disclosure provides engineered immune cells expressing a polynucleotide or vector encoding any one of the chimeric antigen receptors described herein.

[0038] In some embodiments, the manipulated 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 manipulated immune cells are autologous T cells.

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

[0041] In some embodiments, the manipulated immune cells are knocked-out TCRs (e.g., TCRα, TCRβ).

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

[0043] In some embodiments, the present disclosure provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a pharmaceutical composition comprising engineered immune cells or engineered immune cells expressing a chimeric antigen receptor as described herein to the subject.

[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 embodiments, the Disclosure provides a product comprising a pharmaceutical composition comprising engineered immune cells or engineered immune cells expressing a chimeric antigen receptor as described herein.

[0047] In some embodiments, the present disclosure provides anti-DLL3 binders disclosed herein.

[0048] In some embodiments, the anti-DLL3 conjugate 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 binder is a monoclonal antibody containing an IgG constant region.

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

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

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

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

[0054] In some embodiments, the Disclosure provides a pharmaceutical composition comprising an anti-DLL3 binder and a pharmaceutically acceptable excipient disclosed herein.

[0055] In some embodiments, the Disclosure provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering an anti-DLL3 binder, or a pharmaceutical composition containing an anti-DLL3 binder, to the subject, 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 explanation of the drawing]

[0058] [Figure 1A-1B] This series of plots shows that the purified anti-DLL3 antibody described herein binds to three DLL3-expressing small cell lung cancer cell lines (SHP-77, DMS 273, and DMS 454). Solid and dashed lines represent staining with the anti-DLL3 antibody or the mouse IgG2A isotype control antibody, respectively. [Figure 2A-2D] The results of the epitope mapping experiment are shown. Figure 2A is a schematic diagram of full-length and truncated human DLL3 proteins expressed on CHO cells for epitope mapping; 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 the 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, with the x-axis showing the signal from the PE channel and the y-axis showing the count. [Figure 3A-3D] Figure 3A is a series of plots and tables showing the structure, transduction efficiency of cells from two different donors, and cytotoxic activity of the anti-DLL3 CAR. Figure 3A is a schematic diagram of the construct encoding the anti-DLL3 CAR, including the anti-DLL3 scFv from the N-terminus to the C-terminus, the hinge and transmembrane regions from human CD8α, the cytoplasmic region from human 41BB, and the cytoplasmic region from CD3ζ. Figure 3B shows experimental data showing that the anti-DLL3 CAR is expressed on the surface of primary T cells and can recognize recombinant DLL3, with the plots gated with living 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 the anti-DLL3 CAR, including the scFv sequence described herein. [Figure 4A-4C]Figure 4A shows a series of plots illustrating the killing data of several anti-DLL3 CARs. Figure 4A shows experimental data demonstrating that anti-DLL3 CAR-T cells specifically killed human DLL3-expressing HEK-293 T cells but not parental HEK-293 T 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 demonstrating that anti-DLL3 CAR-T cells killed endogenous DLL3-expressing SHP-77 and WM266.4 cells in a 3-day cytotoxicity assay at the indicated effector:target ratio. Figure 4C shows experimental data demonstrating that anti-DLL3 CAR-T cells killed endogenous DLL3-expressing DMS 454 and DMS 273 small cell lung cancer cells in a 3-day cytotoxicity assay at the indicated effector:target ratio. For all plots in Figures 4A-4C, target cell viability was evaluated using the One-glo assay system (n=3). [Figure 5] This is a series of bar graphs showing that anti-DLL3 CAR-T cells released cytokines after co-incubation with DLL3-expressing SHP-77 cell lines when CAR-T cells and SHP-77 cells were incubated for 24 hours in 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 Pro-inflammatory 9Plex Kit from MSD (n=3). [Figure 6A-6C]These plots show experimental data from serial killing assays of DLL3+ cell lines after repeated exposure to anti-DLL3 CAR-T cells. Figure 6A shows serial killing of anti-DLL3 CAR-T cells against DLL3+ WM266.4 cells. Some 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 cells against the DMS 273 small cell lung cancer line. Target cell viability was assessed for all plots in Figures 6A-6C using a one-glo assay or CellTiter-glo system (n=3-5). [Figures 7A-7B] This plot shows that anti-DLL3 CAR-T cells dose-dependently eliminated established SHP-77 small cell lung cancer subcutaneous tumors in mice. [Figure 8] This plot shows 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) from day 14 to day 28, n=4–5. *, p<0.05. **, p<0.01. [Figures 9A-9C] The primary T cell structure, transduction efficiency, and cytotoxic activity of anti-DLL3 CARs with safety switches are shown. 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 a flow cytometry plot showing that the anti-DLL3 CARs with safety switches shown in Figure 9A are expressed on the surface of primary T cells and can recognize recombinant DLL3. The plot is gated with living CD3+ cells, and the numbers on the plot 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 a serial kill assay. [Figure 10A]This plot shows that two xenograft (PDX) models derived from small cell lung cancer patients 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 are presented showing that anti-DLL3 CAR-T exhibits cytotoxic activity against the same two PDX models. [Figures 11A-11B] The safety switch enables the detection and depletion of DLL3 CAR-T cells by rituximab. Figure 11A shows 8E11-SR2 and 26C8-R2S anti-DLL3 CAR-T cells stained with recombinant DLL3 and PE conjugate rituximab 14 days after proliferation and analyzed using flow cytometry. The numbers in the quadrants represent the percentage of total T cells. Figure 11B shows the rituximab-mediated complement-dependent cytotoxicity (CDC) of 8E11-SR2 and 26C8-R2S anti-DLL3 CAR-T cells. CAR-T cells were incubated with 25% young rabbit complement and rituximab for 3 hours, and cytotoxicity was assessed using flow cytometry. [Figures 12A-12B] The plots show that anti-DLL3 CAR-T cells with a safety switch inhibited the growth of subcutaneous or IV-injected small cell lung cancer tumors. Figure 12A shows that DLL3 CAR-T cells with a safety switch eliminated established SHP-77 subcutaneous tumors of small cell lung cancer in mice. Figure 12B is a plot showing that anti-DLL3 CAR-T cells inhibited the growth of IV-injected DMS 273-DLL3 small cell lung cancer tumors. [Figure 13A] This image shows a representative image of mouse DLL3 RNA expression in the brain of NSG mice. The circles indicate clusters of mouse DLL3 RNA. [Figure 13B] Anti-human CD3 staining is shown for spleen, pituitary, and brain samples from animals treated with untransduced T cells, 10G1-K DLL3 CAR-T cells, or 2G1 DLL3 CAR-T cells. Arrows indicate CD3-positive cells in the spleen. [Figures 14A-14F]This document describes the experimental design and results of a mouse safety study using a subcutaneous LN229-mDLL3 tumor model. Figure 14A shows the study group and experimental design. Figure 14B shows the timing of tissue sampling and tumor volume from animals that received either non-transduced T cells or DLL3 CAR-T cells. Figure 14C is a table showing the human CD3 staining scores of brain and pituitary samples. Figure 14D is a table showing the histological analysis of the collected brain and pituitary samples. Figure 14E shows images of pituitary samples stained with anti-vasopressin antibody. Figure 14F shows images of pituitary samples stained with anti-oxytocin antibody. [Figures 15A-15D] This document presents the experimental design and results of a mouse safety study using an intracranial LN229-mDLL3 tumor model. Figure 15A shows the study group and experimental design. Figure 15B shows the timing of tissue sampling and tumor volume from animals treated with untransduced T cells, DLL3 CAR-T cells, or EGFRvIII CAR-T cells. Figure 15C is a table showing human CD45 staining scores for brain, pituitary, and spleen samples. Figure 15D is a table showing histological analysis of brain and pituitary samples. [Figure 16A-16C] The following shows experimental data on the in vitro cytotoxicity of dissociated mouse pituitary cells. Figure 16A shows the cytotoxic readout of target cells after 3 days of co-culture with DLL3 CAR-T cells, demonstrating that DLL3 CAR-T is not cytotoxic to mouse pituitary cells in vitro. Figure 16B shows flow cytometry analysis of surface staining for the activation markers CD25 and 41BB of T cells co-cultured with the target, demonstrating that mouse pituitary cells do not activate DLL3 CAR-T in vitro. Figure 16C shows cytokines secreted in the cell culture medium, analyzed by MSD, demonstrating that no cytokines are secreted after DLL3 CAR-T cells are co-cultured with mouse pituitary cells in vitro for 3 days. [Modes for carrying out the invention]

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

[0060] I. DLL-3 binder This disclosure provides DLL-3 conjugates (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 containing enough canonical immunoglobulin sequence elements to give specific binding to a particular target antigen (e.g., DLL-3). As known in the art, naturally produced intact antibodies are tetramers of about 150 kD, consisting of two identical heavy-chain polypeptides (each about 50 kD) and two identical light-chain polypeptides (each about 25 kD), which associate with each other in what is commonly called a “Y-shaped” structure. Each heavy chain consists 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). Short regions, known as “switches,” connect the heavy-chain variable and constant regions. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. The two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in the intact antibody. Each light chain consists of two domains, an amino-terminal variable (VL) domain separated from each other by another "switch," followed by a carboxy-terminal constant (CL) domain. Those skilled in the art are familiar with antibody structures and sequence elements and recognize the "variable" and "constant" regions in the given sequence, and understand that there may be some flexibility in defining the "boundaries" between such domains, and that different presentations of the same antibody chain sequence may indicate such boundaries at positions shifted by one or several residues relative to different presentations of the same antibody chain sequence.

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

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

[0063] An intact antibody tetramer consists of two heavy-light chain dimers, where the heavy and light chains are linked to each other by a single disulfide bond, with two other disulfide bonds connecting the heavy chain hinge regions, resulting in the dimers being linked to each other and forming 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., 3, 4, or 5-strand 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 naturally occurring antibody folds, the FR region forms 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 so that a single hypervariable antigen-binding site is created, with them located at the tip of the Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also 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 to Fc receptors can be modulated by glycosylation or other modifications. In some embodiments, the antibody produced and / or utilized according to the present invention comprises a glycosylated Fc domain, which includes an Fc domain having such modified or manipulated glycosylation.

[0064] For the purposes of the present invention, in certain embodiments, any polypeptide or polypeptide complex containing sufficient immunoglobulin domain sequences, as found in natural antibodies, may be referred to and / or used as “antibody,” whether such polypeptides are naturally produced (e.g., by organisms reacting to an antigen) or produced by recombinant operations, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal; in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primated, chimeric, etc., as known in the art.

[0065] Furthermore, as used herein, the term “antibody” may, in appropriate embodiments (unless otherwise stated or evident from the context), refer to any construct or format known or developed in the art for utilizing the structural and functional characteristics of an antibody in an alternative presentation. For example, in some embodiments, the antibodies utilized according to the present invention include intact IgA, IgG, IgE, or IgM antibodies; bi- or multi-specific antibodies (e.g., Zybodies®); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camel antibodies; masked antibodies (e.g., Probodies®); small modular immunotherapies ("SMIP®"); single-chain or tandem diabodies (e.g., TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or The format is selected from, but is 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 covalent modifications (e.g., glycan attachments) that it would have if it were naturally produced. In some embodiments, the antibody may contain covalent modifications (e.g., glycan attachments), a payload (e.g., a detectable portion, a therapeutic portion, a catalytic portion, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0066] Antibodies contain antibody fragments. Antibodies can be polyclonal, monoclonal, chimeric dAb (domain antibody), single-chain, or F abF a F (ab)2 Fragments, scFv, and F ab Expression libraries are included, but not limited to, antibody libraries. Antibodies may be whole antibodies, immunoglobulins, or antibody fragments.

[0067] As detailed above, all antibodies consist of two pairs: a "light chain" (LC) and a "heavy chain" (HC) (such a light chain (LC) / heavy chain pair is abbreviated as LC / HC herein). The light and heavy chains of such antibodies are polypeptides consisting of several domains. In all antibodies, each heavy chain contains a heavy chain variable region (abbreviated as HCVR or VH herein) and a heavy chain constant region. The heavy chain constant region contains 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 contains a light chain variable domain VL and a light chain constant domain CL. The variable domains VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl 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). The two pairs of heavy and light chains (HC / LC) can specifically bind to the same antigen. Thus, the entire antibody is a bivalent monospecific antibody. Such “antibodies” include, for example, mouse antibodies, human antibodies, chimeric antibodies, humanized antibodies, and genetically modified antibodies (variant or mutant antibodies) as long as their characteristic properties are retained. In some embodiments, the antibody or binder is a humanized antibody, in particular as a recombinant human or humanized antibody.

