TET-on system with low leakage and low immunogenicity for inducible transgene expression

Abstract

The present invention relates to nucleic acids and modified immune cells or precursors thereof, comprising a first polynucleotide encoding an NF-κB p65-rTetO fusion protein comprising one or more transactivation domains (TAD) of human NF-κB p65 fused to a reverse Tet operator (rTetO) binding domain; a second polynucleotide comprising a Tet operator region (TetOR) for providing inducible expression of one or more transgene(s) operatively linked thereoto; and a third polynucleotide comprising a promoter directing transcription of p65-rTetO from a first strand of DNA, where the p65-rTetO is configured to induce transcription of a transgene of interest operatively linked to the TetOR in the presence of tetracycline or doxycycline (Dox) from an opposite strand of DNA.

Description

[0001] Atorney Docket No. 046483 -7474WO 1(04010)

[0002] TET-ON SYSTEM WITH LOW LEAKAGE AND LOW IMMUNOGENICITY FOR INDUCIBLE TRANSGENE EXPRESSION

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 693,474 filed on September 11, 2024, which is herein incorporated by reference in its entirety.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The Sequence Listing submitted herewith as an XML file named "046483- 7474xx_3.xml," created on September 11, 2025 and having a size of 472,979 bytes, is herein incorporated by reference in its entirety.

[0007] BACKGROUND

[0008] Conventional Tet-ON systems for inducible gene expression typically consist of three components: 1) a reverse tetracycline-controlled transactivator (rtTa), 2) a Tet Response Element (TRE) combined with a minimal promoter and 3) tetracycline (Tet) or its more stable derivatives, including doxycycline (Dox). rtTA is composed of a reverse Tet repressor (rTetR) fused to one or more VP16 transactivation domains derived from the herpes simplex virus (HSV). TRE generally consists of 7 repeats of an 19bp bacterial TetO sequence separated by short spacer sequences. In the presence of Tet or Dox, rtTA binds TetO sequences inside the TRE, thereby activating the adjacent minimal promoter and leading to the expression of target genes.

[0009] Inducible gene expression systems are becoming increasingly important for their potential usage in cellular immunotherapies such as chimeric antigen receptor (CAR) T cell immunotherapy. However, clinical translation of conventional Tet-ON systems has been impeded by their inert leakiness and immunogenicity which could cause unwanted and toxic side effects in patients.

[0010] Thus, there is a need in the art for novel gene expression systems with low leakage and immunogenicity for clinical translation. The present invention addresses this need.

[0011] SUMMARY OF THE INVENTION Atorney Docket No. 046483 -7474WO 1(04010)

[0012] In one aspect, the present invention provides a nucleic acid comprising: (1) a first polynucleotide encoding an NF-KB p65-rTetO fusion protein comprising one or more transactivation domains (TAD) of human NF-KB p65 fused to a reverse Tet operator (rTetO) binding domain; (2) a second polynucleotide comprising a Tet operator region (TetOR) for providing inducible expression of one or more transgene(s) operatively linked thereto; and (3) a third polynucleotide comprising a promoter directing transcription of p65-rTetO from a first strand of DNA, where p65-rTetO is configured to induce transcription of a transgene of interest operatively linked to the TetOR in the presence of tetracycline or doxycycline (Dox) from an opposite strand of DNA.

[0013] In an embodiment, the nucleic acid comprises a spacer between the TetOR and the promoter directing transcription of p65-rTetO. In one embodiment, the spacer comprises the nucleotide sequence of SEQ ID NO: 4.

[0014] In an embodiment, the nucleic acid further comprises a fourth polynucleotide comprising a transgene encoding a protein of interest, which is operatively linked to the TetOR.

[0015] In an embodiment, a portion of the first polynucleotide encoding p65-rTetO encodes p65 TAD2. In certain embodiments, the portion of the first polynucleotide encoding p65 TAD2 comprises the nucleic acid sequence of SEQ ID NO: 1 and / or the amino acid sequence of SEQ ID NO: 2. In another embodiment, a portion of the first polynucleotide encoding p65-rTetO encodes p65 TAD1 and p65 TAD2. In certain embodiments, the portion of the first polynucleotide encoding p65 TAD1 and p65 TAD2 comprises the nucleic acid sequence of SEQ ID NO: 21 or 23 and / or the amino acid sequence of SEQ ID NO: 22 or 24.

[0016] In an embodiment, a portion of the first polynucleotide encoding p65-rTetO encodes an rTetO, where the portion of the first polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 6 and / or encodes an amino acid sequence of SEQ ID NO: 7. In some embodiments, a portion of the first polynucleotide encoding rTetO is codon-optimized to enhance expression of p65-rTetO.

[0017] In an embodiment, the second polynucleotide comprises a Tet operator region (TetOR) comprising one or more copies of a nucleotide sequence of SEQ ID NO: 1. In certain embodiments, the second polynucleotide comprising the TetOR comprises the nucleotide sequence of SEQ ID NO: 2. Atorney Docket No. 046483 -7474WO 1(04010)

[0018] In an embodiment, the nucleotide sequence between the transgene of interest and the p65- rTetO comprises the nucleotide sequence is set forth in SEQ ID NO: 3.

[0019] In an embodiment, the third polynucleotide comprises a constitutive promoter. In certain embodiments, the constitutive promoter is selected from the group consisting of an EF-1 alpha promoter, a phosphoglycerate kinase- 1 (PGK) promoter, a Rous sarcoma virus (RSV) promoter, a cytomegalovirus (CMV) immediate-early promoter (CMV), a human Ubiquitin C promoter (UBC), a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV), a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus (EBV) immediate early promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. In one embodiment, the constitutive promoter is an EF-1 alpha promoter. In certain embodiments, the EF-1 alpha promoter comprises the nucleotide sequence of SEQ ID NO: 82.

[0020] In another embodiment, the third polynucleotide comprises a cell type-specific promoter. In certain embodiments, the cell type-specific promoter is selected from the group consisting of light and / or heavy chain immunoglobulin gene promoters, a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, an NK cell-specific promoter.

[0021] In some embodiments, the protein of interest is a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the CAR comprises an antigen binding domain selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a nanobody. In some embodiments, the CAR comprises an antigen binding domain that specifically binds a B cell protein. In certain embodiments, the B cell protein is selected from the group consisting of CD10, CD19, CD20, CD22, CD79b, CD34, CD52, CD123, FLT-3, ROR1, CD179b, and CD79a. In one embodiment, the CAR comprises an anti-CD19 antigen binding domain.

[0022] In some embodiments, the CAR comprises an antigen binding domain that specifically binding a tumor associated antigen. In certain embodiments, the tumor associated antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, Atorney Docket No. 046483 -7474WO 1(04010) glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof. In one embodiment, the tumor associated antigen is mesothelin.

[0023] In an embodiment, the CAR comprises a transmembrane domain from a protein selected from the group consisting of alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In certain embodiments, the CAR comprises a transmembrane domain selected from the group consisting of CD8, CD28, ICOS, and 0X40.

[0024] In an embodiment, each intracellular domain comprises a costimulatory domain and an intracellular signaling domain. In certain embodiments, the costimulatory domain is from a protein selected from the group consisting of CD28, 4-1BB (CD137), ICOS (CD278), 0X40, CD5, CD27, LFA-1 (CD1 la / CD18), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, ITGB7, NKG2D, NKG2C, 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, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, BTLA, an MHC class I molecule, and a ligand that specifically binds with CD8.

[0025] In an embodiment, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif. In some embodiments, the intracellular signaling domain is from a protein selected from the group consisting of CD3 zeta, common FcR gamma, FcyRIII Fc gamma Rlla, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12. In one embodiment, the intracellular signaling domain is from CD3 zeta.

[0026] In an embodiment, each antigen-binding domain is connected to the transmembrane domain by a hinge region. In certain embodiments, the hinge region is from a protein selected Atorney Docket No. 046483 -7474WO 1(04010) from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof. In one embodiment, the hinge region is from CD8 or CD28.

[0027] In an embodiment, the CAR comprises a leader sequence encoding an N-terminal signal peptide. In one embodiment, the leader sequence encodes a CD8a signal peptide.

[0028] In some embodiments, the fourth polynucleotide further encodes a second transgene encoding a second protein of interest. In certain embodiments, the first and second transgenes are operatively linked to the TetOR.

[0029] In an embodiment, the second protein of interest is a second CAR, a checkpoint inhibitor, a cytokine, a chemokine, a cytokine inhibitor, or a switch receptor.

[0030] In some embodiments, the second protein of interest is a checkpoint inhibitor antagonizing the activity of a checkpoint protein. In certain embodiments, the checkpoint inhibitor is selected from the group consisting of PD1, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG 3, TIGIT, TIM-3, and VISTA.

[0031] In some embodiments, the second protein of interest is a cytokine. In certain embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-9, IL-12, IL- 15, IL-18, IL-21 IL-22, IL-23, CXCL9, and CCL19.

[0032] In some embodiments, the second protein of interest is a chemokine. In certain embodiments, the chemokine is CXCL9 or CCL19.

[0033] In some embodiments, the second protein of interest is a cytokine inhibitor. In certain embodiments, the cytokine inhibitor is selected from the group consisting of anti-IL-6, anti-IL-1, anti-fFN-y, anti-TNFa, anti-IL-8, anti-ILlO, anti-GM-SCM, including scFvs and / or nanobodies thereof.

[0034] In some embodiments, the second protein of interest is a switch receptor selected from the group consisting of PD1-CD28, PD1A132L-CD28, TIM3-CD28, PD1-4-1BB, PDlA132L-4- 1BB, TGFPRI-IL-12RP1, and TGFPRII-IL-12RP2.

[0035] In some embodiments, the nucleic acid encodes a self-cleaving 2A peptide domain between the first and second protein of interest. In certain embodiments, the self-cleaving peptide domain is selected from the group consisting of T2A, P2A, E2A and F2A. Atorney Docket No. 046483 -7474WO 1(04010)

[0036] In some embodiments, the nucleic acid further encodes a furin cleavage site between the C-terminal end of the protein of interest and the self-cleaving 2A peptide domain.

[0037] In another aspect, the present invention provides an expression vector comprising any of the nucleic acids described herein. As used herein, the term “expression vector” may be used interchangeably with the term “expression construct”, which refers to an expression vector operatively linked to one or more polynucleotides or transgenes to be expressed in a cell. In some embodiments, the expression vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In some embodiments, the expression vector is a lentiviral vector. In certain embodiments, the lentiviral vector is a self-inactivating HIV vector comprising an LTR comprising a U3 deletion. In certain embodiments, the HIV vector comprises a partial gag encoded sequence, such as one comprising the nucleotide sequence of SEQ ID NO: 77. In certain embodiments, the HIV vector further comprises a central polypurine tract (cPPT), such as a cPPT encoded by the nucleotide sequence of SEQ ID NO: 78. In some embodiments, the expression vector or, more particularly, the HIV vector comprises a Woodchuck Hepatitis virus post-transcriptional regulatory sequence element (WPRE), such as a WPRE element encoded by the nucleotide sequence of SEQ ID NO: 83. In certain embodiments, the lentivirus expression vectors are lentivirus expression constructs comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 127-132, 139, 141, 144, and 157-160.

[0038] In another aspect, the present invention provides a cell comprising any of the nucleic acids or expression constructs described herein. In some embodiments, the cell is an immune cell. In certain embodiments, the immune cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell (Treg). In certain embodiments, the cell is a T cell. In certain embodiments, the cell is an autologous cell, which may be obtained from e g., a human subject.

[0039] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a modified (or transformed) cell described herein.

[0040] In another aspect, the present invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering a cell described herein or a pharmaceutical composition thereof to the subject, and further administering doxycycline in an Atorney Docket No. 046483 -7474WO 1(04010) amount sufficient to induce expression of one or more transgenes. In some embodiments, the doxycycline is administered after at least 12, 24, 36, or 48 hours have elapsed following administration of the modified cell or pharmaceutical composition.

[0041] In some embodiments, administration of doxycycline is stopped if or when the subject demonstrates a side effect.

[0042] In some embodiments, the disease or disorder is a cancer.

[0043] In certain embodiments, the cancer is a liquid tumor.

[0044] In certain embodiments, the cancer is a hematological malignancy.

[0045] In other embodiments, the cancer is a leukemia, a lymphoma, or a myeloma.

[0046] In certain embodiments, the cancer is a solid tumor. In certain embodiments, the solid tumor is selected from the group consisting of the solid tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, melanoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, hepatic cancer and brain cancer. In certain embodiments, the subject is a human subject.

[0047] In another aspect, a method for generating a modified cell according to the present invention comprises introducing into the cell any nucleic acid or expression construct described herein. In certain embodiments, the cell is cultured in the presence of IL-2, IL-7, IL- 15, IL-21, or a combination thereof.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments, which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0050] FIGs. 1 A-1B are schematics describing the mechanism of action and design of the low leakage and immunogenicity Tet-ON system. FIG. 1A shows the mechanism of action of the Tet-ON system; top: the reverse tetracycline-controlled transactivator (rtTA) is comprised of the reverse Tet repressor (rTetR) and a transcriptional activation domain (VP48) consisting of three Atorney Docket No. 046483 -7474WO 1(04010) subunits (VP16); bottom left: in the absence of doxycycline, rtTA cannot bind to the Tet response element (TRE) rendering the adjacent promoter (P) inactive; botom right: upon binding of doxycycline, rtTA binds to TRE which activates the promoter and induces gene expression. FIG. IB shows a schematic layout of the low-leakage Tet-On system; constitutive and inducible expression cassettes are inserted back-to-back into a third-generation self-inactivating (SIN) lentiviral vector. The constitutive EFla promoter expresses cargo 1 followed by a 2A selfcleaving peptide (P2A) and rtTA-M2ml (rtTA). The doxycycline-inducible promoter (YB-TRE) controls the expression of cargo2. A spacer (S) separates both promoters to reduce crosstalk. LTR = viral long-terminal repeat sequences; \|t = viral packaging sequence; RRE = viral Rev response element; cPPT = central polypurine tract; P2A = 2A sequence from porcine teschovirus-1; WPRE = woodchuck hepatitis virus post-transcriptional regulatory element.