[0068] In some embodiments, the antibody or conjugate may be "symmetrical." "Symmetrical" means that the antibody or conjugate has the same type of Fv region (for example, the antibody has two Fab regions). In some embodiments, the antibody or conjugate may be "asymmetrical." "Asymmetrical" means that the antibody or conjugate has at least two different types of Fv region (for example, the antibody has Fab and scFv regions, Fab and scFv2 regions, or Fab-VHH regions). Various asymmetric antibody or conjugate 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 a drug that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex containing sufficient immunoglobulin structural elements to provide specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may comprise one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may comprise one or more sequence elements that are humanized, primated, chimeric, etc., as known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, antibody agents used in accordance with the present invention include intact IgA, IgG, IgE, or IgM antibodies; bi- or multi-specific antibodies (e.g., Zybodies®); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camel antibodies; masked antibodies (e.g., Probodies®); small modular immunotherapies ("SMIP®"); single-chain or tandem diabodies (e.g., TandAb (Registered Trademark)); VHH; Anticalins(Registered Trademark); Nanobodies(Registered Trademark) Minibodies; BiTE(Registered Trademark); Ankyrin Repeat Protein 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) are selected and are not limited to these formats.

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

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

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

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

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

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

[0076] DLL3 conjugates (e.g., antibodies) capable of binding to various regions or domains of the DLL3 target are provided herein. The epitope may be, for example, an adjacent amino acid of the DLL3 target (linear or adjacent epitope) or may be composed of two or more non-adjacent regions of the DLL3 target (conformational, nonlinear, discontinuous, or non-adjacent epitopes). The epitope to which the DLL3 antigen-binding domain binds can be determined by various assays, e.g., 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 mutagenic mapping (e.g., site-directed mutagenic mapping).

[0077] A typical DLL3 region or domain is shown in Figure 2. The exemplary DLL3 antibodies described herein bind to the DLL3 domains provided in Table 1a. [Table 1]

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

[0079] In some embodiments, the DLL3 binder comprises a variable light chain (VL), the amino acid sequence of the VL being selected from the VL sequences presented in Table 1c. In some embodiments, the anti-DLL-3 binder comprises an immunoglobulin light chain having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences presented in Table 1c. [Table 3-1] [Table 3-2] [Table 3-3]

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

[0081] In some embodiments, the DLL-3 binder includes heavy chains CDR1, CDR2, and CDR3. In some embodiments, the heavy chain sequences CDR1, CDR2, and CDR3 are selected from the heavy chain CDRs presented in Table 1e. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0082] In some embodiments, the DLL-3 binder includes light chains 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] This disclosure encompasses modifications to DLL3 antibody agents including the sequences shown in Tables 1b-1e, and includes functionally equivalent DLL3 antibody agents with modifications that do not significantly affect their properties, as well as variants with enhanced or reduced activity and / or affinity. For example, amino acid sequences may be mutated to obtain a DLL3 antigen conjugate with a desired binding affinity to DLL3. Polypeptide modifications are routine practice in the art and do not need to be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, those with deletions or additions of one or more amino acids that do not significantly and detrimentally alter functional activity, or those that mature (enhance) the polypeptide's affinity to its ligand, or polypeptides with the use of chemical analogues.

[0084] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus 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 antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include the fusion of enzymes or polypeptides to the N or C terminus of an antibody that increases the antibody's half-life in the bloodstream.

[0085] Substitutional variants have at least one amino acid residue in the removed antigen-binding domain and a different residue inserted in its place. In some embodiments, the target site for substitutional mutagenesis includes a hypervariable region / CDR, but FR changes are also intended. Conservative substitutions are shown in Table 2 under the heading "Conservative Substitutions". If such substitutions result in changes in biological activity, more substantial changes, referred to as "exemplary substitutions" in Table 2 or further described below in reference to amino acid classes, may be introduced and the product may be screened. [Table 6]

[0086] i.Antibody fragment In one embodiment, the anti-DLL-3 antibody agent according to any of the above embodiments may be an antibody fragment. The 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, diabody, linear antibody, antibody fragment antibody, and multispecificity formed from scFv fragments, as well as other fragments described below. In some embodiments, the antibody is a full-length antibody, such as an intact IgG1 antibody, or another antibody class or isotype as described herein. (See, for example, Hudson et al., Nat. Med., 9:129-134 (2003), Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, pp. 269-315 (1994), Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), WO93 / 01161, and U.S. Patents No. 5,571,894, No. 5,869,046, No. 6,248,516, and No. 5,587,458). A full-length antibody, intact antibody, or whole antibody is an antibody having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein. Antibody fragments can be prepared by a variety of techniques, including, but not limited to, the proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as is known in the art.

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

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

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

[0090] Fv and scFv are species that have intact binding sites that do not contain a constant region. Therefore, they may be suitable for reduced nonspecific binding during in vivo use. Single-chain Fv (sFv or scFv) is a V attached to a single polypeptide chain. H and V L It is an antibody fragment containing an antibody domain. The sFv polypeptide allows the sFv to form the desired structure for antigen binding. H Domain and V L The scFv fusion protein may further include a polypeptide linker between the domain and the fusion protein (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, see below). The scFv fusion protein can be constructed to result in the fusion of the effector protein at either the amino or carboxyl terminus of the sFv. The 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 includes an scFv containing the light chain variable (VL) and heavy chain variable (VH) regions of a DLL3-specific monoclonal antibody bound by a flexible linker. Single-chain variable region fragments can be prepared by binding the light chain and / or heavy chain variable regions using a linking peptide. An example of a linking peptide is its amino acid sequence (GGGGS). x The linker is a GS linker having , 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). Generally, the linker can be a short, flexible polypeptide, and in some embodiments, it consists of about 20 or fewer amino acid residues. The linker can then be modified for additional functions, such as drug attachment or attachment to a solid support. Single-stranded variants can be produced either recombinantly or synthetically. Automated synthesizers can be used for the synthetic production of scFv. For recombinant production of scFv, a suitable plasmid containing the polynucleotide encoding scFv can be introduced into a suitable host cell, either a eukaryote such as yeast, plant, insect, or mammalian cell, or a prokaryote such as E. coli. The polynucleotide encoding the desired scFv can be prepared by routine operations such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0092] In exemplary embodiments, a DLL3 antigen-binding domain is provided herein, comprising a VH region including VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in Table 1b, and / or a VL region including 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 may be encoded by a DNA sequence comprising GGCGGTGGAGGCTCCGGAGGGGGGGGCTCTGGCGGAGGGGGCTCC (SEQ ID NO: 564). In some embodiments, the linker may be encoded by a DNA sequence comprising gggggcggcggctctggaggaggaggcagcggcggaggaggctccggaggcggcggctct (SEQ ID NO: 630). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 534). In some embodiments, the linker is an scFv Whitlow linker which may comprise the amino acid sequence GTSTGSGKPGSGEGSTKG (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 scFv can be oriented with the VH sequence located at the N-terminus of the scFv, followed by the linker, and then the VL sequence, while in other embodiments, the scFv can be oriented with the VL sequence at the N-terminus, followed by the linker, and then the VH sequence.

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

[0094] In some embodiments, the anti-DLL-3 antibody agents provided herein may be chimeric antibodies (see, for example, U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). A chimeric antibody may be an antibody in which a portion of the heavy chain and / or light chain originates from a particular source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species. In one example, a chimeric antibody may 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 further examples, a chimeric antibody may be a “class-switched” antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody contains its antigen-binding fragment.

[0095] In some embodiments, the chimeric antibody may be a humanized antibody (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. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005), Padlan, Mol. Immunol, 28:489-498 (1991), Dall'Acqua et al. See 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). Humanized antibodies are chimeric antibodies containing amino acid residues from non-human hypervariable regions and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, with all or substantially all of the hypervariable regions (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody.

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

[0097] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the “best fit” method, framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions, human mature (somatic mutation) framework regions or human germline framework regions, and framework regions derived from screening FR libraries (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. See al., J. Biol. Chem., 271:22611-22618 (1996).

[0098] iii. Human antibodies In some embodiments, the anti-DLL-3 antibody agents provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art (see, 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 may be produced by humans or human cells, or may have amino acid sequences corresponding to the amino acid sequences of antibodies derived from the human antibody repertoire or non-human sources that utilize sequences encoding other human antibodies. This definition of human antibodies specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be prepared by administering an immunogen (e.g., DLL-3 protein) to an intact human antibody or a transgenic animal modified to produce an intact antibody having a human variable region in response to an antigen challenge (see, for example, Lonberg, Nat. Biotech., 23:1117-1125 (2005), U.S. Patents 6,075,181, 6,150,584, 5,770,429, and 7,041,870, and U.S. Patent Application Publication US2007 / 0061900). The human variable region from the intact antibody produced by such an animal can be further modified, for example, by combining it with a different human constant region.

[0099] Human antibodies can also be produced by hybridoma-based methods. For example, human antibodies can be produced from human myeloma and mouse-human heterozygous myeloma cell lines using human B-cell hybridoma technology and other methods (e.g., Kozbor, J. Immunol, 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (1987), Boerner et al., J. Immunol, 147:86 (1991), Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), U.S. Patent No. 7,189,826, Ni, Xiandai Mianyixue, 26(4):265-268 (2006), Vollmers and Brandlein, Histology and See 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 region.

[0100] Modification of oligosaccharides in antibodies can be performed, for example, to create antibody variants with specific improved properties. For example, an antibody glycosylated variant may have improved CDC function. In some embodiments, this disclosure may envision antibody variants with some, but not all, effector functions, which are desirable candidates for applications where the half-life of the antibody in vivo is important, but 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 preparations provided herein may be further modified to include additional non-proteinoid moieties known in the art and readily available. Moieties suitable for antibody derivatization 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 homopolymers, oxidized polypropylene / oxidized ethylene copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water.

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

[0103] In some embodiments, an antibody-nonproteinate conjugate is provided that can be selectively heated by exposure to radiation. In some embodiments, the nonproteinate portion may be a carbon nanotube (see, for example, Kam et al., Proc. Natl. Acad. Sci. USA, 102:11600-11605 (2005)). The radiation may be of any wavelength that does not harm normal cells, but may include, but is not limited to, wavelengths that heat the nonproteinate portion to a temperature in which cells proximal to the antibody-nonproteinate portion are killed.

[0104] A DLL3 binder (e.g., a molecule containing an antigen-binding domain) is said to "specifically bind" to its target antigen (e.g., human, cynomolgus monkey, or mouse DLL3) if its dissociation constant (Kd) is approximately 1 nM. The antigen-binding domain specifically binds to the antigen with "high affinity" when its Kd is 1-5 nM, and with "very high affinity" when its Kd is 0.1-0.5 nM. In one embodiment, the antigen-binding domain has a Kd of approximately 1 nM. In one embodiment, the offrate is <1 × 10⁻⁶ -5 In other embodiments, the antigen-binding domain is approximately 1 × 10⁶ to human DLL3. -7 M~1×10 -12 In yet another embodiment, the antigen-binding domain is approximately 1 x 10 -5 M~1x10 -12 It will likely be joined by M's Kd.

[0105] As provided herein, the antigen-binding domain of this 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 this disclosure binds to mammalian DLL3 at a rate of 1 × 10⁻¹⁶. -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, or 1 × 10 -9 It binds with a Kd of less than M. 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 rate of 1 × 10⁻¹⁶ -7 It binds with a Kd of less than M. In another embodiment, the DLL3 antigen-binding domain binds to mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3, or mouse DLL3) at a rate of 1 × 10⁻¹⁶ -8 It binds with a Kd of less than M. In some embodiments, the DLL3 antigen-binding domain binds to mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3) at approximately 1 × 10⁻¹⁶ values. -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 is joined by Kd. In certain embodiments, Kd is K off / K on It is calculated as the quotient of K on and K off This is determined, for example, using a monovalent antibody such as a Fab fragment, measured by BIAcore® surface plasmon resonance technology. In other embodiments, Kd is K off / K on It is calculated as the quotient of K on and K off This 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 configured to bind mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3, or mouse DLL3) to 1 × 10⁶ -4 M -1 s- 1 Less than 2 × 10 -4 M -1 s- 1 Less than 3 x 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 x 10 -6 M -1 s- 1 Less than 8 x 10 -6 M -1 s- 1 Less than 9 x 10 -6 M -1 s- 1 Less than, or 1 × 10⁻⁶ -7 M -1 s- 1 Meeting rate less than (k on ) is joined. In certain embodiments, k on This is determined, for example, using a monovalent antibody such as a Fab fragment, as measured by BlAcore® surface plasmon resonance technology. In other embodiments, k on This is determined, for example, using a bivalent antibody, such as one measured by BlAcore® surface plasmon resonance technology.