[0051] FIGs. 2A-2B show constitutive CAR expression and doxycycline-inducible surface tag expression. FIG. 2A shows a schematic layout of PM352xl73 (CARCD19-Tet-LNGFR) for obtaining constitutive expression of the anti-CD19 CAR (CARCD19) and rtTA-M2ml (rtTA) in combination with Dox-inducible expression of the truncated low-affinity nerve growth factor receptor (tLNGFR) surface tag. FIG. 2B depicts a flow cytometry (FCM) analysis showing expression of tLNGFR in primary human T cells transduced with PM352xl73 after 5, 9, 12, and 19 days in the presence or absence of Ipg / ml doxycyline.

[0052] FIGs. 3A-3C show Dox-inducible expression of a mesothelin-targeting CAR (CARM5) and killing of mesothelin-expressing SKOV3 tumor cells. FIG. 3A shows a schematic layout of PM346xl43 (Tet-CARM5) illustrating the constitutive expression of the blue fluorescent protein reporter (BFP2) and rfTA-M2ml (rtTA) in combination with Dox-inducible expression of CARM5. FIG. 3B shows killing of mesothelin-expressing SKOV3 tumor cells by PM346xl43 or untransduced (UTD) T cells at different effector-to-target ratios (5:1, 2.5: 1, 1.25: 1, 0.625: 1) in the presence or absence of Ipg / ml doxycyline. FIG. 3C shows killing of mesothelin-expressing SKOV3 tumor cells by PM346xl43 or untransduced (UTD) T cells in the presence or absence of different amounts of doxycyline.

[0053] FIGs. 4A-4B show doxycyline-inducible expression of a CD19-targeting CAR (CARCD19) and killing of CD19-expressing NALM6 tumor cells. FIG. 4A is a schematic layout of PM346xl42 (Tet-CARCD19) with constitutive expression of the blue fluorescent protein reporter (BFP2) and rtTA-M2ml (rtTA) in combination with dox-inducible expression of Atorney Docket No. 046483 -7474WO 1(04010)

[0054] CARCD19. FIG. 4B shows killing of CD19-expressing NALM6 tumor cells by PM346xl42 T cells at different effector-to-target ratios (4: 1, 2:1. 1 :1, 0.5:1) in the presence or absence of Ipg / ml doxycyline.

[0055] FIGs. 5A-5B show the design of a partially humanized rtTA (rTA-p65). FIG. 5A shows the original reverse tetracycline-controlled transactivator (rtTA) comprised of the reverse Tet repressor (rTetR) and a transcriptional activation domain (VP48) consisting of three subunits (VP16) each derived from the herpes simplex virus (HSV). To reduce immunogenicity, VP48 is replaced with transactivation domains (TAD) from human p65 to generate rtTA-p65. FIG. 5B, top shows a simplified overview of p65 protein domains including the Rel homology domain and selected TADs; FIG. 5B, bottom shows truncated p65 mutants (vl / 2 / 3 / 4) selected for incorporation into rtTA; numbers indicate amino acid positions of human p65.

[0056] FIGs. 6A-6B show the expression and functional validation of rtTA-p65 mutants. FIG. 6A shows expression of CARCD19 by Tet-CARCD19 T cells with original rtTA-M2ml (rtTA- VP48; PM346xl42) or rtTA-p65 mutants (rtTA-p65vl / 2 / 3 / 4; PM492 / 493 / 494 / 495xl42) in the presence or absence of Ipg / ml doxycyline after 22h. FIG. 6B is a bar graph showing % killing of CD19-expressing NALM6 tumor cells by Tet-CARCD19 T cells with original rtTA-M2ml (rtTA-VP48; PM346xl42) or selected rtTA-p65 mutants (rtTA-p65v2 / 3; PM493 / 494xl42) in the presence or absence of Ipg / ml Dox after 48h.

[0057] FIGs. 7A-7B shows a comparison of the in vitro killing of mesothelin-expressing SKOV3 tumor cells by T cells expressing either a doxycycline-inducible or constitutive mesothelin- specific CAR. FIG. 7A is a schematic layout of the doxycycline-inducible vector PM346xl43 (Tet-CAR-MSLN, top) and the constitutive control vector PM359 (MSLN CAR, bottom). FIG. 7B shows killing of mesothelin-expressing SKOV3 tumor cells by PM346xl43, PM359 or untransduced (UTD) T cells at an effector-to-target (ET) ratio of 1 : 1 in the presence or absence of Ipg / ml doxycycline.

[0058] FIGs. 8A-8D show the doxycycline-inducible expression of a mesothelin-targeting CAR (MSLN CAR) alone or combined with secretion of mutant IL- 18 (IL- 18m) and the resulting cytotoxicity against mesothelin-expressing SKOV3 tumor cells. FIG. 8A presents a schematic layout of doxycycline-inducible vectors expressing MSLN CAR alone (PM346xl43, top) or with secretion of IL-18m (PM346xl87, bottom). FIG. 8B shows IL18m secretion from PM346xl87 T cells in the presence or absence of O.lpg / ml doxycycline. FIG. 8C shows killing of mesothelin- Atorney Docket No. 046483 -7474WO 1(04010) expressing SKOV3 tumor cells by PM346xl43, PM346xl 87 or untransduced (UTD) T cells with an effector-to-target (ET) ratio of 1: 1 in the presence or absence of Ipg / ml doxycycline. FIG. 8D shows killing of mesothelin-expressing SKOV3 tumor cells by the different T cell groups from FIG. 8C during a second round of co-culture in the presence or absence of Ipg / ml doxycycline.

[0059] FIGs. 9A-9D show the doxycycline-inducible expression of a constitutive IL-9 signaling mediator (cIL9s) and its effects on STAT signaling and in vivo cytotoxicity. FIG. 9A is a schematic layout of vector PM466xl74 (CD19-CAR Tet-cIL9s) expressing a constitutive CD19- specific CAR (blue) and doxycycline-inducible cIL9s (red). FIG. 9B is a flow cytometry analysis of PM466xl74 T-cells that were intracellularly stained for phosphorylated STAT1, STAT3 and STAT5. FIG. 9C shows the in vivo killing of B cell lymphoma cells (Ramos) by PM466xl74 T cells in the presence or absence of doxycycline compared to untransduced (UTD) T cells. FIG. 9D shows the corresponding overall survival.

[0060] FIG. 10 is a schematic depiction of a constitutive IL-2 receptor expressed in an NK92 non-Hodgkin-lymphoma cell line and the resulting in vitro cytotoxicity of the NK-transformed cell line against U266GFP (multiple myleoma) and U251 (glioblastoma) tumor cell lines.

[0061] FIG. 11 is a schematic depiction of a recombinant lentivirus construct (PM357x90) according to the present disclosure.

[0062] FIG. 12 shows the expression of PM357x90 and an mEGFP control (PM173) in T cells.

[0063] FIG. 13 depicts a phenotypic characterization of T cells expressing PM357x90 and an mEGFP control (PM173) at days 18, 25, and 41 post-transduction.

[0064] FIG. 14 depicts a phenotypic characterization of T cells expressing PM357x90 and an mEGFP control (PM 173) at days 18, 25, and 41 post-transduction.

[0065] FIG. 15 is a schematic depiction of a Tet-inducible recombinant lentivirus construct (PM346x90) according to the present disclosure.

[0066] FIG. 16 shows the results of experiments utilizing CAR T cells in combination with Tet- inducible recombinant lentivirus constructs.

[0067] FIG. 17 is a schematic design of a Tet-inducible recombinant lentivirus construct in combination with CAR expression.

[0068] FIG. 18 shows an exemplary TIL-specific IL-2 mutant for use in accordance with the present disclosure. Atorney Docket No. 046483 -7474WO 1(04010)

[0069] FIG. 19 shows the results of experiments utilizing Tet-inducible recombinant lentivirus CAR constructs.

[0070] FIG. 20 shows a platform for constitutive cytokine or CAR signaling.

[0071] FIG. 21 shows a stratgey for boosting CAR-T cells with IL-9 signaling.

[0072] FIG. 22 shows exemplary Tet-inducible lentivirus constructs for CAR-T / IL-9 signaling.

[0073] FIG. 23 shows STAT-signaling mediated by cIL9s.

[0074] FIG. 24 shows the results of CAR-T cells armored with Tet-inducible IL-9 signaling demonstrating enhanced tumor control in vivo.

[0075] DETAILED DESCRIPTION

[0076] Definitions

[0077] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0078] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0079] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0080] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0081] “Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division. Atorney Docket No. 046483 -7474WO 1(04010)

[0082] As used herein, the term “adaptor molecule” refers to a polypeptide with a sequence that permits interaction with two or more molecules, and in certain embodiments, promotes activation or inactivation of a cytotoxic cell.

[0083] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0084] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0085] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0086] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, a and P light chains refer to the two major antibody light chain isotypes.

[0087] By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0088] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the Atorney Docket No. 046483 -7474WO 1(04010) activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0089] The term “anti-tumor effect” as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.

[0090] The term “auto-antigen” means, in accordance with the present invention, any selfantigen which is recognized by the immune system as being foreign. Auto-antigens comprise, but are not limited to, cellular proteins, phosphoproteins, cellular surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0091] The term “autoimmune disease” as used herein is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen. Examples of autoimmune diseases include but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid Atorney Docket No. 046483 -7474WO 1(04010) arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among others.

[0092] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0093] “Allogeneic” refers to a graft derived from a different animal of the same species.

[0094] “Xenogeneic” refers to a graft derived from an animal of a different species.

[0095] The term “broadly neutralizing antibody (bnAb)” refers to an antibody that defends a cell from multiple strains of a particular virus by neutralizing its effect. In some embodiments, broadly neutralizing HIV-1 Antibodies (bnAbs) are neutralizing antibody which neutralize multiple HIV- 1 viral strain.

[0096] The term “cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

[0097] By the term "CD4" as used herein is meant any amino acid sequence specifying CD4 from any source, including an amino acid sequence of CD4 that has been generated through codon optimization of the nucleic acid sequence encoding CD4. Codon optimization may be accomplished using any available technology and algorithms designed to optimize codons in an amino acid sequence.

[0098] The term “chimeric antigen receptor” or “CAR,” as used herein, refers to an artificial T cell receptor that is engineered to be expressed on an immune effector cell and specifically bind an antigen. CARs may be used as a therapy with adoptive cell transfer. T cells are removed from a patient and modified so that they express the receptors specific to a particular form of antigen. In some embodiments, the CARs have been expressed with specificity to a tumor associated antigen, for example. CARs may also comprise an intracellular activation domain, a transmembrane domain and an extracellular domain comprising a tumor associated antigen binding region. In some aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived monoclonal antibodies, fused to transmembrane and intracellular domain. The specificity of CAR designs may be derived from ligands of receptors (e.g., peptides). In some Atorney Docket No. 046483 -7474WO 1(04010) embodiments, a CAR can target HIV infected cells by redirecting the specificity of a T cell expressing the CAR specific for HIV associated antigens.

[0099] The term “chimeric intracellular signaling molecule” refers to recombinant receptor comprising one or more intracellular domains of one or more co-stimulatory molecules. The chimeric intracellular signaling molecule substantially lacks an extracellular domain. In some embodiments, the chimeric intracellular signaling molecule comprises additional domains, such as a transmembrane domain, a detectable tag, and a spacer domain.

[0100] As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for the ability to bind antigens using the functional assays described herein.

[0101] “Co-stimulatory ligand,” as the term is used herein, includes a molecule on an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, Atorney Docket No. 046483 -7474WO 1(04010)

[0102] CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4- 1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0103] A “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (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, NKp44, NKp30, NKp46, NKG2D, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combination thereof.

[0104] A “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0105] The term “cytotoxic” or “cytotoxicity” refers to killing or damaging cells. In one embodiment, cytotoxicity of the modified cells is improved, e.g. increased cytolytic activity of T cells. Atorney Docket No. 046483 -7474WO 1(04010)

[0106] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0107] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0108] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0109] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0110] As used herein “envelope glycoprotein gp!20” or “gpl20” refers to a 120 kDa glycoprotein on the surface of the HIV envelope. gp !20 binds to a CD4 receptor on a host cell, such as a CD4 T lymphocyte. This starts the process by which HIV fuses its viral membrane with the host cell membrane and enters the host cell.

[0111] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0112] The term “expand” as used herein refers to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number Atorney Docket No. 046483 -7474WO 1(04010) relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term "ex vivo," as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0113] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0114] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0115] By the terms "Human Immunodeficiency Virus" or HIV" as used herein is meant any HIV strain or variant that is known in the art or that is heretofore unknown, including without limitation, HIV-1 and HIV-2.

[0116] “Homologous” as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. As applied to the nucleic acid or protein, "homologous" as used herein refers to a sequence that has about 50% sequence identity. More preferably, the homologous sequence has about 75% sequence identity, even more preferably, has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

[0117] “Humanized” forms of non-human (e g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or Atorney Docket No. 046483 -7474WO 1(04010) other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 : 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0118] “Fully human” refers to an immunoglobulin, such as an antibody, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody.