[0107] In some embodiments, the DLL3 antigen-binding domain is configured to bind mammalian DLL3 (e.g., human DLL3, cynomolgus monkey DLL3, or mouse DLL3) to 1 × 10⁶ -2 s -1 Less than 2 × 10 -2 s -1 Less than 3 x 10 -2 s -1 Less than 4 x 10 -2 s -1 Less than 5 x 10 -2 s -1 Less than 6 × 10 -2 s -1 Less than 7 x 10 -2 s -1 Less than 8 x 10 -2 s -1 Less than 9 x 10 -2 s -1 Less than 1 × 10 -3 s -1 Less than 2 × 10 -3 s -1 Less than 3 x 10 -3 s -1 Less than 4 x 10 -3 s -1 Less than 5 x 10 -3 s-1 Less than 6 × 10 -3 s -1 Less than 7 x 10 -3 s -1 Less than 8 x 10 -3 s -1 Less than 9 x 10 -3 s -1 Less than 1 × 10 -4 s -1 Less than 2 × 10 -4 s -1 Less than 3 x 10 -4 s -1 Less than 4 x 10 -4 s -1 Less than 5 x 10 -4 s -1 Less than 6 × 10 -4 s -1 Less than 7 x 10 -4 s -1 Less than 8 x 10 -4 s -1 Less than 9 x 10 -4 s -1 Less than 1 × 10 -5 s -1 Less than, or 5 x 10 -4 s -1 Dissociation rate less than (k off ) is joined. In certain embodiments, k off This is determined, for example, using a monovalent antibody such as a Fab fragment, as measured by BlAcore® surface plasmon resonance technology. In other embodiments, k off This is determined, for example, using a bivalent antibody, such as one measured by BlAcore® surface plasmon resonance technology.

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

[0109] The chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising 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., anti-DLL3 scFv), a hinge and transmembrane region, and one or more consecutive 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 containing the DLL3 variable heavy chain and / or variable light chain described herein, a CD8α hinge and transmembrane region, a 41BB cytoplasmic signaling domain, and a CD3ζ cytoplasmic signaling domain (Figure 4, Table 7).

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

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

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

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

[0114] Variants of antigen-binding domains (e.g., variants of CDRs, VH, and / or VL) 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 more 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). One of ordinary 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, one of ordinary skill in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not considered important for activity.

[0115] In certain embodiments, the polypeptide structure of the antigen-binding domain is antibody-based and includes, without limitation, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. In some embodiments, the antigen-binding domain comprises or consists of an affimer.

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

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

[0118] In some embodiments, the DLL3-specific CAR comprises a leader or signal peptide, and in some embodiments, the leader peptide 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, this disclosure relates to isolated polynucleotides encoding any one of the DLL3 antigen-binding domains described herein. In some embodiments, this disclosure relates to isolated polynucleotides encoding the DLL3 CARs listed in Table 10. A vector containing a polynucleotide and a method for constructing the same are also provided herein. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] Table 8-7 Table 8-8 Table 8-9 Table 8-10 Table 8-11 Table 8-12 Table 8-13 Table 8-14 Table 8-15 Table 8-16 Table 8-17 Table 8-18 Table 8-19 Table 8-20 [Table 8-21] [Table 8-22] [Table 8-23] [Table 8-24] [Table 8-25] [Table 8-26] [Table 8-27]

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

[0121] According to this disclosure, additional on / off or other types of control switch techniques may be incorporated herein. These techniques may employ the use of dimerization domains and any activators of such domain dimerization. These techniques include, for example, those described by Wu et al., Science 2014 350(6258) utilizing the FKBP / rapalog dimerization system in specific cells, the contents of which are incorporated herein by reference in their entirety. Additional dimerization techniques are described, for example, by Fegan et al., Chem. Rev. 2010, 110, 3315-3336 and U.S. Patents 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 / cyclophylline receptor, estrogen / estrogen receptor (optionally using tamoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, and 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, US Patent No. 8,486,693, US2014 / 0171649, and US2012 / 0130076, the contents of which are further incorporated herein in their entirety by reference.

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

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

[0124] When suicide polypeptides are expressed on the surface of CAR immune cells (CAR-T cells), rituximab binding to the polypeptide's R-epitope causes cell lysis. Two or more molecules of rituximab may bind to each polypeptide expressed on the cell surface. Each R-epitope of the polypeptide may bind to a separate molecule of rituximab. Deletion of DLL3-specific CAR immune cells (e.g., CAR-T cells) can occur in vivo, for example, by administering rituximab to a patient. The decision to delete the transferred cells may arise from undesirable effects detected in the patient attributable to the transferred cells, such as when unacceptable levels of toxicity are detected.

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

[0126] In some embodiments, the extracellular domain of any one of the DLL3-specific CARs disclosed herein may contain 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 their entirety in International Patent Publication WO2016 / 120216, which is incorporated herein. 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). The CAR containing the mAb-specific epitopes may be single-chain or multi-chain.

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

[0128] Accordingly, in some embodiments, the present disclosure relates to methods for sorting and / or depleting engineered immune cells conferred with CARs containing mAb-specific epitopes, as well as methods for promoting the recovery of endogenous DLL3-expressing cells.

[0129] Using several epitope-monoclonal antibody couples, CARs can be generated that include monoclonal antibody-specific epitopes, particularly those already approved for medical use, such as the CD20 epitope / rituximab, as a non-limiting example.

[0130] The disclosure also includes a method for selecting engineered immune cells conjugated with DLL3-specific CARs expressing mAb-specific epitopes, and a therapeutic method in which the activation of these CAR-conjugated engineered immune cells is regulated 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 disclosure. [Table 9]

[0131] In some embodiments, the extracellular binding domain of CAR is 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 Includes, -In the formula, -V1 is V L Therefore, V2 is V H Either V1 is V H Therefore, V2 is V L And, -L1 is V H Chain V L It is a linker suitable for connecting to a chain. - Epitope 1, Epitope 2, Epitope 3, and Epitope 4 are mAb-specific epitopes, are identical, or

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

[0133] The hinge domain may contain up to 300 amino acids, 10 to 100 amino acids in some embodiments, or 25 to 50 amino acids in some embodiments. The hinge domain may originate from all or part of a naturally occurring molecule, such as the extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (in particular, the hinge region of IgG, which will be understood to contain some or all members of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof), or all or part of the constant region of an antibody heavy chain. Alternatively, the A domain may be a synthetic sequence corresponding to a naturally occurring A sequence, or it may be a completely synthetic A sequence. In some embodiments, the A domain is part of a human CD8α chain (e.g., NP_001139345.1). In another specific embodiment, the hinge and transmembrane domains contain part of a human CD8α chain. In some embodiments, the hinge domain of the CAR described herein includes a subsequence of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα, particularly one of the hinge regions of CD8α, CD28, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain includes 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 includes scFv, a human CD8α hinge and a 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 the extracellular domain amino acid sequences listed in Table 5 herein.

[0135] d. Transmembrane domain The CARs of this disclosure are designed with a transmembrane domain fused to the extracellular domain of the CAR. Similarly, it may be fused to the intracellular domain of the CAR. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein in order to minimize interaction with other members of the receptor complex. In some embodiments, a short linker may form a linkage between one or more of the extracellular, transmembrane, and intracellular domains of the CAR.

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

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

[0138] The transmembrane regions of specific use in this disclosure include CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (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, and Lentegrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT Ligands that specifically bind 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 may be derived from (including or corresponding to) these ligands.

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

[0140] In some embodiments, the transmembrane domain in the CAR of the Disclosure is a CD8α transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the Disclosure is a CD8α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 549). In some embodiments, the CD8α transmembrane domain comprises a nucleic acid sequence encoding the transmembrane amino acid sequence of SEQ ID NO: 549. In some embodiments, the hinge and transmembrane domain in the CAR of the 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 Disclosure is a CD28 transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the Disclosure is a CD28 transmembrane domain comprising the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 550). In some embodiments, the CD28 transmembrane domain comprises a nucleic acid sequence encoding the transmembrane amino acid sequence of SEQ ID NO: 550.

[0142] e. intracellular domain The intracellular (cytoplasmic) domain of the CARs of this disclosure can provide activation of at least one normal effector function of immune cells containing the CAR, e.g., signal 1 / activation and / or signal 2 / costimulation. For example, the effector function of a T cell may refer to cytolytic activity or helper activity, including cytokine secretion. In some embodiments, the activated intracellular signaling domain for use in CAR may be, for example, cytoplasmic sequences of T cell receptors and co-receptors that act in coordination to initiate signal transduction after antigen receptor ligation, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0143] Suitable (e.g., activated) intracellular domains are not limited to these, but include CD3 zeta, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (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, etc. Itokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT It will be understood that it contains signaling domains derived from (or corresponding to) ligands that specifically bind 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] The intracellular domains of the CARs of this disclosure may incorporate, in addition to the activation domains described above, a co-stimulatory signaling domain (as interchangeably referred herein with the co-stimulatory molecule) to enhance their efficacy. The co-stimulatory domain can provide a signal in addition to the primary signal provided by the activation molecule described herein.

[0145] Preferred co-stimulatory domains within the scope of this 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, signal transduction lymphocyte activating 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 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 ligands, or fragments or combinations thereof may be derived from (or correspond to) these ligands.It will be understood that any additional co-stimulatory molecules or fragments thereof not listed above are within the scope of this disclosure.

[0146] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to include the 41BB / CD137 domain, either by itself or in combination with any other desired intracellular domain useful in the context of the CARs of this disclosure. The complete native amino acid sequence of 41BB / CD137 is described in NCBI reference sequence:NP_001552.2. The complete native nucleic acid sequence of 41BB / CD137 is described 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 CARs of this 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 may 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 this disclosure. In some embodiments, the intracellular signaling domain of the CAR may include a CD3ζ signaling domain having an amino acid sequence having at least about 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 481 in Table 7. For example, the intracellular domain of the CAR may include a CD3 zeta chain portion and a portion of a co-stimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR of this disclosure may be linked to each other randomly or in a specific order. In some embodiments, the intracellular domain is designed to include a CD3 zeta activation domain and a CD28 signaling domain. In some embodiments, the intracellular domain is designed to include a CD3 zeta activation domain and a 4-1BB co-stimulatory / signaling domain.

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

[0150] In some embodiments, the intracellular domain in CAR is designed to include portions of CD28 and CD3 zeta, and the intracellular CD28 includes the nucleic acid sequence shown in Sequence ID No. 567. AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCC ACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCGCTGCCTATCGGAGC (SEQ ID NO: 567).

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

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

[0153] In exemplary embodiments, the CAR of the present disclosure comprises, from the N-terminus to the C-terminus, a (cleavable) CD8α signaling 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 including CAR a. Immune cells Engineered immune cells expressing the CARs of this disclosure (e.g., CAR-T cells) are provided herein.

[0155] In some embodiments, the manipulated immune cells comprise a population of CARs, each containing a different extracellular antigen-binding domain. In some embodiments, the immune cells comprise a population of CARs, each containing the same extracellular antigen-binding domain.

[0156] Manipulated immune cells can be allogeneic or autologous.

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

[0158] In some embodiments, the manipulated immune cells may be derived from, for example, stem cells, without limitation. Stem cells 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 by nucleic acid vectors using methods selected from the group consisting of electroporation, sonoporation, microparticle guns (e.g., gene guns), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retroviruses, lentiviruses, AAVs), or polyplexes.

[0161] In some embodiments, engineered immune cells expressing the DLL3-specific CAR of the Disclosure on their cell surface membranes include percentages of stem cell memory and central memory cells greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. %, approximately 15%~80%, approximately 15%~70%, approximately 15%~60%, approximately 15%~50%, approximately 15%~40%, approximately 15%~30%, approximately 20%~100%, approximately 20%~90%, approximately 20%~80%, approximately 20%~70%, approximately 20%~60%, approximately 20%~50%, approximately 20%~40%, approximately 20%~30%, approximately 30%~100%, approximately 30%~90 %, approximately 30%~80%, approximately 30%~70%, approximately 30%~60%, approximately 30%~50%, approximately 30%~40%, approximately 40%~100%, approximately 40%~90%, approximately 40%~80%, approximately 40%~70%, approximately 40%~60%, approximately 40%~50%, approximately 50%~100%, approximately 50%~90%, approximately 50%~80%, approximately 50%~70%, approximately 50%~60 This includes percentages of stem cell memory and central memory cells in the following ranges: %, approximately 60% to 100%, approximately 60% to 90%, approximately 60% to 80%, approximately 60% to 70%, approximately 70% to 90%, approximately 70% to 80%, approximately 80% to 100%, approximately 80% to 90%, approximately 90% to 100%, approximately 25% to 50%, approximately 75% to 100%, or approximately 50% to 75%.

[0162] In some embodiments, the immune cells are inflammatory T lymphocytes expressing any one of the CARs described herein. In some embodiments, the immune cells are cytotoxic T lymphocytes expressing any one of the CARs described herein. In some embodiments, the immune cells are regulatory T lymphocytes expressing any one of the CARs described herein. In some embodiments, the immune cells are helper T lymphocytes expressing any one of the CARs described herein.

[0163] Prior to proliferation and genetic modification, the cell source can be obtained from the subject through a variety of non-limiting methods. Cells can be obtained from several non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In some embodiments, any number of T cell lines available to those skilled in the art and known can be used. In some embodiments, cells may originate from healthy donors, patients diagnosed with cancer, or patients diagnosed with infections. In some embodiments, cells may be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0164] Cell lines obtained from immune cells (e.g., T cells) transformed according to any of the methods described above are also provided herein. Modified cells resistant to immunosuppressive therapy are also provided herein. In some embodiments, the cells isolated according to this disclosure contain polynucleotides encoding CARs.

[0165] The immune cells of this disclosure can be activated and proliferated either before or after genetic modification of the immune cells using commonly known methods. Generally, the engineered immune cells of this disclosure can be proliferated, for example, by contacting them with agents that stimulate the CD3 TCR complex and costimulatory molecules on the surface of T cells to create activation signals to T cells. Chemicals such as the calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitotic lectins such as phytohemagglutinin (PHA) may be used to create activation signals to T cells.

[0166] In some embodiments, a population of T cells may be stimulated in vitro by contact with an anti-CD3 antibody, such as an OKT3 antibody, or its antigen-binding fragment, or an anti-CD2 antibody immobilized on its surface, or by contact with a protein kinase C activator (e.g., bryostatin) combined with a calcium ionophore. For co-stimulation of accessory molecules on the surface of T cells, ligands that bind the accessory molecules are used. For example, a population of T cells may be contacted with an anti-CD3 antibody (e.g., an OKT3 antibody) and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. The anti-CD3 and anti-CD28 antibodies may be placed on beads, such as plastic or magnetic beads, or on a plate or other substrate. Suitable conditions for T cell culture include a suitable medium (e.g., Minimal Essential Media or RPMI Media 1640, or X-vivo 15 (Lonza)) that may contain factors necessary for proliferation 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 additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, either supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, as well as / or sufficient amounts of cytokines (e.g., IL-7 and / or IL-15) for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture and not in the cell cultures injected during the study.The target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air and 5% CO2). T cells exposed to different stimulation times may exhibit different characteristics. In some embodiments, the cells of this disclosure may be grown by co-culturing with tissue or other cells. The cells may also be grown in vivo in the blood of a subject, for example, after administration of the cells to the subject.

[0167] In some embodiments, the immune cells manipulated according to this disclosure may contain one or more disrupted or inactivated genes. In some embodiments, the immune cells manipulated according to this 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 CAR, multi-stranded CAR, and / or pTα transgenes. In some embodiments, isolated cells contain polynucleotides encoding polypeptides containing multi-stranded CARs. In some embodiments, the isolated cells according to this disclosure are 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 method 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 CAR, multistranded CAR, and pTα transgenes. In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing an endonuclease into the cell that can selectively inactivate genes by selective DNA cleavage.In some embodiments, the endonuclease may 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 cells in accordance with this disclosure by disrupting or inactivating the TCRα gene and / or TCRβ gene. In some embodiments, methods are provided for obtaining modified cells derived from an individual, the cells being able to proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can proliferate independently of the MHC signaling pathway and are easily obtained by this method are within the scope of this disclosure. 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 therefore, a method for treating a patient in need of treatment for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising treating the patient by administering to the patient an effective amount of modified cells containing disrupted or inactivated TCRα and / or TCRβ genes, is within the scope of this disclosure.

[0169] In some embodiments, immune cells are engineered to be resistant to one or more chemotherapeutic agents. These chemotherapeutic agents may be, for example, purine nucleotide analogs (PNAs), thus making the immune cells suitable for cancer treatment in combination with adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, either 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 pro-apoptosis agents, and are potent inhibitors of ribonucleotide reductase (RNR) involved in trinucleotide production. DLL3-specific CAR-T cells containing disrupted or inactivated dCK genes are provided herein. In some embodiments, dCK knockout cells are produced, for example, by transfection of T cells using polynucleotides encoding specific TAL nucleases directed to the dCK gene, via mRNA electroporation. dCK knockout DLL3-specific CAR-T cells are resistant to PNAs, for example, chloropharabine and / or fludarabine, and maintain their T-cell cytotoxic activity against DLL3-expressing cells.

[0170] In some embodiments, the isolated cells or cell lines of this disclosure may contain 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] This disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein. In some embodiments, the CARs may be introduced into immune cells as transgenes via a plasmid vector. In some embodiments, the plasmid vector may also include a selection marker that provides, for example, identification and / or selection of the vector-receiving cells.

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

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

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

[0175] Various known techniques can be used when preparing polynucleotides, polypeptides, vectors, antigen-binding domains, immune cells, compositions, etc., as described in this disclosure.

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

[0177] i. Source material In some embodiments, 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, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL® isolation.

[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 leukocytes, erythrocytes, and platelets. In certain embodiments, cells collected by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for further processing.

[0179] In certain embodiments, T cells are isolated from PBMCs by lysing erythrocytes and depleting monocytes, for example, using centrifugation with a PERCOLL® gradient. Specific subpopulations of T cells (e.g., CD28+, CD4+, CDDS+, CD45RA-, CD45RO+, CDDS+, 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 T cell subpopulation is CD45RO+, CD95+, IL-2Rβ+, CCR7+, CD62L+. In another example, the T cell subpopulation is CD45RO+, CD95+, IL-2Rβ+, CCR7-, CD62L-. In yet another example, the T cell subpopulation is CD45RA+, CD95+, IL-2Rβ+, CCR7-, CD62L-. For example, enrichment of a T cell population by negative selection can be achieved with a combination of antibodies directed to surface markers specific 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 to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate the cell population of interest for use in this disclosure.

[0180] PBMCs can be directly used for genetic modification by immune cells (such as CARs or TCRs) 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 proliferation.

[0181] In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying characteristic cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers in 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 characteristic cell surface antigens.

[0183] iii. Stem cell-derived immune cells In some embodiments, immune cells may be derived from stem cells, such as progenitor cells, bone marrow stem cells, inducible pluripotent stem cells, iPSCs, hematopoietic stem cells, and mesenchymal stem cells. iPS cells and other types of stem cells may be cultured immortal cell lines or isolated directly from patients. Various methods for isolating, developing, and / or culturing stem cells are known in the art and may be used to carry out 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 may be induced pluripotent stem cells (iPSCs) derived from T cells or non-T cells. Alternatively, the source material may 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 proliferated (or differentiated in the case of progenitor cells) in vitro before genetic modification. In some embodiments, isolated immune cells are genetically modified to reduce or eliminate the expression of endogenous TCRα and / or CD52. In some embodiments, cells are genetically modified using gene editing techniques (e.g., CRISPR / Cas9, CRISPR / CAS12, zinc finger nucleases (ZFNs), TALENs, MegaTAL, meganucleases) to reduce or eliminate the expression of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, immune cells, such as T cells, are optionally further genetically modified with chimeric antigen receptors described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding CARs), and then activated and / or proliferated in vitro.

[0186] Methods for activating and proliferating 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. Generally, such methods involve contacting PBMCs or isolated T cells with stimulating and co-stimulating molecules, such as anti-CD3 and anti-CD28 antibodies attached to plastic or magnetic beads or other surfaces, in a culture medium containing a suitable cytokine such as IL-2. The anti-CD3 and anti-CD28 antibodies attached to the same beads function as “surrogate” antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other embodiments, T cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent No. 6,040,177, U.S. Patent No. 5,827,642, and WO2012 / 129514, whose entirety is incorporated herein by reference.

[0187] Specific methods for constructing the constructs and manipulated immune cells of this 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 may further contain other cytotoxic lymphocytes such as NK cells or NKT cells. Expression vectors possessing the coding sequence of 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 possessing the expression vector can be sorted using flow cytometry to isolate CD3-positive T cells, which can then be further proliferated 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 proliferation of T cells are carried out in the absence of non-human animal-derived products such as fetal calf serum and fetal bovine serum. In one embodiment, cryopreservation may include freezing in a suitable culture medium such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions).

[0189] For polynucleotide cloning, a vector is introduced into a host cell (an isolated host cell) to allow the vector itself to replicate, thereby amplifying copies of the polynucleotides it contains. Cloning vectors generally contain, but are not limited to, a replication origin, promoter sequence, transcription start sequence, enhancer sequence, and selectable markers, as well as other sequence components. These elements can be appropriately selected by those skilled in the art. For example, the replication origin may be selected to promote autonomous replication of the vector in the host cell.

[0190] In certain embodiments, this disclosure provides isolated host cells containing the vectors provided herein. These vector-containing host cells may be useful for the expression or cloning of polynucleotides contained in the vectors. Suitable host cells may 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 vectors can be introduced into host cells using any preferred 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 impingement, receptor-mediated gene delivery, and delivery mediated by polylysine, histones, chitosan, and peptides. Standard methods for viral transfection and transformation of cells for expression of the vector of interest are well known in the art. In further embodiments, a mixture of different expression vectors can be used to genetically modify a donor population of immunoeffector cells, each vector encoding a different CAR as disclosed herein. The resulting transduced immunoeffector cells form a mixed population of engineered cells, in which a certain percentage of engineered cells express two or more different CARs.

[0192] In one embodiment, the present disclosure provides a method for preserving genetically engineered cells expressing a CAR that targets the DLL3 protein. In one embodiment, this includes cryopreserving immune cells so that the cells remain viable upon thawing. In one embodiment, cryopreservation may include freezing in a suitable medium such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions). A fraction of immune cells expressing the CAR can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients with malignant tumors. If necessary, the cryopreserved transformed immune cells can be thawed, grown, and proliferated for more such cells.

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

[0194] iv. Allogeneic CAR T cells In short, the process for producing allogeneic CAR T therapy, or AlloCAR®, involves harvesting healthy, selected, screened, and tested T cells from a healthy donor. 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 lymphocyte depletion of the host immune system, allowing the CAR T cells to remain engrafted and achieve a complete therapeutic effect. The T cells are then engineered to express CARs that recognize specific cell surface proteins (e.g., DLL-3) expressed in hematological malignancies or solid tumors. The engineered T cells then undergo a purification step and are cryopreserved in vials for final delivery to the patient.

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

[0196] IV. Treatment Methods This disclosure includes methods for treating or preventing conditions associated with undesirable and / or elevated DLL3 levels in patients, which include administering an effective amount of at least one CAR, or immune cells containing the CARs disclosed herein, to patients in need thereof.