[0119] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e. , five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical. Atorney Docket No. 046483 -7474WO 1(04010)

[0120] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0121] The guide nucleic acid sequence may be complementary to one strand (nucleotide sequence) of a double stranded DNA target site. The percentage of complementation between the guide nucleic acid sequence and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%,

[0122] 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,

[0123] 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The guide nucleic acid sequence can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

[0124] 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more nucleotides in length. In some embodiments, the guide nucleic acid sequence comprises a contiguous stretch of 10 to 40 nucleotides. The variable targeting domain can be composed of a DNA sequence, a RNA sequence, a modified DNA sequence, a modified RNA sequence (see for example modifications described herein), or any combination thereof.

[0125] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3and e'100indicating a closely related sequence.

[0126] The term “immunoglobulin” or “Ig,” as used herein is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins Atorney Docket No. 046483 -7474WO 1(04010) are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

[0127] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0128] As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and / or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and / or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

[0129] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0130] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo. Atorney Docket No. 046483 -7474WO 1(04010)

[0131] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0132] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0133] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0134] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0135] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0136] The term “overexpressed” tumor antigen or “overexpression” of a tumor antigen is intended to indicate an abnormal level of expression of a tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors or a Atorney Docket No. 046483 -7474WO 1(04010) hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.

[0137] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0138] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0139] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0140] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0141] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory Atorney Docket No. 046483 -7474WO 1(04010) sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0142] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0143] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0144] A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0145] The term “resistance to immunosuppression” refers to lack of suppression or reduced suppression of an immune system activity or activation.

[0146] A “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell.

[0147] “Single chain antibodies” refer to antibodies formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered span of amino acids. Various methods of generating single chain antibodies are known, including those described in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242: 1038-1041.

[0148] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity Atorney Docket No. 046483 -7474WO 1(04010) does not itself alter the cl ssification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0149] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and / or reorganization of cytoskeletal structures, and the like.

[0150] A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

[0151] A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

[0152] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

[0153] As used herein, the term “substantially lacks an extracellular domain” refers to a molecule that is essentially free of a domain that extrudes extracellularly. In one embodiment, Atorney Docket No. 046483 -7474WO 1(04010) the chimeric intracellular signaling molecule lacks any function performed by an extracellular domain, such as antigen binding. In another embodiment, the chimeric intracellular signaling molecule includes a transmembrane domain but lacks a functional extracellular domain.

[0154] As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0155] As used herein, the term “switch receptor” or “chimeric switch receptor” refers to a molecule designed to switch a negative signal transduction signal into a positive signal. In some embodiments, the switch receptor is a chimeric protein comprising a first protein or fragment thereof associated with a negative signal, and a second protein or fragment thereof associated with a positive signal. Examples of proteins associated with a negative signal include, without limitation, CTLA-4, PD-1 , BTLA, TIM-3 and the like. Examples of proteins associated with a positive signal include, without limitation, CD28, ICOS, 4-1 BB, TGFpR and the like.

[0156] A “target site” or “target sequence” refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0157] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (0) chain, although in some cells the TCR consists of gamma and delta (y / 6) chains. TCRs may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell. Atorney Docket No. 046483 -7474WO 1(04010)

[0158] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0159] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0160] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0161] The term “tumor” as used herein, refers to an abnormal growth of tissue that may be benign, pre-cancerous, malignant, or metastatic.

[0162] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0163] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0164] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 Atorney Docket No. 046483 -7474WO 1(04010) to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0165] Nucleic Acids and Expression Vectors

[0166] In one aspect, the present invention provides a nucleic acid comprising: a first polynucleotide encoding an NF-KB p65-rTetO fusion protein (p65-rTetO or rtTA-p65) comprising one or more transactivation domains of human NF-KB p65 fused to a reverse Tet operator (rTetO) binding domain; a second polynucleotide comprising a Tet operator region (TetOR) for providing inducible expression of one or more transgene(s) operatively linked thereto; a third polynucleotide comprising a constitutive promoter directing transcription of p65- rTetO from a first strand of DNA, wherein p65-rTetO is configured to induce transcription of a transgene operatively linked to the TetOR in the presence of tetracycline or doxycycline (Dox) from an opposite strand of DNA. In an embodiment, the nucleic acid further comprises a fourth polynucleotide comprising an transgene encoding a protein of interest that is operatively linked to the TetOR and transcriptionally induced in the presence of tetracycline or Dox.

[0167] Human NF-KB p65 is known to comprise three TADs (N-ter to C-ter), p65 TAD3, p65 TAD1, and p65 TAD2 (FIG. 5B). In an embodiment, a portion of the first polynucleotide encoding p65-rTetO encodes p65 TAD2. In another embodiment, a portion of the first polynucleotide encoding p65-rTetO encodes p65 TAD1 and p65 TAD2. In an embodiment, p65 TAD1 comprises the nucleotide sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 15. In one embodiment, the portion of the first polynucleotide encoding p65 TAD2 comprises the nucleic acid sequence of SEQ ID NO: 16 and / or the amino acid sequence of SEQ ID NO: 17. In another embodiment, the portion of the first polynucleotide encoding p65 TAD1 and p65 TAD2 comprises the nucleic acid sequence of SEQ ID NO: 18 and / or the amino acid sequence of SEQ ID NO: 19. In another embodiment, a portion of the first polynucleotide encoding p65-rTetO encodes p65 TAD3, p65 TAD1, and p65 TAD2. In certain embodiments, the portion of the first polynucleotide encoding p65 TAD3, p65 TAD1, and p65 TAD2 comprises the nucleic acid sequence of SEQ ID NO: 133 and / or the amino acid sequence of SEQ ID NO: 134. In some embodiments, a portion of the first polynucleotide encoding p65-rTetO may comprise 1, 2, 3 or more copies of either one of TAD 3, TAD1, TAD2, or any combination thereof in any order. Atorney Docket No. 046483 -7474WO 1(04010)

[0168] In an embodiment, a portion of the first polynucleotide encoding p65-rTetO encodes the rTetO binding domain comprising the nucleic acid sequence of SEQ ID NO: 6 and / or encoding the amino acid sequence of SEQ ID NO: 7. In one embodiment, a portion of the first polynucleotide encoding rTetO is codon-optimized to enhance expression of p65-rTetO.

[0169] In one embodiment, the first polynucleotide encodes rtTA-p65, which comprises the nucleotide sequence of SEQ ID NOs: 21 or 23 and / or the amino acid sequence of SEQ ID NOs: 22 or 24, respectively.

[0170] In an embodiment, the second polynucleotide comprises a Tet operator region (TRE) comprising one or more copies of a nucleotide sequence comprising a Tet operator binding sequence (SEQ ID NO: 1) operatively linked to a minimal promoter. In one embodiment, the TRE comprises the nucleotide sequence of SEQ ID NO: 2 and a minimal promoter comprising the nucleotide sequence of SEQ ID NO: 3.

[0171] In some embodiments, the third polynucleotide comprises one or more transcriptional control elements (e.g., a promoter, enhancer) for promoting transcription of p65-rTetO. Suitable promoter and enhancer elements are known to those of skill in the art. In some embodiments, the promoter is a constitutive promoter. In one embodiment, the constitutive promoter is an EF- 1 alpha promoter. An exemplary EF-1 alpha comprises the nucleotide sequence of SEQ ID NO: 82. In another embodiment, the constitutive promoter is a phosphoglycerate kinase-1 (PGK) promoter. An exemplary PGK promoter comprises the nucleotide sequence of SEQ ID NO: 80. In another embodiment, the constitutive promoter is a Rous sarcoma virus (RSV) promoter. An exemplary RSV promoter comprises the nucleotide sequence of SEQ ID NO: 75. Other constitutive promoter sequences may also be used, including, but not limited to a cytomegalovirus (CMV) immediate-early promoter (CMV), a human Ubiquitin C promoter (UBC), a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV), a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus (EBV) immediate early promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.

[0172] Other suitable promoters include, but are not limited to cell-type specific promoters, including light and / or heavy chain immunoglobulin gene promoters, a CD8 cell-specific Atorney Docket No. 046483 -7474WO 1(04010) promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, an NK cell-specific promoter, and various art-known cell-type specific promoters.

[0173] In some embodiments, the nucleic acid comprises a bidirectional promoter, wherein the Tet operator is configured to induce transcription of the transgene in an opposite direction relative to the promoter directing transcription of p65-rTetO. In other embodiments, the Tet operator is configured to induce transcription of the transgene in the same direction relative to the promoter directing transcription of p65-rTetO.

[0174] In some embodiments, the nucleic acid comprises a spacer region between the TetOR directing inducible transcription of the transgene and the promoter directing transcription of p65- rTetO. In some embodiments, the spacer region comprises the nucleotide sequence of SEQ ID NO: 4.

[0175] In one embodiment, the p65-rTetO-induced transgene encoding the protein of interest is a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain.

[0176] In one embodiment, the antigen binding domain is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a nanobody.

[0177] In some embodiments, the CAR comprises an antigen binding domain that specifically binds a tumor associated antigen, such as an antigen that is specific for a tumor or cancer of interest. In some embodiments, the tumor associated antigen is overexpressed in a solid tumor. In some embodiments, the tumor associated antigen is overexpressed in a liquid tumor. Exemplary tumor associated antigens, include but are not limited to, alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD 19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof. In one embodiment, the tumor associated antigen is mesothelin. Atorney Docket No. 046483 -7474WO 1(04010)

[0178] In some embodiments, the CAR comprises an antigen binding domain that specifically binds a B cell protein. In some embodiments, the B cell protein is selected from the group consisting of CD10, CD19, CD20, CD22, CD79b, CD34, CD52, CD123, FLT-3, ROR1, CD 179b, and CD79a. In one embodiment, the CAR comprises an anti-CD19 antigen binding domain.

[0179] In some embodiments, the CAR comprises a transmembrane domain from a protein selected from the group consisting of alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the CAR comprises a transmembrane domain selected from the group consisting of CD8, CD28, ICOS, and 0X40.

[0180] In some embodiments, each intracellular domain comprises a costimulatory domain and an intracellular signaling domain. In some embodiments, the costimulatory domain is from a protein selected from the group consisting of CD28, 4-1BB (CD137), ICOS (CD278), 0X40, CD5, CD27, LFA-1 (CD1 la / CD18), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, BTLA, an MHC class I molecule, and a ligand that specifically binds with CD8. In one embodiment, the costimulatory domain is from a protein selected from the group consisting of CD28, 4-1BB, ICOS, and 0X40.

[0181] In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif. In some embodiments, the intracellular signaling domain is from a protein selected from the group consisting of CD3 zeta, common FcR gamma, FcyRIII Fc gamma Rlla, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12. In one embodiment, the intracellular signaling domain is from CD3 zeta. Atorney Docket No. 046483 -7474WO 1(04010)

[0182] In some embodiments, each antigen-binding domain is connected to the transmembrane domain by a hinge region. In some embodiments, the hinge region is from a protein selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof. In one embodiment, the hinge region is from CD8 or CD28.

[0183] In some embodiments, a nucleic acid of the present disclosure further comprises a leader sequence encoding a signal peptide at the N-terminal end of a cell surface protein (e g., CAR, marker protein etc.) or secreted protein, which is cleaved from the protein (e.g., antigen binding domain) during cellular processing and localization of the CAR and / or marker protein to the cellular membrane or secretion of a protein therefrom. Suitable leader sequences are known to those of skill in the art. In one embodiment, each CAR is encoded by a codon-altered polynucleotide encoding a CD8a signal peptide, which shares no significant homology to one another. In a particular embodiment, each CD8a signal peptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 36-39 and comprises an amino acid sequence of SEQ ID NO: 40.

[0184] In some embodiments, the third of fourth polynucleotide may further encode one or more additional transgenes encoding one or more additional proteins of interest. In certain embodiments, the additional protein(s) of interest may include another CAR with a different antigen binding specificity, an immune checkpoint inhibitor, a cytokine, a cytokine antagonist, a chemokine, a marker protein, or a combination thereof. In some embodiments, the additional protein(s) of interest is / are inducibly expressed in the presence of Tet or Dox. In other embodiments, the additional protein(s) of interest is / are operatively linked to the promoter in the third polynucleotide.

[0185] In some embodiments, an additional protein of interest is an immune checkpoint inhibitor antagonizing the activity of a checkpoint protein. Exemplary immune checkpoint inhibitor include, but are not limited to, antagonists of a protein selected from the group consisting of the Programmed Death 1 receptor (PD-1), the Adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA / CD272), CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4 / CD152), Indoleamine 2,3 -dioxygenase (IDO), the Killer-cell Immunoglobulin-like Receptor (KIR), the Lymphocyte Activation Gene-3 Atorney Docket No. 046483 -7474WO 1(04010)

[0186] (LAG3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), the V-domain Ig suppressor of T cell activation, and combinations thereof.

[0187] Exemplary immune checkpoint inhibitors include, but are not limited to PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta.

[0188] Exemplary cytokines include, but are not limited to IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21 IL-22, and IL-23. In an embodiment, the cytokine is a mutant IL-18 comprising the amino acid sequence set forth in SEQ ID NO: 143 and / or encoded by the nucleotide sequence of SEQ ID NO: 142.

[0189] Exemplary cytokine antagonists include, but are not limited to, those inhibiting cytokine release syndrome (CRS), such as anti-IL-6, anti-IL-1, anti-fFN-y, anti-TNFa, anti-IL-8, anti- IL10, anti-GM-SCM, including scFvs and / or nanobodies thereof.

[0190] Exemplary chemokines include, but are not limited to CXCL9 and CCL19.