[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 an effective amount of the engineered immune cells of the present invention to the subject. In some embodiments, the T cell-mediated immune response is directed toward target cells. In some embodiments, the engineered immune cells comprise a chimeric antigen receptor (CAR). In some embodiments, the target cells are tumor cells. In some embodiments, the disclosure includes a method for treating or preventing malignancies, the method comprising administering an effective amount of at least one isolated antigen-binding domain described herein to a subject in need. In some embodiments, the disclosure includes a method for treating or preventing malignancies, the method comprising administering an effective amount of at least one immune cell to a subject in need, the immune cell comprising at least one chimeric antigen receptor and / or an isolated antigen-binding domain described herein. CARs containing the immune cells of the disclosure can be used to treat malignancies accompanied by DLL3 aberration. In some embodiments, CARs containing the immune cells of this disclosure can be used to treat malignancies such as small cell lung cancer, melanoma, low-grade glioma, glioblastoma, medullary thyroid carcinoma, carcinoid, dispersive 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 the anti-DLL3 CAR-T cells of this disclosure, are used to treat small cell lung cancer.

[0198] Methods for reducing tumor size in a subject are also provided, including administering manipulated cells to the subject, wherein the cells comprise a chimeric antigen receptor containing a DLL3 antigen-binding domain and bind to the 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 manipulated cells are delivered to a tumor bed, such as a tumor bed found in small cell lung cancer. In some embodiments, the cancer is present in the bone marrow of the subject. In some embodiments, the manipulated cells are autoimmune cells, e.g., autoimmune cells. In some embodiments, the manipulated cells are alloimmune cells, e.g., alloimmune cells. In some embodiments, the manipulated cells are heteroimmune cells, e.g., heterogeneous T cells. In some embodiments, the manipulated cells are transfected or transduced ex vivo. As used herein, the term “in vitro cells” refers to any cells cultured ex vivo.

[0200] A therapeutic agent, such as a "therapeutic effective dose," "effective dose," "effective amount," or "therapeutic effective dosage" of engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-asymptomatic periods, or the prevention of functional impairment or disability due to the distress of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to skilled practitioners (e.g., physicians or clinicians), such as in human subjects during clinical trials or in animal model systems predicting efficacy in humans, or by assaying the activity of the drug 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 formally diagnosed disabilities, subjects without formally recognized disabilities, subjects receiving medical treatment, subjects at risk of developing disabilities, etc.

[0202] The terms “to treat” and “treatment” include therapeutic actions, preventive actions, and uses that reduce the risk of an object developing a disorder or other risk factor. Treatment does not require a complete cure of the disorder and includes embodiments that reduce symptoms or potential risk factors. The term “preventive” does not require 100% elimination of the possibility of an event; rather, it indicates that the likelihood of the event occurring is reduced in the presence of the compound or method.

[0203] The desired total therapeutic amount of cells in the composition comprises at least two cells (e.g., at least one CD8+ T cell and at least one CD4+ T cell, or two CD8+ T cells, or two CD4+ T cells), or more typically 10 2 More than 10 cells, and up to 10 6 pieces, maximum 10 8 or 10 9 It is a single cell, 10 10 or 10 12 There may be more than 10 cells. The number of cells will depend on the intended use of the composition and the type of cells contained therein. The desired cell density is typically 10 6 It is greater than cells / ml, and is generally 10 7 It is greater than cells / ml, and is generally 10 8 The number of cells / ml is greater than 10. A clinically relevant number of immune cells. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 The cells can be distributed to multiple injections that are cumulatively equal to or greater than a single cell. In some aspects of this disclosure, in particular, all injected cells are redirected to a specific target antigen (e.g., DLL3), so 10 6 / kilogram (10 per patient) 6 ~10 11A lower number of cells within the range of ) may be administered. CAR therapy may be administered multiple times in doses within these ranges. The cells may be autologous, allogeneic, or heterogeneous to the patient receiving the therapy.

[0204] In some embodiments, the therapeutically effective dose of CAR T cells is approximately 1 x 10⁻¹⁰ 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 9 x 10 7 It is cells / kg.

[0205] In some embodiments, the target dose for CAR+ / CAR-T+ cells is approximately 1 × 10⁻⁶. 6 ~Approx. 1×10 10 Cells / kg range, for example, about 1 × 10⁻⁶ 6 cells / kg, approximately 1×10 7 cells / kg, approximately 1×10 8 cells / kg, approximately 1×10 9cells / kg, or approximately 1 × 10⁶ 10 The dose is given as cells / kg. It will be understood that doses above and below this range may be appropriate for specific subjects, and the appropriate dose level may be determined by the healthcare provider as needed. In addition, multiple doses of cells may be provided in accordance with this disclosure.

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

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

[0208] Pharmaceutical compositions (solutions, suspensions, etc.) may include one or more of the following: sterile diluents, e.g., water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride; fixing oils, e.g., synthetic mono or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can serve as solvents or suspension media; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates; and agents for adjusting tonicity, e.g., sodium chloride or dextrose. Parenteral preparations can be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. For therapeutic use, injectable pharmaceutical compositions are preferably sterile.

[0209] In some embodiments, 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 upon administration to a patient. In one embodiment, the percentage reduction or lysis of DLL3-expressing endogenous cells or DLL3-expressing cell lines 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 DLL3-expressing cell lines 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, endogenous DLL3-expressing cells are endogenous DLL3-expressing myeloid cells.

[0210] In one embodiment, the percentage reduction or lysis of target cells, such as a cell line expressing DLL3, by engineered immune cells expressing the DLL3-specific CAR of this disclosure on their cell surface membranes can be measured using the assay disclosed herein.

[0211] The method may further include administering one or more chemotherapeutic agents to the patient before administering the manipulated cells provided herein. In certain embodiments, the chemotherapeutic agent is a lymphocyte depletion (preconditioning) chemotherapeutic agent. For example, a method for conditioning a patient requiring T-cell therapy involves administering a specific beneficial dose of cyclophosphamide (200 mg / m²) to the patient. 2 / day~2000mg / m 2 / day, about 100mg / m2 / day~about 2000mg / m 2 For example, approximately 100 mg / m² per day. 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 / m² 2 ( / day), and specific doses of fludarabine (20 mg / m²). 2 / day~900mg / m 2 / day, about 10mg / m 2 / day~about 900mg / m 2 For example, about 10 mg / m² per day. 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, about 90mg / m 2 / day, about 100mg / m 2 / day, about 500mg / m 2 / day, or approximately 900 mg / m² 2 This includes administering approximately 30 mg / m² / day to the patient three days prior to administering a therapeutically effective dose of engineered T cells. An exemplary dosing regimen involves administering approximately 30 mg / m² / day. 2 Approximately 300 mg / m² in combination with, or before or after, the administration of fludarabine per day. 2 Treatment of the subject involves administering cyclophosphamide to the patient daily.

[0212] In some embodiments, particularly when the manipulated cells provided herein have been gene-edited to eliminate or minimize CD52 surface expression, lymph depletion further includes the administration of an anti-CD52 antibody such as alemtuzumab. In some embodiments, the CD52 antibody is administered IV for 1, 2, or 3 days or more at a dose of about 1 to 20 mg / day, for example, about 13 mg / day IV. The antibody may be administered before or after the administration of other components of the lymph depletion regimen (e.g., cyclophosphamide and / or fludarabine).

[0213] In other embodiments, the antigen-binding domain, transduced (or otherwise manipulated) cells, and chemotherapeutic agents are each administered in amounts effective to treat the disease or condition in the subject.

[0214] In certain embodiments, compositions comprising CAR-expressing immunoeffector 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®); alkyl sulfonates such as busulfan, improsulfan, and pigosulfan; aziridines such as benzodopa, carboquan, metsuredopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethyloromelamelamine; chlorambucil, Lornafadine, chlorophosphamide, estramustine, ifosfamide, mechloretamine, oxidized mechloretamine hydrochloride, melphalan, nobenbitin, phenesterine, prednimustine, trophosphamide, uracil mustard and other nitrogen mustards; carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine and other nitrosureas; acrasinomycin, actinomycin, ausramycin, azaserine, bleomycin, kakutinomycin, calyke Amycin, Carabicin, Carminomycin, Cardinophilin, Chromomycin, Dactinomycin, Daunorubicin, Detorubicin, 6-Diazo-5-Oxo-L-Norleucine, Doxorubicin, Epirubicin, Esolubicin, Idarubicin, Marcelomycin, Mitomycin, Mycophenolic acid, Nogaramycin, Olibomycin, Peplomycin, Potophyllomycin, Promycin, Keramycin, Rhodolubicin, Streptonigrin, Streptozocin, Tubercidine, Ubenimec Antibiotics such as dinostatin and zolubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU;Androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; acegraton; aldofrosphamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisantren; edatrexate; defofamine; demecolsin; diazion; elformitin; eriptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; ronidamine; mitoglucon; mitoxantrone; mopida Mole; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK®; Lazoxane; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®, Bristol-Myers Squibb), and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RF S2000; Difluoromethylomitin (DMFO); Retinoic acid derivatives such as Targretin (trademark) (bexarotene), Panretin (trademark), (alitretinoin); ONTAK (trademark) (deniloikin difutox); Esperamicin; Capecitabine;This also includes any pharmaceutically acceptable salts, acids, or derivatives of the above. This definition includes, for example, anti-estrogens including tamoxifen, raloxifene, the aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); as well as anti-hormonal agents that act to modulate or inhibit the hormonal effects on tumors, such as anti-androgens like flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any pharmaceutically acceptable salts, acids, or derivatives of the above. If necessary, combinations of chemotherapeutic agents, including but not limited to CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, may also be administered.

[0215] In some embodiments, the chemotherapeutic agent is administered concurrently with or within one week thereafter with the administration of the engineered cells, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered over a period of approximately 1 to 7 days, approximately 1 to approximately 4 weeks, approximately 1 week to approximately 1 month, approximately 1 week to approximately 2 months, approximately 1 week to approximately 3 months, approximately 1 week to approximately 6 months, approximately 1 week to approximately 9 months, or approximately 1 week to approximately 12 months after the administration of the engineered cells, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least one month before the administration of the cells, polypeptide, or nucleic acid. In some embodiments, the method further includes 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, pizilizumab, and atezolizumab.

[0217] Additional therapeutic agents suitable for use in combination with this disclosure include ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), and imatinib (G Leevec (registered trademark), cetuximab (Erbitux (registered trademark), panitumumab) (Vectibix (registered trademark)), catumakisomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib Sorafenib, Toceranib, Restaurtinib, Axitinib, Sediranib, Lenvatinib, Nintedanib, Pazopanib, Regorafenib, Semaxanib, Sorafenib, Sunitinib, Tibozanib, Toceranib, Vandetanib, Entrectinib, Cabozantinib, Imatinib, Dasatinib, Nilotinib, Ponatinib, Radotinib, Bosutinib, Restaurtinib, Ruxolitinib, Paxol This includes, but is not limited to, mTOR inhibitors such as litinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin difutitox, everolimus, and temsirolimus; hedgehog inhibitors such as sonidecib and bismodegib; and CDK inhibitors such as palbociclib.

[0218] In some embodiments, compositions containing CAR-containing immune cells may be administered in conjunction with therapeutic regimens to prevent or reduce cytokine release syndrome (CRS) or neurotoxicity. Therapeutic regimens to prevent cytokine release syndrome (CRS) or neurotoxicity may include rengliumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, and iNOS inhibitors (e.g., L-NIL or 1400W). In additional embodiments, compositions containing CAR-containing immune cells may be administered in conjunction with anti-inflammatory agents. 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, and triamcinolone), aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and nonsteroidal anti-inflammatory drugs (NSAIDs) including mycophenolates. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialates. Exemplary analgesics include acetaminophen, oxycodone, and tramadol (proporxiphene hydrochloride). Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed at cell surface markers (e.g., CD4, CD5), cytokine inhibitors, e.g., TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers also 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 include 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; Müllerian inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF) such as NGF-beta; platelet growth factor; and transforming growth factors such as TGF-alpha and TGF-beta. These include factors such as TNF-α (TGF); insulin-like growth factor-I and -II; erythropoietin (EPO); bone induction factor; interferons such as interferon-alpha, beta, and gamma; colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1-alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, and 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 recombinant cell cultures, and their bioactive equivalents to naturally occurring 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, wherein a subset of the immune cell population includes engineered immune cells expressing one of a DLL3-specific CARs containing an epitope specific to a monoclonal antibody (e.g., an exemplary mimotope sequence). The method includes contacting the immune cell population with an epitope-specific monoclonal antibody and selecting immune cells that bind to the monoclonal antibody to obtain a population of cells rich in engineered immune cells expressing a DLL3-specific CAR.

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

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

[0223] In some embodiments, the mAbs used in the method for sorting immune cells expressing CAR include alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tocitumomab, absiximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol, and dacrizuma. The mAb is selected from 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 includes at least 70%, 75%, 80%, 85%, 90%, and 95% of the 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 includes at least 85% of the CAR-expressing immune cells.

[0225] In some embodiments, the population of CAR-expressing immune cells obtained when using the methods for in vitro sorting of CAR-expressing immune cells described above exhibits increased cytotoxic activity in vitro compared to the initial (unsorted) cell population. In some embodiments, this in vitro cytotoxic activity 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] CAR-expressing immune cells administered to recipients can be enriched in vitro from a source population. Methods for expanding the source population may include selecting cells expressing antigens 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. Generally, flow cytometry is a method for quantifying the components or structural characteristics of cells, primarily by optical means. Because different cell types can be distinguished by quantifying structural characteristics, flow cytometry and cell sorting can be used to count and sort cells with different phenotypes in a mixture.

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

[0230] In some embodiments, the method used to sort T cells expressing CAR is magnetically activated cell sorting (MACS). Magnetically activated cell sorting (MACS) is a method for separating different 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 rapid 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 an 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 purifying 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 may involve administering a treatment to a mammalian organism (e.g., a molecule that binds to an epitope on the CAR) aimed at stopping the proliferation of CAR-expressing immune cells by inhibition or elimination.

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

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

[0235] In some embodiments, the step of contacting the manipulated immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb includes 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, the step of contacting the manipulated immune cells or the CAR-expressing immune cells with at least one epitope-specific mAb is performed on the patient at a dose of approximately 375 mg / m². 2 This involves administering rituximab once or several times. In some embodiments, the mAb (e.g., rituximab) is administered once a week.

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

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

[0239] According to another specific embodiment, an injected bispecific mAb can bind to both an mAb-specific epitope on engineered immune cells expressing a chimeric scFv and to surface antigens on effector and cytotoxic cells (e.g., immune cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), and cytotoxic T lymphocytes (CTLs)). In doing so, the depletion of engineered immune cells caused by the BsAb can occur through antibody-dependent 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) enable sensitive differentiation between healthy and diseased tissues compared to the use of drugs alone. Several ADCs have received market approval, and technologies for producing them, particularly on linkers, are listed (Payne, G. (2003) Cancer Cell 3:207-212, Trail et al (2003) Cancer Immunol.Immunother. 52:328-337, Syrigos and Epenetos (1999) Anticancer Research 19:605-614, Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172, U.S. Patent No. 4,975,278).

[0241] In some embodiments, the injected epitope-specific mAbs are pre-conjugated with molecules that can promote complement-dependent cytotoxicity (CDC). Thus, the complement system assists or complements the antibody's ability to eliminate pathogens from an organism. When stimulated, an activation cascade is triggered as a large-scale amplification of the response and activation of the cell-killing membrane invasion complex. mAbs can be conjugated using different molecules such as glycans (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 This application provides a kit comprising either a DLL3-containing CAR or a DLL3 CAR-containing immune cell as 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 kit of the present disclosure comprises allogeneic DLL3 CAR-containing T cells, as well as a CD52 antibody for administering lymphocyte depletion regimens and CAR-T regimens to subjects.

[0244] This application also provides a product comprising any one of the therapeutic compositions or kits described herein. An example of a product is a vial (e.g., a sealed vial). [Examples]

[0245] Example 1: Production and testing of DLL3-targeted antibody The monoclonal antibodies used in accordance with the present invention can be produced by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Anti-DLL3 antibodies were first screened by Flag-DLL3 (adipogen) ELISA, and then by FACS to determine their binding to human DLL3-expressing or non-expressing HEK-293T cells.

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

[0247] Example 2: Determination of the rate and affinity of an anti-DLL3 antibody against DLL3. This example determines the binding rate 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 scFvs, the variable region of the anti-DLL3 antibody derived from their respective hybridomas was cloned adjacent to a (GGGGS)3 (SEQ ID NO: 472) or (GGGGS)4 (SEQ ID NO: 478) linker, followed by a hinge and portion of the Fc from the human IgG2 sequence, resulting in scFv-Fc fusions which were expressed using Expi293. 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 rates were determined by surface plasmon resonance (Biacore™ surface plasmon resonance (SPR) system, GE Healthcare Bio-Sciences, Pittsburg PA). Antibodies diluted with HBS-T+ running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% vol / vol Tween20, 1 mg / mL BSA) were captured on a CM4 chip immobilized with an antibody specific to the constant domain of the anti-DLL3 antibody. Purified DLL3 was serially diluted with HBS-T+ and injected at 30 uL / min for 2 minutes. After a 10-minute dissociation time, the surface was regenerated between injections with either 10 mM glycine-HCl pH 1.7 or phosphoric acid. The kinetic association rate (kon) and dissociation rate (koff) are obtained simultaneously by fitting the data holistically to a 1:1 Langmuir coupled 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 The value is k off / k on It is calculated as follows.

[0249] Table 8 shows the rate and affinity parameters of the tested anti-DLL3 antibodies. Specifically, Table 8 shows the affinity of anti-DLL3 antibodies (either IgG or scFv-Fc fusion) against human, cynomolgus monkey, and mouse DLL3. The last column indicates which extracellular domain of human DLL3 each anti-DLL3 antibody recognizes. [Table 11]

[0250] Example 3: Generation of CHO cells expressing full-length and truncated DLL3 Using a panel of CHO cells expressing full-length and various truncated forms of human DLL3, we determined which domains each DLL3-targeting antibody recognizes. The extracellular domain of human DLL3 can be subdivided into different 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 for use in epitope mapping, the sequences of each of the eight extracellular domains of human DLL3 (signal peptide and N-terminus, DSL, EGF1, EGF2, EGF3, EGF4, EGF5, and EGF6) were deleted one by one from the antigen, starting from the N-terminus. Table 6 shows the generated truncated DLL3 proteins (see also Figures 2A–2D). [Table 12-1] [Table 12-2]

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

[0253] Expression of full-length and truncated DLL3 was validated by a FACS assay using a PE-conjugated anti-HA antibody (Biolegend, catalog no. 901518). As a negative control, cells were incubated with an isotype-matched, PE-labeled antibody (Biolegend, catalog no. 400111) instead of the 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-targeted antibody CHO cells expressing full-length and truncated DLL3 were stained with hybridoma supernatant in PBS + 1% BSA or with purified DLL3 antibody. The bound DLL3 antibody was detected with PE-labeled anti-mouse IgG antibody (Biolegend, catalog number 405307). Samples were analyzed by flow cytometry. The binding domain of each clone was determined using the panel of CHO cells expressing full-length or truncated DLL3 described in Example 2. Flow cytometry analysis showed that, for example, a clone recognizes EGF3 if it binds to all truncated proteins including EGF3 but not to any truncated proteins that do not contain EGF3. As shown in the representative image in Figure 2D, the anti-DLL3 antibody recognizes the DSL, EGF1, and EGF3 domains, respectively. The signal from the PE channel is shown on the x-axis, and the count is shown on the y-axis.

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

[0256] The anti-DLL3 antibodies listed in Table 1a were reformatted to CAR. Using the amino acid sequences of the heavy chain and light chain variable regions of these antibodies (Tables 1b and 1c), a single-chain variable fragment (scFv) (Table 1d) with the following general structure: heavy chain variable region -- linker -- light chain variable region was designed. 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 include the following elements from 5' to 3': the CD8α signal sequence (SEQ ID NO: 477), 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 [Table 13-19] [Table 13-20] [Table 13-21] [Table 13-22] [Table 13-23] [Table 13-24] [Table 13-25] [Table 13-26] [Table 13-27]

[0258] A schematic diagram of the CAR structure is shown in Figure 3A. Representative CAR sequences reformatted from anti-DLL3 clones are included in SEQ ID NOs: 482-533. Codon-optimized DLL3 CAR sequences were synthesized and subcloned into the following lentiviral vector pLVX-EF1a-DLL3 CAR (Clontech) using the 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 then purified from the PBMCs using a commercially available T cell isolation kit (Miltenyi Biotec, catalog number 130-096-535). Alternatively, primary human T cells can be purified directly from LeukoPak (StemCell Technologies).

[0260] To produce lentivirus encoding DLL3 CAR, HEK-293T cells were seeded on day 0 at a rate of 400,000 cells per mL in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone or JR Scientific) per well of a 6-well plate. On day 1, lentiviruses were prepared by mixing 1.5 ug of lentivirus packaging vector psPAX2, 0.5 ug of pMD2G, and 0.5 ug of appropriate transfer CAR vector in 250 uL of Opti-MEM (Gibco) per well of a 6-well plate ("DNA mix"). 10 uL of Lipofectamine 2000 (Invitrogen) in 250 uL of Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mix. The mixture was incubated at room temperature for 20 minutes, and a total volume of 500 uL was slowly added to the side of the wells containing 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, catalog no. 130-111-160, 1:100 dilution). On day 2, the medium in each well of a 6-well plate was replaced with 2 mL of T cell transduction medium per well, i.e., X-Vivo-15 supplemented with 10% FBS. On day 3, the T cells were resuspended in 1 mL of T cell transduction medium per well of a Grex-24 plate (Wilson Wolf, catalog no. 80192M) at a rate of 500,000 cells per mL. Lentiviral supernatant from HEK293 T cells was collected, filtered through a 0.45 micron filter (EMD Millipore) to remove cell fragments, and then added to T cells with 100 IU / mL of human IL-2. On day 5, 4.5 mL of T cell proliferation medium, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), was added to each well of a Grex-24 plate.Transduction efficiency was determined on days 9 and 13 by detecting the percentage of T cells recognizing recombinant DLL3 (Adipogen) using flow cytometry. Cells were grown in T cell growth medium in larger flasks or G-Rex containers (Wilson Wolf) as needed. On day 14, DLL3 CAR-T cells were cryopreserved. The percentage of cells stained with recombinant DLL3 was normalized across clones immediately before cryopreservation.

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

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

[0263] Example 6: In vitro feature analysis 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 cells, HEK-293T cells were transduced using a lentivirus encoding full-length human DLL3.

[0265] To test DLL3-specific killing, HEK-293T cells expressing human DLL3 or non-expressing firefly luciferase were subsequently 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 cells expressing human DLL3 or non-expressing in T cell proliferation medium, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), at effector:target (E:T) ratios ranging from 1:9 to 9:1. Cell viability was measured after 72 hours using a one-glo assay kit (Promega). Representative DLL3 CAR-T cells showed potent killing of HEK-293T-DLL3 cells, but no detectable activity was observed 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 in T cell proliferation medium, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), at effector:target (E:T) ratios ranging from 1:9 to 9:1. Cell viability was measured after 72 hours using a one-glo assay kit (Promega). Each condition was assayed with three replicates. The mean percentage and standard deviation of viable cells were seeded (Figures 4B and 4C).

[0267] Figure 4A shows experimental data demonstrating that anti-DLL3 CAR-T cells specifically killed human DLL3-expressing HEK-293 T cells but not parental HEK-293 T cells 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 a negative control.

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

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

[0270] To measure cytokines secreted from DLL3 CAR-T cells, DLL3 CAR-T cells were incubated with DLL3+SHP-77 cells in T cell proliferation medium, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), at effector:target (E:T) ratios of 1:1 or 1:9. After 24 hours, tissue culture supernatant was collected, and the levels of three cytokines in the supernatant [interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and IL-2] were measured using the Human Pro-Inflammatory 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 DLL3-expressing SHP-77 cell lines. CAR-T cells and SHP-77 cells were incubated for 24 hours at effector:target ratios of 1:1 or 1:9 (n=3).

[0271] Example 7: Serial Killing Assay The serial killing assay involves repeated exposure of CAR-T cells to their targets, allowing them to proliferate and, in certain cases, differentiate and deplete. Using this assay, we selected optimal clones with high target cell lysis and proliferation capabilities after several rounds of exposure to target cells.