[0191] The term “switch receptor” refers to a molecule designed to switch a negative signal transduction signal into a positive signal. In some embodiments, the switch receptor is a chimeric protein comprising a first protein or fragment thereof associated with a negative signal, and a second protein or fragment thereof associated with a positive signal. Examples of proteins associated with a negative signal include, without limitation, CTLA-4, PD-1 , BTLA, TIM-3 and the like. Examples of proteins associated with a positive signal include, without limitation, CD28, ICOS, 4-1BB, IL-12R, an extracellular domain of a TGF£R (e g., TGF0RI or TGF0RII) and the like. Exemplary switch receptors include, but are not limited to, PD1-CD28, TIM3- CD28, PD1-4-1BB, PDlA132L-4-lBB, PD1A132LcD28, TGFPRI-IL-12R01 , TGF0RII-IL-12RP2, and the dominant-negative TGFpRII switch receptor, TGFpRIIDN.

[0192] Other suitable switch receptors for use in the present invention are described in US Patent No. 10,981,969 B2, the disclosure of which is incorporated herein by reference.

[0193] Exemplary marker proteins include, but are not limited to, minimal low affinity nerve growth factor (mLNGFR), truncated LNGFR (tLNGFR), truncated EGFR (tEGFR), truncated CD 19 (tCD19), truncated CD34 (tCD34), blue fluorescent protein (BFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and combinations thereof. Atorney Docket No. 046483 -7474WO 1(04010)

[0194] In some embodiments, the fourth polynucleotide encodes a self-cleaving 2A peptide domain fused in-frame between one or more proteins of interest and / or comprises an internal ribosome binding site (IRES) between one or more proteins of interest. As used herein, a “selfcleaving peptide” or “2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various selfcleaving or 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV), and porcine tescho virus- 1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.

[0195] In one embodiment, the nucleic acid of the present disclosure comprises the nucleotide sequence of SEQ ID NOs: 69 or 70, which encodes the T2A self-cleaving peptide of SEQ ID NO: 71. In another embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 72, which encodes the P2A self-cleaving peptide of SEQ ID NO: 73. Those of skill in the art would be able to select the appropriate self-cleaving peptide for use in the present invention.

[0196] In some embodiments, one or more transgenes are separated or additionally separated from one another by a nucleotide sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-golgi and processes protein precursors before their secretion. Furin cleaves at the COOH- terminus of its consensus recognition sequence. Various furin consensus recognition sequences (or “furin cleavage sites”) are known to those of skill in the art, including, without limitation, R(X1)KR (SEQ ID NO: 85) or R(X1)RR (SEQ ID NO: 86), (X2)R(X1)(X2)R (SEQ ID NO: 87) and R(X1)(X1)R (SEQ ID NO: 88), such as RRKR (SEQ ID NO: 89), RQKR (SEQ ID NO: 8390), where XI is any naturally occurring amino acid, X2 is Lys or Arg. Those of skill in the art would be able to select the appropriate Furin cleavage site for use in the present invention.

[0197] In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding a combination of a Furin cleavage site and a 2A peptide. In one embodiment, the Furin cleavage site is positioned between the C-terminal end of the protein of interest and the self-cleaving 2A peptide domain. Examples include, without limitation, a linker comprising a nucleic acid Atorney Docket No. 046483 -7474WO 1(04010) sequence encoding Furin and F2A, a linker comprising a nucleic acid sequence encoding Furin and E2A, a linker comprising a nucleic acid sequence encoding Furin and P2A, a linker comprising a nucleic acid sequence encoding Furin and T2A. Those of skill in the art would be able to select the appropriate combination for use in the present invention.

[0198] In such embodiments, the nucleic acid comprises a spacer sequence between the Furin cleavage site and the 2A peptide. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers such as (GS)n, GSG, (GSGGS)n (SEQ ID NO: 91), (GGGS)n (SEQ ID NO: 92 (GGGGS)n (SEQ ID NO: 93), where n represents an integer of at least 1. Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 94), GGSGG (SEQ ID NO: 95), GSGSG (SEQ ID NO: 96), GSGGG (SEQ ID NO:97), GGGSG (SEQ ID NO:98), GSSSG (SEQ ID NO:99), GGGGS (SEQ ID NO: 100), (G4S)3 (SEQ ID NO: 101), (G4S)4 (SEQ ID NO: 102), and the like. Those of skill in the art would be able to select the appropriate spacer sequence for use in the present invention.

[0199] In some embodiments, the linker comprises a nucleic acid sequence that encodes for an internal ribosome entry site (IRES). As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art, including, without limitation, IRES obtainable from viral or cellular mRNA sources, e.g., immunogloublin heavychain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). An exemplary IRES, encephalomyocarditis virus (EMCV) IRES, comprises the nucleotide sequence of SEQ ID NO: 103. Those of skill in the art would be able to select the appropriate IRES for use in the present invention.

[0200] A nucleic acid of the present disclosure may be present within an expression vector and / or a cloning vector.

[0201] In one aspect, the present invention provides an expression vector comprising a nucleic acid of the present invention as exemplified above. An expression vector can include a Atorney Docket No. 046483 -7474WO 1(04010) selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example, and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0202] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, e.g., viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, those based on lentiviruses, such as human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); other retroviruses, including gamma-retrovirus vectors derived from Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or Murine Stem Cell Virus (MSCV), spleen necrosis virus, Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, myeloproliferative sarcoma virus, and mammary tumor virus); adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); vaccinia virus; Atorney Docket No. 046483 -7474WO 1(04010) poliovirus; SV40; herpes simplex virus; and the like. In certain embodiments, the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0203] In some embodiments, the expression vector (e.g., a lentiviral vector) may be used to introduce and express one or more transgene(s) for expressing e g., one or more CARs and / or other therapeutically active products in an immune cell or precursor thereof (e.g., a T cell). In some embodiments, the expression vector comprises a mammalian promoter, such as a constitutive promoter described above. In one embodiment, the vector further comprises an elongation-factor- 1 -alpha promoter (EF- la promoter). Use of an EF-la promoter may increase the efficiency in expression of downstream transgenes (e.g., a chimeric receptor encoding nucleic acid sequence). Physiologic promoters (e.g., an EF-la promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of a lentivirus or retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector (e.g., lentiviral vector) are known to those of skill in the art and may be incorporated into a vector of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises a non-requisite cis acting sequence that may improve titers and gene expression. One non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention.

[0204] In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). In one embodiment, the WPRE comprises the nucleotide sequence of SEQ ID NO: 83. Accordingly, in some embodiments a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention.

[0205] A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal Atorney Docket No. 046483 -7474WO 1(04010) repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and to viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector (e.g., lentiviral vector) of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, an RRE sequence, a packaging signal, a central polypurine tract (cPPT) sequence, a 5’LTR, and a 3’ U3 deleted LTR in addition to the polynucleotides encoding the transgenes of the present disclosure.

[0206] Vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). See Zufferey, R. et al., J. Virol. 73(12):9873-9880, Dec. 1998; and Dull et al., J. Virol. 72(11):8463-8471, Nov. 1998). A selfinactivating vector is engineered to prevent viral transcription beyond the first round of viral replication. Consequently, a self-inactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors virtually eliminate the risk of creating a replication-competent virus.

[0207] In one embodiment, the lentiviral vector is a self-inactivating HIV vector comprising a 5’ U3 LTR (e.g., SEQ ID NO: 76), a 3’ U3 deleted LTR (e.g., SEQ ID NO: 84), a partial HIV gag sequence comprising an RRE sequence and packaging signal (t|t) (e.g., SEQ ID NO: 77), and an HIV central polypurine tract (cPPT) sequence (SEQ ID NO: 78). In some embodiments, the expression vector and / or HIV vector comprises a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE). In an exemplary embodiments, the WPRE comprises the nucleotide sequence of SEQ ID NO: 83.

[0208] Chimeric Antigen Receptors (CARs)

[0209] In one aspect, the present invention provides compositions and methods for making and using modified immune cells or precursor cells thereof, e.g., modified T cells, comprising one or Atorney Docket No. 046483 -7474WO 1(04010) more chimeric antigen receptors (CARs), each having specific binding affinity for a different antibody. A subject CAR of the invention comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. A subject CAR of the invention may additionally comprise a signal peptide domain and / or a hinge domain. Accordingly, in an embodiment, a subject CAR of the invention comprises (N- terminal to C terminal) a signal peptide domain, an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory signaling domains, and an intracellular signaling domain, such as the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). In some embodiments, the CAR comprises two costimulatory domains, such as two costimulatory domains back-to-back between the transmembrane domain and the intracellular signaling domain. In some embodiments, one or more of the CAR functional domains are separated by a linker or spacer.

[0210] The antigen binding domain may be operably linked to another domain of the CAR, such as the transmembrane domain, the costimulatory signaling domain or the intracellular signaling domain, each described elsewhere herein, for expression in the cell. In one embodiment, a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding a transmembrane domain, and further operably linked to a third a nucleic acid sequence encoding a costimulatory signaling domain.

[0211] The antigen binding domains described herein can be combined with any of the transmembrane domains, any of the costimulatory signaling domains, any of the intracellular signaling domains or any of the other domains described herein for inclusion in a CAR of the present invention.

[0212] Antigen Binding Domain

[0213] The antigen binding domain of a CAR is an extracellular region of the CAR for binding to a specific target antigen including proteins, carbohydrates, and glycolipids. In some embodiments, the CAR comprises affinity to a target antigen (e.g., a tumor associated antigen) on a target cell (e.g., a cancer cell). The target antigen may include any type of protein, or epitope thereof, associated with the target cell. For example, the CAR may comprise affinity to a target antigen on a target cell that indicates a particular status of the target cell. Atorney Docket No. 046483 -7474WO 1(04010)

[0214] The choice of antigen binding domain depends upon the type and number of antigens that are present on the surface of a target cell. For example, the antigen binding domain may be chosen to recognize an antigen that acts as a cell surface marker on a target cell associated with a particular status of the target cell.

[0215] In one embodiment, a CAR of the invention comprises an extracellular domain having an antigen binding domain that targets a tumor antigen. In one embodiment, the tumor antigen is selected from the group consisting of EGFRvIII, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, B7H4, CEACAM5, KIT, IL-13Ra2, interleukin- 11 receptor a (IL-l lRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, Folate receptor alpha (FRa), ERBB2 (Her2 / neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE- la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6- AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1

[0216] In some embodiments, the tumor antigen is a B cell antigen. Exemplary B cell antigens include but are not limited to CD5, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD33 (IL3Ra), CD34, CD37, CD38, CD40, CD52, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269. Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-L CXCR4, 5, 7, IL- 7 / 3R, IL7 / 4 / 3R, and L4R, and variants thereof. Atorney Docket No. 046483 -7474WO 1(04010)

[0217] In one embodiment, the B cell antigen is selected from the group consisting of CD10, CD19, CD20, CD22, CD33 (IL3Ra), CD34, CD52, CD79b, CD123, CD179b, FLT-3, ROR1, and any variant thereof. In a particular embodiment, the B cell antigen is selected from the group consisting of CD 19, CD20, CD22, and CD79b.

[0218] As described herein, a CAR of the present disclosure having affinity for a specific target antigen on a target cell may comprise a target-specific binding domain. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the targetspecific binding domain is of human origin.

[0219] Depending on the desired antigen to be targeted, the CAR of the invention can be engineered to include the appropriate antigen binding moiety that is specific to the desired antigen target. For example, if CD 19 is the desired antigen that is to be targeted, an antibody for CD 19 can be used as the antigen binding moiety for incorporation into the CAR of the invention. For example, in an exemplary embodiment, a CAR of the present disclosure having affinity for CD 19 on a target cell may comprise a human CD 19 binding domain in the form of an scFv. In some embodiments, the binding domain is a murine binding domain. In some embodiments, the binding domain is a chimeric or partially humanized binding domain. In some embodiments, the binding domain is a human binding domain.

[0220] For example, in an exemplary embodiment, a CAR of the present disclosure having affinity for CD 19 on a target cell may comprise a human CD 19 binding domain in the form of an scFv. In some embodiments, the binding domain is a murine binding domain. In some embodiment, the binding domain is a chimeric or partially humanized binding domain. In some embodiments, the binding domain is a human binding domain.

[0221] In one embodiment, the CD 19 binding domain is an scFv expressed from a polynucleotide comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 115, 117, and 119, and / or an amino acid sequence selected from the group consisting of SEQ ID NOs: 116, 118, and 120, respectively.

[0222] In another embodiment, the CD20 binding domain is a nanobody expressed from a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 121 and / or the amino acid sequence of SEQ ID NO: 122. Atorney Docket No. 046483 -7474WO 1(04010)

[0223] In another embodiment, the CD22 binding domain is a nanobody expressed from a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 123 and / or the amino acid sequence of SEQ ID NO: 124.

[0224] In another embodiment, the CD79b binding domain is an scFv expressed from a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 125 and / or the amino acid sequence of SEQ ID NO: 126.

[0225] The antigen binding domain can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a nanobody, or any fragment thereof. Thus, in one embodiment, the antigen binding domain portion comprises a mammalian antibody or a fragment thereof. In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), a single-chain variable fragment (scFv), and a nanobody.

[0226] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH:VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker or spacer, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The terms “linker” and “spacer” are used interchangeably herein. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N- terminus to C-terminus, VH - linker - VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL - linker - VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

[0227] The linker is typically rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, GSG, (GSGGS)n (SEQ ID NO: 91), (GGGS)n (SEQ ID NO: 92), and (GGGGS)n (SEQ ID NO: 93), Atorney Docket No. 046483 -7474WO 1(04010) where n represents an integer of at least 1 . Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 94), GGSGG (SEQ ID NO: 95), GSGSG (SEQ ID NO: 96), GSGGG (SEQ ID NO: 97), GGGSG (SEQ ID NO: 98), GSSSG (SEQ ID NO: 99), GGGGS (SEQ ID NO: 100), (G4S)3 (SEQ ID NO: 101), (G4S)4 (SEQ ID NO: 102), and the like. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention.

[0228] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3): 173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8)768-71; Ledbetter et al., CritRev Immunol 1997 17(5-6):427-55; Ho et al., Biochim Biophys Acta 2003 1638(3) :257-66).

[0229] As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0230] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab’) (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0231] In some instances, the antigen binding domain may be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding Atorney Docket No. 046483 -7474WO 1(04010) domain of the CAR may comprise a human antibody as described elsewhere herein, or a fragment thereof.

[0232] Tolerable variations of the antigen binding domain will be known to those of skill in the art, while maintaining specific binding to its target. For example, in some embodiments the an antigen binding domain comprises an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to another binding domain corresponding to the same target..

[0233] In some embodiments, the binding domain is encoded by a nucleic acid sequence comprising a nucleotide sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to another binding domain corresponding to the same target.

[0234] The antigen binding domain may be operably linked to another domain of the CAR, such as the signal peptide domain or transmembrane domain both described elsewhere herein. In one embodiment, a nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding a signal peptide domain and a nucleic acid encoding a transmembrane domain.

[0235] The antigen binding domains described herein, such as the antibody or fragment thereof can be combined and operatively linked to any of the transmembrane domains described herein, any of the costimulatory domains, any of the intracellular signaling domains described herein, or any of the other domains described herein that may be included in the CAR.

[0236] Transmembrane Domain

[0237] With respect to the transmembrane domain, a CAR of the present invention can be designed to comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain. The transmembrane domain of a subject CAR is a region that is capable of spanning the plasma membrane of a cell (e.g., an immune cell or precursor thereof). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell Atorney Docket No. 046483 -7474WO 1(04010) membrane. In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR.

[0238] In one embodiment, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0239] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane regions of particular use in this invention include, without limitation, transmembrane domains derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.

[0240] A transmembrane domain may be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains or intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in the CAR. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0241] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In one embodiment, a subject CAR comprises a CD8a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 50.

[0242] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In one embodiment, a subject CAR comprises a CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 48. Atorney Docket No. 046483 -7474WO 1(04010)

[0243] In some embodiments, a subject CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. The hinge region is capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells and facilitating proper protein folding for the CAR (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible, hydrophilic domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell. The flexibility of the hinge region permits the hinge region to adopt many different conformations.

[0244] The hinge region is an optional component for the CAR. In some embodiments, the CAR may include a hinge region selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4). In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0245] In some embodiments, the hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 103); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof.

[0246] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.

[0247] Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino Atorney Docket No. 046483 -7474WO 1(04010) acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0248] In some embodiments, the hinge region may include glycine polymers (G)n, glycineserine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 91) and (GGGS)n (SEQ ID NO: 92), and (GGGGS)n (SEQ ID NO: 93), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 94), GGSGG (SEQ ID NO: 95), GSGSG (SEQ ID NO: 96), GSGGG (SEQ ID NO: 97), GGGSG (SEQ ID NO: 98), GSSSG (SEQ ID NO: 99), GGGGS (SEQ ID NO: 100), (G4S)3 (SEQ ID NO: 101), (G4S)4 (SEQ ID NO: 102), and the like.

[0249] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 104); CPPC (SEQ ID NO: 105); CPEPKSCDTPPPCPR (SEQ ID NO: 106) (see, e.g, Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO: 107); KSCDKTHTCP (SEQ ID NO: 108); KCCVDCP (SEQ ID NO: 109); KYGPPCP (SEQ ID NO: 110); EPKSCDKTHTCPPCP (SEQ ID NO: 111) (human IgGl hinge); ERKCCVECPPCP (SEQ ID NO: 112) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 113) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 114) (human IgG4 hinge); and the like.

[0250] In some embodiments, a subject CAR comprises a CD8a hinge domain and a CD8a transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 50 and the CD8a hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 46. Atorney Docket No. 046483 -7474WO 1(04010)

[0251] Tolerable variations of the transmembrane and / or hinge domain will be known to those of skill in the art, while maintaining its intended function. For example, in some embodiments a transmembrane domain or hinge domain comprises an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least

[0252] 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

[0253] 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least

[0254] 97%, at least 98%, at least 99% sequence identity to any amino acid sequence corresponding to a particular transmembrane domain or hinge region.

[0255] The transmembrane domain may be combined with any hinge region and / or may comprise one or more transmembrane domains described herein.

[0256] In some embodiments, a subject CAR may further comprise, between the extracellular domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR, a spacer domain. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the intracellular domain in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, e.g., 10 to 100 amino acids, or 25 to 50 amino acids. In some embodiments, the spacer domain may be a short oligo- or polypeptide linker, e.g., between 2 and 10 amino acids in length as described above. In some embodiments, a glycineserine doublet, a glycine-serine-glycine triplet, or any of the glycine-serine rich linkers described above can provide a suitable linker between the transmembrane domain and the intracellular signaling domain of the subject CAR.

[0257] Accordingly, a subject CAR of the present disclosure may comprise any of the transmembrane domains, hinge domains, or spacer domains described herein.

[0258] Intracellular Domain

[0259] A subject CAR of the present invention also includes an intracellular domain. The intracellular domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., immune cell). The intracellular domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and / or destroying a target cell. Atorney Docket No. 046483 -7474WO 1(04010)

[0260] The intracellular domain or otherwise the cytoplasmic domain of the CAR is responsible for activation of the cell in which the CAR is expressed. Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co- stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.

[0261] In certain embodiments, the intracellular domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In certain embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain.

[0262] Examples of the intracellular signaling domain include, without limitation, the chain of the T cell receptor complex or any of its homologs, e g., q chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 8 and s), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptors, and combinations thereof.

[0263] Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but are not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma R1 la, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD lib, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME Atorney Docket No. 046483 -7474WO 1(04010)

[0264] (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combination thereof.

[0265] Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0266] Intracellular signaling domains suitable for use in a subject CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) IT AM motifs as described below. In some embodiments, the intracellular signaling domain includes DAP10 / CD28 type signaling chains. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.

[0267] Intracellular signaling domains suitable for use in a subject CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, an IT AM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the IT AM motif are separated from one another by 6 to 8 amino acids. In one embodiment, the intracellular signaling Atorney Docket No. 046483 -7474WO 1(04010) domain of a subject CAR comprises 3 IT AM motifs. In some embodiments, intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. (2014) Immunol. 5:254).

[0268] A suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0269] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcR gamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3 -DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 delta chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T- cell surface antigen T3 / Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein Atorney Docket No. 046483 -7474WO 1(04010)

[0270] CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig- alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In one embodiment, an intracellular signaling domain suitable for use in a subject CAR of the present disclosure includes a DAP10 / CD28 type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in a subject CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 zeta.

[0271] In one embodiment, the intracellular domain of a subject CAR comprises a CD3 zeta intracellular signaling domain. In some embodiments, the CD3z intracellular signaling domain is encoded by a nucleotide sequence of any one of SEQ ID NOs: 63-66 or an amino acid sequence of SEQ ID NO: 67 or 68. In some embodiments, two or more of the CARs in an expression construct may comprise a CD3z intracellular signaling domain that is encoded by a different codon-altered polynucleotide sharing no significant homology therebetween to prevent or reduce recombination between otherwise identical nucleotide sequences.

[0272] While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0273] In some embodiments, the intracellular domain of a subject CAR comprises a costimulatory signaling domain which includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, 0X40, ICOS, 4- IBB, PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof. Atorney Docket No. 046483 -7474WO 1(04010)

[0274] In certain embodiments, the costimulatory domain is derived from CD28, 4-1BB, ICOS, or 0X40. In an embodiment, a polynucleotide encoding the CD28 costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 55 and / or amino acid sequence of SEQ ID NO: 56. In an embodiment, a polynucleotide encoding the 4-1BB costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 57 and / or amino acid sequence of SEQ ID NO: 58. In an embodiment, a polynucleotide encoding the ICOS8 costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 59 and / or amino acid sequence of SEQ ID NO: 60. In an embodiment, a polynucleotide encoding the 0X40 costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 61 and / or amino acid sequence of SEQ ID NO: 62.

[0275] The intracellular signaling domains described herein can be combined with any of the costimulatory signaling domains described herein, any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR.

[0276] Tolerable variations of the intracellular domain will be known to those of skill in the art, while maintaining specific activity. For example, in some embodiments the intracellular domain, costimulatory domain, and / or intracellular signaling domain comprises an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to another corresponding amino acid sequence for the particular intracellular domain, costimulatory domain, and / or intracellular signaling domain.

[0277] In an exemplary embodiment, an anti-CD19 CAR (CAR18) with a CD 19 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 26 and / or the amino acid sequence of SEQ ID NO: 27.

[0278] In another exemplary embodiment, an anti-CD20 CAR (C ARI 9ml) with a CD20 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 28 and / or the amino acid sequence of SEQ ID NO: 29. Attorney Docket No. 046483 -7474WO 1(04010)

[0279] In another exemplary embodiment, an anti-CD22 CAR (CAR13) with a CD22 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 30 and / or the amino acid sequence of SEQ ID NO: 31.

[0280] In another exemplary embodiment, an anti-CD79b CAR (CAR14) with a CD79b antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 32 and / or the amino acid sequence of SEQ ID NO: 33.

[0281] In another exemplary embodiment, an anti-CD79b CAR (CAR14ml) with a CD79b antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 137 and / or the amino acid sequence of SEQ ID NO: 138 Exemplary nucleotide and amino acid sequences corresponding to various rtTA components, CARs and their functional domains, including vector sequences, and expression constructs are shown in Table 1.

[0282] Table 1 : Nucleotide and Amino Acid Sequences Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) Atorney Docket No. 046483 -7474WO 1(04010) 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[0283] Modified Immune Cells

[0284] The present invention provides a modified immune cell or precursor cell thereof (e.g., a modified T cell, a modified natural killer {NK) cell, a modified natural killer T (NKT) cell), comprising an above-described nucleic acid or expression vector encoding one or more CARs, Atorney Docket No. 046483 -7474WO 1(04010) marker proteins, or therapeutically active proteins. Accordingly, such modified cells generally possess the binding specificity of the antigen binding domain corresponding to a particular CAR expressed therein.

[0285] Any modified cell comprising a CAR may be envisioned to comprise any antigen binding domain, any hinge, any transmembrane domain, any intracellular costimulatory domain, and any intracellular signaling domain described herein, and can readily be understood and made by a person of skill in the art in view of the disclosure herein.

[0286] In some embodiments, the modified cell is an immune cell or precursor cell thereof. In an exemplary embodiment, the modified cell is a T cell. In an exemplary embodiment, the modified cell is an autologous cell. In an exemplary embodiment, the modified cell is an autologous immune cell or precursor cell thereof. In an exemplary embodiment, the modified cell is an autologous T cell. Additional examples of modified immune cells are further described below.

[0287] In some embodiments, a modified cell of the present invention is genetically edited to disrupt the expression of one or more endogenously expressed genes to reduce the risk of autoreactivity and / or increase the efficacy of the modified cells. In certain embodiments, the gene-edited immune cells (e.g., T cells) have a reduction, deletion, elimination, knockout, or disruption in expression of one or more endogenously expressed receptor. In one embodiment, the modified cell is genetically edited to disrupt the expression of an endogenous TCR gene product (e.g., gene products of T Cell Receptor Alpha Constant (TRAC) and T Cell Receptor Beta Constant (TRBC)), Beta-2-microglobulin (B2M), and Class II Major Histocompatibility Complex Transactivator (CIITA).

[0288] In another embodiment, the modified cell of the present disclosure is genetically edited to disrupt the expression of endogenous PD-1 gene products. In certain embodiments, disrupting the expression of endogenous PD-1 may create “checkpoint” resistant modified cells, resulting in increased tumor control. Checkpoint resistant modified cells may also be created by disrupting the expression of, for example, without limitation, the Adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA / CD272), CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4 / CD152), Indoleamine 2,3- dioxygenase (IDO), the Killer-cell Immunoglobulin-like Receptor (KIR), the Lymphocyte Activation Gene-3 (LAG3), the T cell immunoreceptor with Ig and ITIM domains (TIGIT), T- Atorney Docket No. 046483 -7474WO 1(04010) cell Immunoglobulin domain and Mucin domain 3 (TIM-3), or the V-domain Ig suppressor of T cell activation (VISTA).

[0289] Pharmaceutical Compositions and Formulations

[0290] Also provided are populations of modified immune cells of the invention, compositions containing such cells and / or enriched for such cells, such as in which cells expressing dual chimeric receptors make up at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[0291] Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0292] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers Atorney Docket No. 046483 -7474WO 1(04010) are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0293] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincot Williams & Wilkins; 21st ed. (May 1, 2005).

[0294] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the Atorney Docket No. 046483 -7474WO 1(04010) disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

[0295] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0296] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0297] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, Atorney Docket No. 046483 -7474WO 1(04010) for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0298] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e. ., by fdtration through sterile filtration membranes.

[0299] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0300] In certain aspects, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the modified cells disclosed herein.

[0301] Methods of Treatment

[0302] The modified cells (e.g., T cells) described herein may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified T cells may be administered.

[0303] In one aspect, the present invention provides a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a modified T cell of the present invention. In another aspect, the invention includes a method of treating a disease or condition in a subject comprising administering to a subject in need thereof a population of modified T cells as described herein.

[0304] In one aspect, the present invention provides a method of treating cancer by providing to the subject in need thereof a modified immune effector cell (e.g., T cells, NK cells), or a modified immune or precursor cell generated by the methods of the present invention, wherein the modified immune or precursor cells are engineered to express one or more CARs as described herein. In some embodiments, the method of treating cancer in a subject in need thereof comprises administering to the subject a modified cell comprising a three, four, or five exogenous CARs as described herein. In some embodiments, the modified precursor cell is Atorney Docket No. 046483 -7474WO 1(04010) selected from the group consisting of a bone marrow stem cell, a hematopoietic progenitor cell, or a cord blood stem cell.