[0272] On day one of the assay, 5,000 firefly luciferase-labeled WM266.4, DMS 454, or DMS 273 cells known to express DLL3 were seeded in 100 μl of X-Vivo-15 medium containing 5% human serum in 96-well plates with white walls and flat, clear bottoms. After the target cells adhered to the bottom of the plate, DLL3 CAR-T cells were thawed and added to the seeded target cells in X-VIVO medium containing 5% human serum at a 1:1 effector:target (E:T) ratio. Thereafter, every two days, 100 μl of medium containing DLL3 CAR-T cells was transferred to the newly seeded target cells, and the lysis percentage of the pre-seeded target cells was measured using a one-glo assay system or a CellTiter-glo system (Promega). Each condition was assayed with 3–6 replicates. The mean percentage and standard deviation of lysis were seeded (Figures 6A–6C). The optimal clone was the one with the highest target cell lysis throughout the entire assay on day 12. These data, along with experimental data from a serial kill assay, demonstrate 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 indicated time point (n=3–6).

[0273] Example 8: In vivo activity To test the antitumor activity of DLL3 CAR-T cells, SHP-77 tumor-carrying NSG mice were used. SHP-77 cells were obtained from frozen stock vials, thawed, and counted according to standard procedures. Cells were placed in full growth medium (RPMI + 10% FBS) at a rate of 50 × 10⁶. 6 Diluted to 100 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 (catalog no. 354234) and 5x10⁶ cells per mouse. 6A 200 μL cell / Matrigel suspension containing 1 SHP-77 cell was subcutaneously injected. Tumor growth was monitored by caliper measurements using a digital caliper, starting on day 5 post-transplant. Tumor size was calculated using the formula: tumor volume = (width^2 × length / 2). Approximately two weeks post-transplant, mice were randomized into groups of 5 based on tumor volume. The average tumor volume per group was 314 mm². 3 The following was performed: One day after randomization of mice, untransduced T cells and DLL3 CAR-T cells were thawed and counted according to a standard procedure. The cells were resuspended in RPMI + 10% FBS and injected by tail vein IV injection of 200 μL per mouse, with 2 million or 5 million CAR+ cells per mouse. Tumors were monitored every 3-4 days until the end of the study. 26C8 and 10G1-K DLL3 CAR-T cells induced dose-dependent tumor inhibition (Figures 7A-7B). Figures 7A-7B show experimental data demonstrating 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 exhibiting metastasis similar to that of human disease, SHP-77 tumors were established by tail vein injection. Tumors were observed in the lungs, liver, brain, kidneys, and spleen. Specifically, SHP-77 cells were thawed and incubated in full growth medium (RPMI + 10% FBS) at a rate of 40x10⁶ cells. 6The cells were diluted to 10G1-K anti-DLL3 CAR-T cells / mL. The cell suspension was kept on ice until transplantation, and 200 μL of the cell suspension per mouse was injected via tail vein IV. On post-transplant day 7, 200 μL of luciferin (15 mg / mL) was injected, and tumor growth was monitored by IVIS imaging. On post-transplant day 11, mice were randomized into groups of 5 based on total light flux. On post-transplant day 12, CAR-T cells were thawed and counted according to a standard procedure. The cells were resuspended in RPMI + 10% FBS, and 2 million or 7 million CAR+ cells per mouse were injected via tail vein IV injection in volumes of 200 μL per mouse. Tumors were 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 structure with 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 include the different safety switch structures listed below (Table 8). [Table 14]

[0276] Table 9 shows protein sequences encoding anti-DLL3 CAR constructs, including safety switches. Exemplary safety switch constructs may include the CD8α signal sequence (SEQ ID NO: 477), the anti-DLL3 scFv described herein, the CD20 mimotope (SEQ ID NO: 536), the QBEND-10 epitope (SEQ ID NO: 544), the hinge and transmembrane region of the human CD8α molecule (SEQ ID NO: 479), the cytoplasmic portion of the 4-1BB molecule (SEQ ID NO: 291), and the cytoplasmic portion of the CD3ζ molecule (SEQ ID NO: 292). [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6]

[0277] CAR-T cells 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 structures 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 are expressed on the surface of primary T cells and can recognize recombinant DLL3. The plots were gated with living 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 the DLL3+ WM266.4 cell line.

[0278] Example 10: Cytotoxicity in a small cell lung cancer PDX model Small cell lung cancer PDX models were purchased from Crown Bioscience. To investigate DLL3 expression on the cell surface, frozen vials of the PDX models were thawed, and 200,000 cells were used in each stained sample. DLL3 expression was validated by a FACS assay using PE conjugate anti-DLL3 antibody. Human and mouse lymphocytes were excluded by adding Brilliant violet 421 conjugate anti-human CD45 and anti-mouse CD45 antibodies to the same stained samples.

[0279] Figure 10A shows experimental data demonstrating that DLL3 is expressed on the surface of two small cell lung cancer PDX models. Figure 10B shows experimental data demonstrating 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 a negative control.

[0280] Example 11: In vitro detection and depletion of DLL3 CAR-T cells using a rituximab-based safety switch To deplete or switch off CAR T cells in undesirable activity events, rituximab off-switches were developed by inserting rituximab mimotopes at various locations in the extracellular region of CARs, as described in Example 9. Rituximab-dependent in vitro depletion of DLL3 CAR-T cells was evaluated using a complement-dependent cytotoxicity assay. In this assay, frozen CAR-T cells were thawed and 1 x 10⁶ cells were used. 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% rabbit complement (Cedarlane, CL3441-S) and rituximab antibody (in-house manufactured, 100 mg / mL). Cells were stained with recombinant DLL3 (Adipogen), and cytotoxicity was analyzed by flow cytometry. Figure 11A shows experimental data demonstrating that anti-DLL3 CAR-T cells can be detected by both recombinant DLL3 and rituximab staining. Figure 11B shows experimental data demonstrating in vitro depletion of DLL3 CAR-T cells in a rituximab-dependent and complement-dependent manner.

[0281] Example 12: In vivo activity of anti-DLL3 CAR-T cells with a safety switch SHP-77 tumor-carrying NSG mice were used to test the antitumor activity of DLL3 CAR-T cells with a safety switch. SHP-77 cells were thawed from frozen vials, counted, and diluted. 50 × 10⁶ cells per mouse in RPMI medium / Matrigel suspension. 6 100 viable cells / mL were injected subcutaneously. Tumor growth was monitored by caliper measurements using a digital caliper, starting on day 5 post-transplant. Tumor size was calculated using the formula: tumor volume = (width^2 × length / 2). Approximately 14 days post-transplant, mice were randomized into groups of 8 based on tumor volume. The average tumor volume per group was 178 mm². 3 On the same day that the mice were randomized, untransduced T cells and DLL3 CAR-T cells were thawed and counted according to standard procedures. The cells were counted at 5 × 10⁶ per mouse. 6 Each CAR+ cell was resuspended in RPMI by tail vein IV injection of 200 μL per mouse. Tumors were monitored every 3–4 days until the end of the study. All groups of DLL3 CAR-T cells with a safety switch induced significant tumor inhibition and complete or near-complete elimination of detectable tumors by day 50 (Figure 12A).

[0282] To test the antitumor activity of DLL3 CAR-T cells in a metastatic model similar to human diseases, we established DMS 273 small cell lung tumors (DMS 273-DLL3) expressing exogenous DLL3 via tail vein injection. Specifically, DMS 273-DLL3 cells were thawed and 5 × 10⁶ cells per mL were added to RPMI medium. 5 The cells were diluted to viable cells. 200 μL of cell suspension was injected per mouse via tail vein IV. On day 3 post-transplant, the mice were randomized into groups of 9. On the same day, DLL3 CAR-T cells were thawed, counted, and 5 x 10⁶ cells were administered per mouse. 6Individual CAR+ cells were resuspended in RPMI medium by tail vein IV injection of 200 μL per mouse. Tumors were monitored every 3–4 days using an IVIS imaging system until the end of the study. As shown in Figure 12B, several different DLL3 CARs with different rituximab-based safety switches were effective against metastatic tumors.

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

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

[0285] Example 14: Mouse safety study using animals carrying subcutaneous tumors To further evaluate the responsibility for potential cerebral and pituitary toxicity, DLL3 CAR-T cells were injected into NSG mice with subcutaneous LN229 tumors expressing exogenous mouse DLL3 (LN229-mDLL3). In this model, CAR-T activation by tumor cells may result in increased sensitivity and activity towards normal tissues potentially expressing DLL3. The experimental design is shown in Figure 14A. Three days prior to tumor transplantation (-3 days), adeno-associated virus (AAV) (Vigene Biosciences) encoding IL-7 and IL-15 was injected via the tail vein to support the proliferation and persistence of CAR-T cells. LN229-mDLL3 cells were then thawed from frozen vials and incubated in full growth medium (RPMI + 10% FBS) at a rate of 4.25 × 10⁶ 7 The cells were diluted to 100 cells / mL. The cell suspension was kept on ice until transplantation. Immediately before transplantation, the cells were mixed 1:1 with BD Matrigel matrix (catalog no. 354234) and administered to 4.25 x 100 mice. 6 A 200 μL cell / Matrigel suspension containing 10 LN229-mDLL3 cells was subcutaneously injected. Tumor growth was monitored by caliper measurements using a digital caliper, starting on day 8 post-transplant. Tumor size was calculated using the formula: tumor volume = (width^2 × length / 2). On day 22 post-transplant, 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), untransduced T cells and mouse cross-reactive 10G1-K DLL3 CAR-T cells were thawed, resuspended in RPMI + 10% FBS, and 1 x 10⁶ cells were administered per mouse. 7 Individual CAR+ cells were injected via tail vein IV injection at a volume of 200 μL per mouse. Tumors were monitored every 3–4 days until the end of the study to observe robust antitumor activity induced by DLL3 CAR T therapy (Figure 14B).

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

[0287] Example 15: Mouse safety study using animals carrying intracranial tumors To promote T cell infiltration into the brain and further understand potential neurotoxicity, NSG mice with intracranial LN229 tumors expressing exogenous mouse DLL3 and human EGFRvIII (LN229-mDLL3-vIII) were used. The experimental design is shown in Figure 15A. LN229-mDLL3 cells were thawed from frozen vials and 1 × 10⁶ cells were saturated in RPMI. 7 Diluted to 10⁴ viable cells / mL. Then, 3 μL of 3 × 10⁴ solution was administered per mouse. 4 A cell suspension containing 10 LN229-mDLL3 cells was injected intracranially. Tumor growth was monitored by the IVIS imaging system. On day 17 post-transplant, 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 1x10⁶ cells were administered. 7 CAR+ cells were injected into mice by tail vein IV injection at a volume of 200 μL / mouse. EGFRvIII CAR-T cells were included as a control to assess potential inflammation induced by tumor lysis in the brain. To support CAR-T cell proliferation and persistence, 0.5 μg of IL-15 (Peprotech AF-200-15) and 3 μg of IL-15Ra Fc fusion protein (R&D Systems 7194-IR) were administered to each animal twice weekly, starting on day 17 until the end of the study. Tumors were continued to be monitored every 3–4 days until the end of the study, and clear antitumor activity was observed (Figure 15B). On days 22 and 38, brain tissue from all animals was trimmed, processed, and embedded to reveal the ventricular system, including the third and fourth lateral ventricles. Three sections were placed in a single block, which was then sequentially sectioned to 4–6 microns and stained with H&E or immunohistochemistry to detect human-specific CD45 (hCD45). Pituitary tissue was processed to include the neural, middle, and anterior lobes and stained with H&E or immunohistochemically to detect hCD45 as a marker for human T cells.

[0288] On day 22, animals treated with non-transduced T cells or 10G1-K DLL3 CAR-T cells had sparse / sparse hCD45-stained T cells in the brain or pituitary gland (data not described). In contrast, in animals treated with EGFRvIII CAR-T cells, hCD45+ staining ranged from sparse / sparse to moderately low or moderate to moderately high in areas of infiltration / gliosis or glioma, consistent with the antitumor activity of this group (data not described). On day 38, animals treated with non-transduced T cells or EGFRvIII CAR-T cells had sparse / sparse hCD45+ staining in the brain and pituitary gland. Animals treated with 10G1-K DLL3 CAR-T cells had minimal or mild mononuclear cell infiltration in the pituitary gland, mainly in the middle and nervous lobes (Figure 15C-D). Furthermore, these animals exhibited slightly more (moderately low) hCD45+ staining, which is associated with small glioma lesions, compared to rare / sparse staining in other areas of the brain (vascular system, choroid plexus, and meninges), and this was consistent with the antitumor activity of this group, as shown in Figure 15B.