[0305] Methods for administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments, the cell therapy, e.g., adoptive T cell therapy is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0306] In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0307] In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g., the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0308] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition Atorney Docket No. 046483 -7474WO 1(04010) over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0309] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0310] The modified immune cells of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease.

[0311] In one embodiment, the cancer to be treated is a solid tumor. In some embodiments, the solid tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, melanoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, hepatic cancer and brain cancer.

[0312] In another embodiment, the cancer to be treated is a hematological cancer. In one embodiment, the cancer is a leukemia. In another embodiment, the cancer is a myeloma. In another embodiment, the cancer is a lymphoma. Exemplary hematological cancers be treated with the compositions of the present invention include those selected from the group consisting of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone lymphoma, Atorney Docket No. 046483 -7474WO 1(04010) myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, a heavy chain disease, plasma cell myeloma, solitary plasmocytoma of bone, extraosseous plasmocytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, or unclassifiable lymphoma.

[0313] In certain exemplary embodiments, the modified immune cells of the invention are used to treat a myeloma, or a condition related to myeloma. Examples of myeloma or conditions related thereto include, without limitation, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undetermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, a method of the present disclosure is used to treat multiple myeloma. In one embodiment, a method of the present disclosure is used to treat refractory myeloma. In one embodiment, a method of the present disclosure is used to treat relapsed myeloma.

[0314] The cells of the invention to be administered may be autologous, with respect to the subject undergoing therapy.

[0315] The administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.

[0316] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, Atorney Docket No. 046483 -7474WO 1(04010) previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0317] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

[0318] In some embodiments, the populations, or sub-types of cells, such as CD8+and CD4+T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4+to CD8+ratio), e.g., within a certain tolerated difference or error of such a ratio.

[0319] In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and / or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or subtype, or minimum number of cells of the population or sub-type per unit of body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and / or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum Atorney Docket No. 046483 -7474WO 1(04010) dose of T cells and a desired ratio of CD4+to CD8+cells, and / or is based on a desired fixed or minimum dose of CD4+and / or CD8+cells.

[0320] In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0321] In some embodiments, the dose of total cells and / or dose of individual sub-populations of cells is within a range of between at or about IxlO5cells / kg to about IxlO11cells / kg 104and at or about 1011cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at or about 1 x 105cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104and at or about 109T cells / kilograms (kg) body weight, such as between 105and 106T cells / kg body weight, for example, at or about 1 x 105T cells / kg, 1.5 x 10?T cells / kg, 2 x 105T cells / kg, or 1 x 106T cells / kg body weight. In other exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO5cells / kg to about IxlO6cells / kg, from about IxlO6cells / kg to about IxlO7cells / kg, from about IxlO7cells / kg about IxlO8cells / kg, from about IxlO8cells / kg about IxlO9cells / kg, from about IxlO9cells / kg about IxlO10cells / kg, from about IxlO10cells / kg about IxlO11cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO8cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO7cells / kg. In other embodiments, a suitable dosage is from about IxlO7total cells to about Atorney Docket No. 046483 -7474WO 1(04010)

[0322] 5x107total cells. In some embodiments, a suitable dosage is from about 1x108total cells to about 5xl08total cells. In some embodiments, a suitable dosage is from about 1.4xl07total cells to about l.lxlO9total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl09total cells.

[0323] In some embodiments, the cells are administered at or within a certain range of error of between at or about 104and at or about 109CD4+and / or CD8+cells / kilograms (kg) body weight, such as between 105and 106CD4+and / or CD8+cells / kg body weight, for example, at or about 1 x 105CD4+and / or CD8+cells / kg, 1.5 x 105CD4+and / or CD8+cells / kg, 2 x 105CD4+and / or CD8+cells / kg, or 1 x 106CD4+and / or CD8 cells / kg body weight. In some embodiments, the cells are administered at or within a certain range of error of, greater than, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD4+cells, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD8+ cells, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106T cells. In some embodiments, the cells are administered at or within a certain range of error of between about 108and 1012or between about 1010and 1011T cells, between about 108and 1012or between about 1010and 101 1CD4+cells, and / or between about 108and IO12or between about 1010and 1011CD8+cells.

[0324] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4+to CD8+cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1:5 and less than about 5: 1), or between at or about 1 :3 and at or about 3 : 1 (or greater than about 1 :3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1 :5 (or greater than about 1 :5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1 : 1, 1 : 1.1, 1 : 1.2, 1 : 1.3, 1: 1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9: 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, or 1 :5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges. Atorney Docket No. 046483 -7474WO 1(04010)

[0325] In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

[0326] In certain embodiments, the subject’s blood comprises at least about 100 modified cells / pL of blood by at least week three after a single administration of the modified T cell.

[0327] In certain embodiments, the subject’s blood comprises at least about 100 modified cells / pL of blood by at least week three after a single administration of the modified T cell.

[0328] In certain embodiments, the modified cell binds to the first and second targets of a cell expressing the first and second targets and kills the cell via granule-mediated cytolysis.

[0329] In certain embodiments, the modified cell is an autologous cell. In certain embodiments, the modified cell is an autologous cell obtained from a human subject. In certain embodiments, the modified cell is a modified T cell.

[0330] Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying expression and / or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[0331] In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic Atorney Docket No. 046483 -7474WO 1(04010) agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent.

[0332] In certain embodiments, the modified cells of the invention may be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PDl, anti-CTLA-4, or anti-PDLl antibody). For example, the modified cell may be administered in combination with an antibody or antibody fragment targeting, for example, PD-1 (programmed death 1 protein). Examples of anti -PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK-3475), and nivolumab (BMS- 936558, MDX-1106, ONO-4538, OPDIVA®) or an antigen-binding fragment thereof. In certain embodiments, the modified cell may be administered in combination with an anti-PD-Ll antibody or antigen-binding fragment thereof. Examples of anti-PD-Ll antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi). In certain embodiments, the modified cell may be administered in combination with an anti-CTLA-4 antibody or antigen-binding fragment thereof. An example of an anti- CTLA-4 antibody includes, but is not limited to, Ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may also be used including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. Immune checkpoint modulators may be administered before, after, or concurrently with the modified cell comprising the CAR. In certain embodiments, combination treatment comprising an immune checkpoint modulator may increase the therapeutic efficacy of a therapy comprising a modified cell of the present invention.

[0333] In certain embodiments, the subject is provided a secondary treatment. Secondary treatments include but are not limited to chemotherapy, radiation, surgery, and medications.

[0334] In some embodiments, the subject can be administered a conditioning therapy prior to CAR T cell therapy. In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning Atorney Docket No. 046483 -7474WO 1(04010) therapy comprises administering an effective amount of fludarabine to the subject. Tn preferred embodiments, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to CAR T cell therapy may increase the efficacy of the CAR T cell therapy. Methods of conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

[0335] In some embodiments, a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the modified T cells. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0336] In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m2 / day. In some embodiments, the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m2 / day.

[0337] In some embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of about 30 mg / m2 / day.

[0338] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m2 / day over three days, and the dosing of fludarabine is 30 mg / m2 / day over three days.

[0339] Dosing of lymphodepletion chemotherapy may be scheduled on Days -6 to -4 (with a -1 day window, i.e., dosing on Days -7 to -5) relative to T cell (e.g., CAR-T, TCR-T, a modified T cell, etc.) infusion on Day 0.

[0340] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous Atorney Docket No. 046483 -7474WO 1(04010) infusion 3 days prior to administration of the modified T cells. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion for 3 days prior to administration of the modified T cells.

[0341] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m2for 3 days.

[0342] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of 30 mg / m2for 3 days.

[0343] Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.

[0344] It is known in the art that one of the adverse effects following infusion of CAR T cells is the onset of immune activation, known as cytokine release syndrome (CRS). CRS is immune activation resulting in elevated inflammatory cytokines. CRS is a known on-target toxicity, development of which likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (constitutional symptoms and / or grade-2 organ toxicity) to severe CRS (sCRS; grade >3 organ toxicity, aggressive clinical intervention, and / or potentially life threatening). Clinical features include: high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation. Dramatic elevations of cytokines including Atorney Docket No. 046483 -7474WO 1(04010) interferon-gamma, granulocyte macrophage colony-stimulating factor, IL-10, and IL-6 have been shown following CAR T-cell infusion. One CRS signature is elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations in clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with the CRS syndrome. The presence of CRS generally correlates with expansion and progressive immune activation of adoptively transferred cells. It has been demonstrated that the degree of CRS severity is dictated by disease burden at the time of infusion as patients with high tumor burden experience a more sCRS.

[0345] Accordingly, the invention provides for, following the diagnosis of CRS, appropriate CRS management strategies to mitigate the physiological symptoms of uncontrolled inflammation without dampening the antitumor efficacy of the engineered cells (e.g., CAR T cells). CRS management strategies are known in the art. For example, systemic corticosteroids may be administered to rapidly reverse symptoms of sCRS (e.g., grade 3 CRS) without compromising initial antitumor response.

[0346] In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the Food and Drug Administration-approved monoclonal antibody tocilizumab, also known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated near-immediate reversal of CRS.

[0347] CRS is generally managed based on the severity of the observed syndrome and interventions are tailored as such. CRS management decisions may be based upon clinical signs and symptoms and response to interventions, not solely on laboratory values alone.

[0348] Mild to moderate cases generally are treated with symptom management with fluid therapy, non-steroidal anti-inflammatory drug (NSAID) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability; with any hemodynamic instability, the administration of tocilizumab is recommended. The first-line management of CRS may be tocilizumab, in some embodiments, at the labeled dose of 8 mg / kg IV over 60 minutes (not to exceed 800 mg / dose); tocilizumab can be repeated Q8 hours. If suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with continued or progressive CRS symptoms, inadequate Atorney Docket No. 046483 -7474WO 1(04010) clinical improvement in 12-18 hours or poor response to tocilizumab, may be treated with high- dose corticosteroid therapy, generally hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be administered high-dose corticosteroid therapy early in the course of the CRS. CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Di scov, 6(6):664-679).

[0349] Features consistent with Macrophage Activation Syndrome (MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007), coincident with clinical manifestations of the CRS. MAS appears to be a reaction to immune activation that occurs from the CRS and should therefore be considered a manifestation of CRS. MAS is similar to HLH (also a reaction to immune stimulation). The clinical syndrome of MAS is characterized by high grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, and a decrease of circulating natural killer (NK) activity.

[0350] Methods of Generating Modified Immune Cells

[0351] The present invention provides methods for producing / generating a modified immune cell or precursor cell thereof (e.g., a T cell / NK cell / NKT cell) comprising one or more transgenes inducibly expressed in the presence of tetracycline or Dox. The cells are generally engineered by introducing a nucleic acid of the present invention as described above. For example, when transducing the cells with a lentivirus vector comprising the nucleic acid of the present invention, the nucleic acid is integrated as a single expression cassette into the host genome.

[0352] Methods of introducing nucleic acids into a cell include physical, biological, and chemical methods. Physical methods for introducing a polynucleotide, such as RNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MA) or the Gene Pulser Atorney Docket No. 046483 -7474WO 1(04010)

[0353] II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg Germany). RNA can also be introduced into cells using cationic liposome mediated transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.. 12(8):861-70 (2001).

[0354] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, especially lentivirus vectors, retroviral vectors, and adeno-associated virus (AAV) vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from poxviruses, herpes simplex virus I, adenoviruses and, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0355] An expression construct of the present invention is generally introduced into a cell by an expression vector. Expression vectors comprising a nucleic acid of the present invention are provided herein. Suitable expression vectors include lentivirus vectors, retrovirus vectors, gamma retrovirus vectors, foamy virus vectors, AAV vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon mediated vectors, such as Sleeping Beauty, Piggyback, and Integrases such as Phi31. Other suitable expression vectors include herpes virus vector, including herpes simplex virus (HSV) and Epstein-Barr virus (EBV) expression vectors.

[0356] Retrovirus expression vectors are capable of integrating into the host genome, delivering a large amount of foreign genetic material, infecting a broad spectrum of species and cell types, and being packaged in special cell lines. The retrovirus vector is constructed by inserting a nucleic acid (e.g., a nucleic acid encoding a subject CAR) into the viral genome at certain locations to produce a virus that is replication defective. Though the retrovirus vectors are able to infect a broad variety of cell types, integration and stable expression of the subject CAR requires the division of host cells.

[0357] Lentivirus vectors are derived from lentiviruses, which are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994, 136). Some examples of lentiviruses include the human immunodeficiency viruses (HIV-1, HIV-2) and the simian immunodeficiency virus (SIV). Lentivirus vectors have been generated by multiply attenuating Atorney Docket No. 046483 -7474WO 1(04010) the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentivirus vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, e.g., of a nucleic acid encoding a subject CAR (see, e.g., U.S. Patent No. 5,994,136).

[0358] Adenovirus expression vectors are based on adenoviruses, which have a low capacity for integration into genomic DNA but a high efficiency for transfecting host cells. Adenovirus expression vectors contain adenovirus sequences sufficient to: (a) support packaging of the expression vector and (b) to ultimately express the subject CAR in the host cell. In some embodiments, the adenovirus genome is a 36 kb, linear, double stranded DNA, where a foreign DNA sequence (e.g., a nucleic acid encoding a subject CAR) may be inserted to substitute large pieces of adenoviral DNA in order to make the expression vector of the present invention (see, e g., Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).

[0359] Another expression vector is based on an adeno associated virus, which takes advantage of the adenovirus coupled systems. This AAV expression vector has a high frequency of integration into the host genome. It can infect non-dividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue cultures or in vivo. The AAV vector has a broad host range for infectivity. Details concerning the generation and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0360] Expression vectors including a nucleic acid of the present disclosure can be introduced into a host cell by any means known to persons skilled in the art. The expression vectors may include viral sequences for transfection, if desired. Alternatively, the expression vectors may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host cell may be grown and expanded in culture before introduction of the expression vectors, followed by the appropriate treatment for introduction and integration of the vectors. The host cells are then expanded and may be screened by virtue of a marker present in the vectors. Various markers that may be used are known in the art, and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. In some embodiments, the host cell is an immune cell or precursor thereof, e.g., a T cell, an NK cell, or an NKT cell.

[0361] The present invention also provides genetically engineered cells which include and stably express a plurality of CARs described herein. In some embodiments, the genetically engineered Atorney Docket No. 046483 -7474WO 1(04010) cells are genetically engineered T-lymphocytes (T cells), regulatory T cells (Tregs), naive T cells (TN), memory T cells (for example, central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), natural killer T cells (NKT cells) and macrophages capable of giving rise to therapeutically relevant progeny. In one embodiment, the genetically engineered cells are autologous cells.

[0362] Modified cells (e.g., comprising a subject CAR) may be produced by stably transfecting host cells with an expression vector including a nucleic acid of the present disclosure. Additional methods to generate a modified cell of the present disclosure include, without limitation, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and / or hydrodynamic delivery) and / or particle-based methods (e.g., impalefection, using a gene gun and / or magnetofection). Transfected cells expressing a subject CAR of the present disclosure may be expanded ex vivo.

[0363] Physical methods for introducing an expression vector into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells including vectors and / or exogenous nucleic acids are well- known in the art. See, e.g., Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0364] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0365] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of Atorney Docket No. 046483 -7474WO 1(04010) enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology' 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.

[0366] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0367] Moreover, the nucleic acids may be introduced by any means, such as transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells. One nucleic acid may be introduced by one method and another nucleic acid may be introduced into the T cell by a different method.

[0368] Sources of Immune Cells

[0369] Prior to expansion, a source of immune cells is obtained from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow. For example, the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject’s bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In certain exemplary embodiments, the subject is a human. Atorney Docket No. 046483 -7474WO 1(04010)

[0370] Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells and / or NKT cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In certain aspects, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.

[0371] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), a natural killer T cell (NK cell) or a dendritic cell. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In an embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell.

[0372] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa- associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, Atorney Docket No. 046483 -7474WO 1(04010) helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any number of T cell lines available in the art, may be used.

[0373] In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and / or engineering them. In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0374] In certain aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0375] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity-based cell separation steps. In some examples, cells are washed, Atorney Docket No. 046483 -7474WO 1(04010) centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0376] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in certain aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in certain aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments , a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In certain embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed, and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0377] In one embodiment, immune cells are obtained from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or another saline solution with or without buffer. In some embodiments, the undesirable Atorney Docket No. 046483 -7474WO 1(04010) components of the apheresis sample may be removed, and the cells directly resuspended in culture media.

[0378] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in certain aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In certain aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0379] In certain exemplary embodiments, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In certain exemplary embodiments, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can Atorney Docket No. 046483 -7474WO 1(04010) simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0380] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker111811) of one or more particular markers, such as surface markers, or that are negative for (marker ) or express relatively low levels (marker1"") of one or more markers. For example, in certain aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L and / or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In certain exemplary embodiments, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0381] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In certain aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long- Atorney Docket No. 046483 -7474WO 1(04010) term survival, expansion, and / or engraftment following administration, which in certain aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0382] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cell population and / or a CD8+ T population is enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in certain aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In certain aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in certain aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0383] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a Atorney Docket No. 046483 -7474WO 1(04010) magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection.

[0384] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In certain aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agents include IL -2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0385] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.

[0386] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD 14, CD 19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody. Atorney Docket No. 046483 -7474WO 1(04010)

[0387] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. An exemplary method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 lb, CD 16, HLA-DR, and CD8.

[0388] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0389] T cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[0390] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In Atorney Docket No. 046483 -7474WO 1(04010) another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0391] Expansion of Immune Cells

[0392] Whether prior to or after modification of cells to express one or more transgenes, the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Publication No. 20060121005. For example, the immune cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the immune cells. In particular, immune cell populations may be stimulated by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the immune cells, a ligand that binds the accessory molecule is used. For example, immune cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the immune cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0393] Expanding the immune cells by the methods disclosed herein can be multiplied by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700 fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000- fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000- fold, 1,000,000-fold, 10,000,000-fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the immune cells expand in the range of about 20-fold to about 50-fold.

[0394] Following culturing, the immune cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal Atorney Docket No. 046483 -7474WO 1(04010) passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. In certain exemplary embodiments, the level of confluence is 70% or greater before passing the cells to another culture apparatus. In particularly exemplary embodiments, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The immune cell medium may be replaced during the culture of the immune cells at any time. In certain exemplary embodiments, the immune cell medium is replaced about every 2 to 3 days. The immune cells are then harvested from the culture apparatus whereupon the immune cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cry opreserving the expanded immune cells. The cryopreserved immune cells are thawed prior to introducing nucleic acids into the immune cell.

[0395] In another embodiment, the method comprises isolating immune cells and expanding the immune cells. In another embodiment, the invention further comprises cryopreserving the immune cells prior to expansion. In yet another embodiment, the cryopreserved immune cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.

[0396] Another procedure for ex vivo expansion cells is described in U.S. Pat. No. 5,199,942 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No. 5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of immune cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3, and c- kit ligand. In one embodiment, expanding the immune cells comprises culturing the immune cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0397] The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0398] Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that Atorney Docket No. 046483 -7474WO 1(04010) facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.

[0399] Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging. Therefore, the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.

[0400] In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for immune cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM- CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of immune cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2). Atorney Docket No. 046483 -7474WO 1(04010)

[0401] The medium used to culture the immune cells may include an agent that can co-stimulate the immune cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. This is because, as demonstrated by the data disclosed herein, a cell isolated by the methods disclosed herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100- fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000- fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater. In one embodiment, the immune cells expand in the range of about 2-fold to about 50-fold, or more by culturing the electroporated population. In one embodiment, human T regulatory cells are expanded via anti- CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating immune cells can be found in U.S. Patent Numbers 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entirety.

[0402] In one embodiment, the method of expanding the immune cells can further comprise isolating the expanded immune cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded immune cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded immune cells, transfecting the expanded immune cells, or electroporating the expanded immune cells with a nucleic acid, into the expanded population of immune cells, wherein the agent further stimulates the immune cell. The agent may stimulate the immune cells, such as by stimulating further expansion, effector function, or another immune cell function.

[0403] EXPERIMENTAL EXAMPLES

[0404] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0405] Example 1 : Design and Construction of a Lentivirus Expression Construct for Providing a Low- Leakage Tet-ON System. Atorney Docket No. 046483 -7474WO 1(04010)

[0406] FIG. 1 is a schematic describing the mechanism of action and design of the low-leakage Tet-ON system. FIG 1A shows the mechanism of action of the Tet-ON system; top: the reverse tetracycline-controlled transactivator (rtTA) is comprised of the reverse Tet repressor (rTetR) and a transcriptional activation domain (VP48) consisting of three subunits (VP16); bottom left: in the absence of doxycycline, rtTA cannot bind to the Tet response element (TRE) rendering the adjacent promoter (P) inactive; botom right: upon binding of doxycycline, rtTA binds to TRE which activates the promoter and induces gene expression. FIG. IB shows a schematic layout of the low-leakage Tet-ON system. To achieve minimal leakage, a synthetic promoter (YB-TATA, PMID: 25331891) with low basal activity was combined with TRE to generate a low-leakage doxycyline-inducible promoter (YB-TRE). YB-TRE was engineered in a back-to-back orientation with the constitutive EFl alpha (EFla) promoter to generate two independent expression cassettes. A spacer sequence (S) separates both promoters to reduce crosstalk. EFla expresses cargol followed by a 2A self-cleaving peptide (P2A) and rtTA-M2ml (rtTA). YB- TRE controls the expression of cargo2. All constructs were generated via restriction cloning of synthesized DNA fragments (IDT) and genes (Genscript) into pTRPE, a third-generation selfinactivating (SIN) lentiviral vector (pTRPE; PMID: 27332733) containing a 5’ Long-Terminal Repeat sequence (SIN 5’LTR), psi sequence for viral packaging (\|t), viral Rev Response Element (RRE), central PolyPurine Tract (cPPT), Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE) and 3’ Long-Terminal Repeat sequence (SIN 3’LTR). Final constructs were validated by Sanger sequencing (Genewiz) and whole-plasmid sequencing (Plasmidsaurus).

[0407] Example 2: Constitutive CAR Expression and Doxycyline-inducible Surface Tag Expression.

[0408] FIG. 2 shows constitutive CAR expression and doxycyline-inducible surface tag expression. FIG. 2A shows a schematic layout of PM352xl73 (CARCD19-Tet-LNGFR) for obtaining constitutive expression of an anti-CD19 CAR (CARCD19) and rfTA-M2ml (rtTA) in combination with Dox-inducible expression of the truncated low-affinity nerve growth factor receptor (tLNGFR) surface tag. FIG. 2B depicts a flow cytometry (FCM) analysis showing expression of tLNGFR in primary human T cells transduced with PM352xl73 after 5, 9, 12, and 19 days in the presence or absence of Ipg / ml doxycyline. The results in FIG. 2B highlight the low-leakage (<0.9%) and high level of activation (85-92%) of PM352xl73. Atorney Docket No. 046483 -7474WO 1(04010)

[0409] Example 3: Doxycycline-Inducible Expression of a Mesothelin-Targeting CAR (CARM5) and Killing of Mesothelin-Expressing SKOV3 Tumor Cells.

[0410] FIG. 3 shows Dox -inducible expression of a mesothelin-targeting CAR (CARM5) and killing of mesothelin-expressing SKOV3 tumor cells. FIG. 3A shows a schematic layout of PM346xl43 (Tet-CARM5) illustrating the constitutive expression of a blue fluorescent protein reporter (BFP2) and rtTA-M2ml (rtTA) in combination with Dox-inducible expression of CARM5. FIG. 3B shows killing of mesothelin-expressing SKOV3 tumor cells by PM346xl43 or untransduced (UTD) T cells at different effector-to-target ratios (5: 1, 2.5: 1, 1.25: 1, 0.625: 1) in the presence or absence of Ipg / ml doxycyline. The results in FIG. 3B show that in the absence of doxycycline no tumor killing compared to the negative control UTD T cells is detected highlighting the low leakage of PM346xl43. FIG. 3C shows killing of mesothelin-expressing SKOV3 tumor cells by PM346xl43 or untransduced (UTD) T cells in the presence or absence of different amounts of doxycyline. The results in FIG. 3C reveal that low doses of doxycycline (>=50ng / ml) are sufficient for tumor clearance highlighting the high sensitivity of PM346xl43.

[0411] Example 4: Doxycycline-Inducible Expression of a CD19-Targeting CAR (CARCD19) and Killing of CD19-Expressing NALM6 Tumor Cells.

[0412] FIG. 4 shows doxycyline-inducible expression of a CD19-targeting CAR (CARCD19) and killing of CD19-expressing NALM6 tumor cells. FIG. 4A is a schematic layout of PM346xl42 (Tet-CARCD19) with constitutive expression of a blue fluorescent protein reporter (BFP2) and rtTA-M2ml (rtTA) in combination with dox-inducible expression of a CD 19- targeting CAR (CARCD19). FIG. 4B shows killing of CD19-expressing NALM6 tumor cells by PM346xl42 at different effector-to-target ratios (4: 1, 2: 1. 1 : 1, 0.5: 1) in the presence or absence of l|ig / ml doxycyline. The results in FIG. 4B show that in the absence of doxycycline no tumor killing compared to the tumor-only control is detected highlighting the low leakage of PM346xl42.

[0413] Example 5: Design of a Partially Humanized rtTA (rTA-p65),

[0414] FIG. 5A shows the original reverse tetracycline-controlled transactivator (rtTA) comprised of the reverse Tet repressor (rTetR) and a transcriptional activation domain (VP48) Atorney Docket No. 046483 -7474WO 1(04010) consisting of three subunits (VP16) each derived from the herpes simplex virus (HSV). To reduce immunogenicity, VP48 is replaced with transactivation domains (TAD) from human p65 to generate rtTA-p65. FIG. 5B, top shows a simplified overview of p65 protein domains including the Rel homology domain and selected TADs; FIG. 5B, bottom shows truncated p65 mutants (vl / 2 / 3 / 4) selected for incorporation into rtTA; numbers indicate amino acid positions of human p65.

[0415] Example 6: Expression and Functional Validation of rtTA-p65 Mutants.

[0416] FIG. 6 shows the expression and functional validation of rtTA-p65 mutants. FIG. 6A shows expression of CARCD19 by Tet-CARCD19 T cells with original rtTA-M2ml (rtTA- VP48; PM346xl42) or rtTA-p65 mutants (rtTA-p65vl / 2 / 3 / 4; PM492 / 493 / 494 / 495xl42) in the presence or absence of Ipg / ml doxycyline after 22h. FIG. 6B is a bar graph showing % killing of CD19-expressing NALM6 tumor cells by Tet-CARCD19 T cells with original rtTA-M2ml (rtTA-VP48; PM346xl42) or rtTA-p65 mutants (rtTA-p65v2 / 3; PM493 / 494xl42) compared to untransduced (UTD) T cells in the presence or absence of Ipg / ml Dox after 48h. The results in FIG. 6B reveal that rtTA-VP48, rtTA-p65v2 and rtTA-p65v3 do not show significant tumor killing in the absence of Dox compared to negative control UTD T cells highlighting the low levels of leakage in these systems. Moreover, rtTA-VP48, rtTA-p65v2 and rtTA-p65v3 all demonstrate similarly high levels of tumor killing (94-96%) in the presence of Dox validating their potency.