[0289] Example 16: In vitro cytotoxicity of dissociated mouse pituitary cells To directly test whether DLL3 CART is active against the pituitary gland, mouse pituitary glands from NSG mice were collected under sterile conditions for in vitro analysis. The tissue was dissociated by 3 rounds of incubation at 37°C in 1 mL of dissociation mix [5 mL DMEM, high glucose, GlutaMax (Gibco, catalog no. 10564), 50 μL enzyme H, 5 μL enzyme R, 6.25 μL enzyme A (Miltenyi Tumor Dissociation Kit no. 130-095-929)], followed by mechanical dissociation using grinding. 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 at RT for 3 minutes, followed by neutralization in complete medium. The cell suspension was filtered through a 70u filter and centrifuged to remove buffer. The cells were counted and placed in complete medium in a 96-well plate, 5 x 10⁴ cells per well. 4 Cells were seeded and allowed to recover for 3 days before CAR-T cells were added. At the time of CAR-T cell addition, the expected target density was 1 × 10⁶ per well. 4 It is a cell. For 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 in E:T ratios of 9:1, 3:1, and 1:1, and co-cultured with the target cells for 3 days. At the end of the 3-day co-culture, the medium was separated from the wells, and the T cells were pelleted by centrifugation. Target cells were treated with 50 μL / well of Cell Titer Glo (Promega, G7570) for 10 minutes, and cytotoxicity reads were analyzed using a SpectraMax plate reader.

[0290] Figure 16A shows experimental data demonstrating that DLL3 CAR-T cells are active against the DLL3+DMS 273 cell line, but are not cytotoxic to mouse pituitary cells in vitro. T cells were pooled and stained for activation markers (41BB and CD25) for flow cytometry analysis. Figure 16B shows that mouse pituitary cells do not activate DLL3 CAR-T cells in vitro. The supernatant was frozen at -80°C and then thawed for cytokine analysis using the Human TH1 / TH2 10-Plex Tissue Culture Kit (Meso Scale Discovery, K15010B). Figure 16C shows that both 10G1-K and 2G1 DLL3 CAR-T cells secrete interferon-gamma (IFNγ), tumor necrosis factor alpha (TNF-α), and IL-2 when co-cultured with the DLL3+DMS 273 cell line, but no cytokine secretion occurs after DLL3 CAR-T cells are co-cultured with mouse pituitary cells. Therefore, DLL3 CAR-T cells were not cytotoxic to pituitary cells in vitro. Another aspect of the present invention may be as follows: [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) Variable heavy chain CDR1 containing 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) Variable heavy chain CDR2 containing 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) Variable heavy chain CDR3 containing 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) Variable light chain CDR1 containing 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) Variable light chain CDR2 containing 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 a variable light chain CDR3 having 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) Variable heavy chain CDR1 containing 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) 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 containing 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) Variable light chain CDR1 containing 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) 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 containing 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) Variable heavy chains containing amino acid sequences 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) comprising at least one variable light chain containing 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, A chimeric antigen receptor in which 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) Variable heavy chains comprising amino acid sequences 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, A chimeric antigen receptor in which the variable heavy chain and the variable light chain are linked by at least one linker. [6] 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 scFv presented in Table 1d. [7] 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 SEQ ID NOs. 632-683. [8] The chimeric antigen receptor according to any one of [1] to [7], wherein the intracellular domain comprises at least one costimulatory domain. [9] The aforementioned costimulatory domains include CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (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 I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, and 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 A chimeric antigen receptor as described in [8], wherein the signaling region is 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 according to [9], wherein the costimulatory domain includes the signaling region of 4-1BB / CD137.

[11] The chimeric antigen receptor according to

[10] , wherein the 4-1BB / CD137 costimulatory domain comprises Sequence ID No. 480 or a fragment thereof.

[12] The chimeric antigen receptor according to any one of [1] to

[11] , wherein the intracellular domain comprises at least one activation domain.

[13] The chimeric antigen receptor according to

[12] , wherein the activating domain contains CD3.

[14] The chimeric antigen receptor according to

[13] , wherein the CD3 comprises CD3 zeta.

[15] The chimeric antigen receptor according to

[14] , wherein the CD3 zeta comprises SEQ ID NO: 481 or a fragment thereof.

[16] The chimeric antigen receptor according to any one of the above [1] to

[15] , wherein the chimeric antigen receptor is encoded by one of the polynucleotide sequences from sequence numbers 570 to 621 and 631.

[17] A chimeric antigen receptor according to any one of the preceding paragraphs [1] to

[16] , further comprising a safety switch.

[18] The chimeric antigen receptor according to

[17] , wherein the safety switch comprises a CD20 mimotope or a QBEND-10 epitope.

[19] The chimeric antigen receptor according to

[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 according to any one of the above

[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 according to any one of the above

[17] to

[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 described in [1] to

[21] above.

[23] A vector comprising the polynucleotide described in

[22] above.

[24] The vector according to

[23] , wherein the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenovirus vector, an adenovirus-related vector, a lentiviral vector, or any combination thereof.

[25] Modified immune cells expressing any one of the chimeric antigen receptors described in [1] to

[21] above.

[26] Manipulated immune cells expressing the polynucleotide described in

[22] or the vector described in

[23] or

[24] .

[27] The manipulated immune cells according to

[25] or

[26] , wherein the immune cells are T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells.

[28] The manipulated immune cells described in

[27] , wherein the cells are autologous T cells.

[29] The manipulated immune cells described in

[27] , wherein the cells are allogeneic T cells.

[30] A pharmaceutical composition comprising the manipulated immune cells described in any one of the above items

[25] to

[29] .

[31] A method for treating a disease or disorder in a subject requiring treatment of the disease or disorder, comprising administering to the subject an engineered immune cell as described in any one of paragraphs

[25] to

[29] , or a pharmaceutical composition as described in paragraph

[30] .

[32] The method according to

[31] , wherein the disease or disorder is cancer.

[33] The method according to

[31] or

[32] , wherein the disease or disorder is small cell lung cancer.

[34] A product comprising the manipulated immune cells described in any one of the preceding paragraphs

[26] to

[325] to

[29] , or the pharmaceutical composition described in paragraph

[30] .

[35] (a) Variable heavy chain CDR1 containing 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) Variable heavy chain CDR2 containing 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) Variable heavy chain CDR3 containing 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) Variable light chain CDR1 containing 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) Variable light chain CDR2 containing 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 conjugate comprising a variable light chain CDR3 containing 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 binder may be an antibody, an antibody conjugate, or an antigen-binding fragment thereof, or optionally, F(ab') 2 The DLL3 binder according to

[35] , which is a fragment, a Fab' fragment, a Fab fragment, an Fv fragment, an scFv fragment, a dsFv fragment, or a dAb fragment.

[37] The anti-DLL3 binder according to

[36] , wherein the binder is a monoclonal antibody containing an IgG constant region.

[38] An anti-DLL3 binder according to any one of the above

[35] to

[37] , comprising a variable weight (VH) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the VH sequence provided in Table 1b.

[39] An anti-DLL3 binder according to any one of the above

[35] to

[38] , comprising a variable light (VL) chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the VL sequence provided in Table 1c.

[40] The anti-DLL3 binder according to any one of the above

[35] to

[39] , wherein the binder comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the scFv sequence presented in Table 1d.

[41] The anti-DLL3 binder according to any one of the above

[35] to

[40] , wherein the binder is a fusion protein comprising an scFv fragment fused to an Fc constant region.

[42] A pharmaceutical composition comprising an anti-DLL3 binder described in any one of the preceding paragraphs

[35] to

[41] and a pharmaceutically acceptable excipient.

[43] A method for treating a disease or disorder in a subject requiring the use of an anti-DLL3 binder as described in any one of the preceding paragraphs

[35] to

[41] , or the pharmaceutical composition as described in

[42] .

[44] The method according to

[43] , wherein the disease or disorder is cancer.

[45] The method according to

[43] or

[44] , wherein the disease or disorder is small cell lung cancer.

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 Variable heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 253, Variable heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 254, Variable heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 255, Variable light chain CDR1 containing the amino acid sequence of SEQ ID NO: 256, Variable light chain CDR2 containing the amino acid sequence of SEQ ID NO: 257, and Variable light chain CDR3 containing the amino acid sequence of SEQ ID NO: 258, A chimeric antigen receptor, including one.

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 Variable heavy chains containing the amino acid sequence of SEQ ID NO: 259, and Variable light chain containing the amino acid sequence of SEQ ID NO: 260, Includes, A chimeric antigen receptor in which the variable heavy chain and the variable light chain are linked by at least one linker.

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

261.

4. A chimeric antigen receptor that specifically binds to DLL3, wherein the chimeric antigen receptor contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 510 or SEQ ID NO: 660, and The aforementioned amino acid sequence is Variable heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 253, Variable heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 254, Variable heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 255, Variable light chain CDR1 containing the amino acid sequence of SEQ ID NO: 256, Variable light chain CDR2 containing the amino acid sequence of SEQ ID NO: 257, and Variable light chain CDR3 containing the amino acid sequence of SEQ ID NO: 258, A chimeric antigen receptor, including one.

5. The chimeric antigen receptor according to claim 4, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 510 or SEQ ID NO:

660.

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

7. The aforementioned costimulatory domains include CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18)), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signal transduction lymphocyte activating molecule (SLAM protein), activated NK cell receptor, BTLA, and 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, ITGAAD, 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 6, wherein the signaling region is 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.

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

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

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

11. The chimeric antigen receptor according to claim 10, wherein the activating domain includes CD3.

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

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

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

598.

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

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

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

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

19. The chimeric antigen receptor according to any one of claims 15 to 18, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least about 80% identical to any one of sequence numbers 626 to 628 and 688 to 690.

20. The chimeric antigen receptor according to any one of claims 15 to 18, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least about 90% identical to any one of sequence numbers 626 to 628 and 688 to 690.

21. The chimeric antigen receptor according to any one of claims 15 to 18, wherein the chimeric antigen receptor comprises one amino acid sequence from sequence numbers 626 to 628 and 688 to 690.

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

23. A vector comprising the polynucleotide described in claim 22.

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

25. Engineered immune cells expressing the chimeric antigen receptor according to any one of claims 1 to 21.

26. Engineered immune cells expressing the polynucleotide described in claim 22 or the vector described in claim 23 or 24.

27. The manipulated immune cells according to claim 25 or 26, wherein the immune cells are T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells.

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

29. The manipulated immune cells according to claim 27, wherein the cells are allogeneic T cells.

30. A pharmaceutical composition comprising manipulated immune cells according to any one of claims 25 to 27.

31. The pharmaceutical composition according to claim 30 for treating a disease or disorder in a subject requiring treatment of a disease or disorder.

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

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

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

35. Variable heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 253, Variable heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 254, Variable heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 255, Variable light chain CDR1 containing the amino acid sequence of SEQ ID NO: 256, Variable light chain CDR2 containing the amino acid sequence of SEQ ID NO: 257, and Variable light chain CDR3 containing the amino acid sequence of SEQ ID NO: 258, An anti-DLL3 binder comprising, The binder is an antibody, an antibody conjugate, or an antigen-binding fragment thereof, and optionally F(ab') 2 An anti-DLL3 binder, which may be a fragment, a Fab' fragment, a Fab fragment, an Fv fragment, an scFv fragment, or a dsFv fragment.

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

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

259.

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

260.

39. The anti-DLL3 binder according to any one of claims 35 to 38, wherein the binder comprises a sequence that is at least about 90% identical to the scFv sequence of Sequence ID No.

261.

40. The anti-DLL3 binder according to any one of claims 35 to 39, wherein the binder comprises the scFv sequence of Sequence ID No.

261.

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

42. A pharmaceutical composition comprising an anti-DLL3 binder according to any one of claims 35 to 41, and optionally comprising pharmaceutically acceptable excipients.

43. The pharmaceutical composition according to claim 42 for treating a disease or disorder in a subject requiring treatment of a disease or disorder.

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

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

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