[0417] Example 7: In Vitro Killing of Mesothelin-Expressing SKOV3 Tumor Cells by T Cells Expressing Either a Doxycycline-Inducible or Constitutive Mesothelin-Specific CAR,

[0418] To compare tumor cell killing between T cells with a doxycycline-inducible CAR targeting mesothelin and conventional T cells with a constitutive mesothelin-specific CAR in vitro SKOV3 tumor cells expressing mesothelin were co-cultured with PM346xl43 and PM359 T cells.

[0419] FIGs. 7A-7B shows a comparison of the in vitro killing of mesothelin-expressing SKOV3 tumor cells by T cells expressing either a doxycycline-inducible or constitutive mesothelin- specific CAR. FIG. 7A depicts a schematic layout of the doxycycline-inducible vector PM346xl43 (Tet-CAR-MSLN, top) and the constitutive control vector PM359 (MSLN CAR, Atorney Docket No. 046483 -7474WO 1(04010) bottom). FIG. 7B shows killing of mesothelin-expressing SKOV3 tumor cells by PM346xl43, PM359 or untransduced (UTD) T cells at an effector-to-target (E:T) ratio of 1 : 1 in the presence or absence of Ipg / ml doxycycline.

[0420] The results in FIG. 7B show that in the absence of doxycycline, PM346xl43 T cells exhibit no detectable tumor killing compared to the negative control UTD T cells. In contrast, PM359 T cells induce tumor lysis in both the presence and absence of doxycycline.

[0421] Example 8: Doxycycline-Inducible Expression of a Mesothelin-Specific CAR and Secretion of mutant IL- 18,

[0422] To facilitate doxycycline-inducible secretion of mutant IL- 18, a lentiviral vector was designed that includes mutant IL-18 alongside the mesothelin-specific CAR. Doxycycline- inducible IL-18 secretion was validated in vitro and SKOV3 tumor killing between T cells expressing a doxycycline-inducible CAR alone (PM346xl43) or in combination with the secreted mutant IL-18 (PM346xl87) was analyzed.

[0423] FIGs. 8A-8D show the doxycycline-inducible expression of a mesothelin-targeting CAR (MSLN CAR) alone or combined with secretion of mutant IL- 18 (IL- 18m) and the resulting cytotoxicity against mesothelin-expressing SKOV3 tumor cells. FIG. 8A presents a schematic layout of doxycycline-inducible vectors expressing MSLN CAR alone (PM346xl43, top) or with secretion of IL-18m (PM346xl87, bottom). FIG. 8B shows IL18m secretion from PM346xl87 T cells in the presence or absence of O.lpg / ml doxycycline. FIG. 8C shows killing of mesothelin- expressing SKOV3 tumor cells by PM346xl43, PM346xl87, or untransduced (UTD) T cells with an effector-to-target (E:T) ratio of 1 : 1 in the presence or absence of 1 pg / ml doxycycline. FIG. 8D shows killing of mesothelin-expressing SKOV3 tumor cells by the different T cell groups from FIG. 8C during a second round of co-culture in the presence or absence of 1 pg / ml doxycycline.

[0424] The results in FIG. 8B show that PM346xl87 T cells secrete mutant IL-18 only in the presence of doxycycline. The results in FIGs. 8C-8D demonstrate that, during initial tumor cocultures in the presence of doxycycline, PM346xl87 T cells (red) exhibit similar levels of tumor killing than PM346xl43 T cells (blue). However, after restimulation with additional SKOV3 tumor cells, PM346xl87 T cells display greater tumor-killing activity than PM346xl43 T cells. Atorney Docket No. 046483 -7474WO 1(04010)

[0425] Example 9: Doxycycline-Inducible Expression of a Constitutive IL-9 Signaling Mediator.

[0426] To facilitate doxycycline-inducible cytokine signaling, a lentiviral vector (PM466xl74) was designed to express a constitutive CD19-specific CAR together with a doxycycline- inducible constitutive IL-9 signaling mediator (cIL9s). The inducible cIL9s signaling was validated in vitro by assessing STAT phosphorylation in T cells. In vivo, tumor killing efficacy in the presence or absence of doxycycline was evaluated using a Ramos B cell lymphoma xenograft model in NSG mice.

[0427] Briefly, NSG mice were engrafted with 0.5E6 Ramos tumor cells expressing the click beetle green luciferase reporter by i.v. injection. Three days later, mice were randomized to receive regular or doxycycline feed (2000 mg / kg). On day 4 post tumor injection, mice were treated with 1E6 PM466xl74 T cells or UTD T cells by i.v. injection.

[0428] FIGs. 9A-9D show the doxycycline-inducible expression of a constitutive IL-9 signaling mediator (cIL9s) and its effects on STAT signaling and in vivo cytotoxicity. FIG. 9A shows a schematic layout of vector PM466xl74 (CD19-CAR Tet-cIL9s) expressing a constitutive CD19- specific CAR (blue) and doxycycline-inducible cIL9s (red). The results in FIG. 9B show that in the presence of doxycycline PM466xl74 T cells exhibit signs of IL9-mediated STAT signaling indicated by a strong increase in the phosphorylation of STAT1 and STAT3. In addition, a mild decrease of STAT5 phosphorylation was observed.

[0429] The results in FIG. 9C show the killing of Ramos tumor cells by PM466xl74 T cells compared to UTD T cells in the presence or absence of doxycycline. To quantify the degree of tumor cell killing, bioluminescent measurements were performed after i.p. injection of D- Luciferin using an IVIS spectrum imager. The results in FIG. 9D show the overall survival of mice in this experiment.

[0430] The results in FIGs. 9C-9D demonstrate that the activation of cIL9s with doxycycline in PM466xl74 T cells leads to reduced tumor growth in vivo in 4 of 7 mice and complete tumor eradication in 3 of 7 mice. Moreover, doxy cy cline-mediated activation of cIL9s increased the overall survival at 75 days from 0% to 43%.

[0431] Enumerated Embodiments

[0432] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance. Atorney Docket No. 046483 -7474WO 1(04010)

[0433] Embodiment 1 provides a nucleic acid comprising: a first polynucleotide encoding an NF-KB p65-rTetO fusion protein comprising one or more transactivation domains (TAD) of human NF-KB p65 fused to a reverse Tet operator (rTetO) binding domain; a second polynucleotide comprising a Tet operator region (TetOR) for providing inducible expression of one or more transgene(s) operatively linked thereto; and a third polynucleotide comprising a promoter directing transcription of p65-rTetO from a first strand of DNA, wherein p65-rTetO is configured to induce transcription of a transgene of interest operatively linked to the TetOR in the presence of tetracycline or doxycycline (Dox) from an opposite strand of DNA.

[0434] Embodiment 2 provides the nucleic acid of embodiment 1, comprising a spacer between the TetOR and the promoter directing transcription of p65-rTetO.

[0435] Embodiment 3 provides the nucleic acid of embodiment 2, wherein the spacer comprises the nucleotide sequence of SEQ ID NO: 4.

[0436] Embodiment 4 provides the nucleic acid of any one of embodiments 1-3, further comprising a fourth polynucleotide comprising a transgene encoding a protein of interest, which is operatively linked to the TetOR.

[0437] Embodiment 5 provides the nucleic acid of any one of embodiments 1-4, wherein a portion of the first polynucleotide encoding p65-rTetO encodes p65 TAD2.

[0438] Embodiment 6 provides the nucleic acid of embodiment 5, wherein the portion of the first polynucleotide encoding p65 TAD2 comprises the nucleic acid sequence of SEQ ID NO: 1 and / or the amino acid sequence of SEQ ID NO: 2.

[0439] Embodiment 7 provides the nucleic acid of any one of embodiments 1-3, wherein a portion of the first polynucleotide encoding p65-rTetO encodes p65 TAD1 and p65 TAD2.

[0440] Embodiment 8 provides the nucleic acid of embodiment 7, wherein the portion of the first polynucleotide encoding p65 TAD1 and p65 TAD2 comprises the nucleic acid sequence of SEQ ID NO: 21 or 23 and / or the amino acid sequence of SEQ ID NO: 22 or 24.

[0441] Embodiment 9 provides the nucleic acid of any one of embodiments 1-8, wherein a portion of the first polynucleotide encoding p65-rTetO encodes an rTetO, wherein the portion of the first polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 6 and / or encodes an amino acid sequence of SEQ ID NO: 7.

[0442] Embodiment 10 provides the nucleic acid of embodiment 9, wherein a portion of the first polynucleotide encoding rTetO is codon-optimized to enhance expression of p65-rTetO. Atorney Docket No. 046483 -7474WO 1(04010)

[0443] Embodiment 1 1 provides the nucleic acid of any one of embodiments 1-10, wherein the second polynucleotide comprises a Tet operator region (TetOR) comprising one or more copies of a nucleotide sequence of SEQ ID NO: 1.

[0444] Embodiment 12 provides the nucleic acid of embodiment 11, wherein the second polynucleotide comprising the TetOR comprises the nucleotide sequence of SEQ ID NO: 2.

[0445] Embodiment 13 provides the nucleic acid of any one of embodiments 1-10, comprising a nucleotide sequence between the transgene of interest and the p65-rTetO, wherein the nucleotide sequence is set forth in SEQ ID NO: 3.

[0446] Embodiment 14 provides the nucleic acid of any one of embodiments 1-13, wherein the third polynucleotide comprises a constitutive promoter.

[0447] Embodiment 15 provides the nucleic acid of embodiment 14, wherein the constitutive promoter is selected from the group consisting of an EF-1 alpha promoter, a phosphoglycerate kinase- 1 (PGK) promoter, a Rous sarcoma virus (RSV) promoter, a cytomegalovirus (CMV) immediate-early promoter (CMV), a human Ubiquitin C promoter (UBC), a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV), a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus (EBV) immediate early promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.

[0448] Embodiment 16 provides the nucleic acid of embodiment 15, wherein the constitutive promoter is an EF-1 alpha promoter, optionally wherein the EF-1 alpha promoter comprises the nucleotide sequence of SEQ ID NO: 82.

[0449] Embodiment 17 provides the nucleic acid of any one of embodiments 1-16, wherein the third polynucleotide comprises a cell type-specific promoter.

[0450] Embodiment 18 provides the nucleic acid of embodiment 17, wherein the cell typespecific promoter is selected from the group consisting of light and / or heavy chain immunoglobulin gene promoters, a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, an NK cell-specific promoter.

[0451] Embodiment 19 provides the nucleic acid of any one of embodiments 2-18, wherein the protein of interest is a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain. Atorney Docket No. 046483 -7474WO 1(04010)

[0452] Embodiment 20 provides the nucleic acid of embodiment 19, wherein the CAR comprises an antigen binding domain selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a nanobody.

[0453] Embodiment 21 provides the nucleic acid of embodiment 19 or 20, wherein the CAR comprises an antigen binding domain that specifically binds a B cell protein.

[0454] Embodiment 22 provides the nucleic acid of embodiment 21, wherein the B cell protein is selected from the group consisting of CD 10, CD 19, CD20, CD22, CD79b, CD34, CD52, CD123, FLT-3, ROR1, CD179b, and CD79a.

[0455] Embodiment 23 provides the nucleic acid of embodiment 22, wherein the CAR comprises an anti-CD19 antigen binding domain.

[0456] Embodiment 24 provides the nucleic acid of embodiment 19 or 20, wherein the CAR comprises an antigen binding domain that specifically binding a tumor associated antigen.

[0457] Embodiment 25 provides the nucleic acid of embodiment 24, wherein the tumor associated antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa)Zfolate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, Pl 6, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

[0458] Embodiment 26 provides the nucleic acid of embodiment 25, where the tumor associated antigen is mesothelin.

[0459] Embodiment 27 provides the nucleic acid of any one of embodiments 19-26, wherein the CAR comprises a transmembrane domain from a protein selected from the group consisting of alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Atorney Docket No. 046483 -7474WO 1(04010)

[0460] Embodiment 28 provides the nucleic acid of embodiment 27, wherein the CAR comprises a transmembrane domain selected from the group consisting of CD8, CD28, ICOS, and 0X40.

[0461] Embodiment 29 provides the nucleic acid of any one of embodiments 19-28, wherein each intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

[0462] Embodiment 30 provides the nucleic acid of embodiment 29, wherein the costimulatory domain is from a protein selected from the group consisting of CD28, 4-1BB (CD137), ICOS (CD278), 0X40, CD5, CD27, LFA-1 (CD1 la / CD18), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, ITGAM, CD1 lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, BTLA, an MHC class I molecule, and a ligand that specifically binds with CD8.

[0463] Embodiment 31 provides the nucleic acid of any one of embodiments 19-30, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif.

[0464] Embodiment 32 provides the nucleic acid of embodiment 31, wherein the intracellular signaling domain is from a protein selected from the group consisting of CD3 zeta, common FcR gamma, FcyRIII Fc gamma Rlla, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12.

[0465] Embodiment 33 provides the nucleic acid of embodiment 32, wherein the intracellular signaling domain is from CD3 zeta.

[0466] Embodiment 34 provides the nucleic acid of any one of embodiments 19-33, wherein each antigen-binding domain is connected to the transmembrane domain by a hinge region.

[0467] Embodiment 35 provides the nucleic acid of embodiment 34, wherein the hinge region is from a protein selected from the group consisting of an Fc fragment of an antibody, a hinge Atorney Docket No. 046483 -7474WO 1(04010) region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof.

[0468] Embodiment 36 provides the nucleic acid of embodiment 35, wherein the hinge region is from CD8 or CD28.

[0469] Embodiment 37 provides the nucleic acid of any one of embodiments 19-36, wherein the CAR comprises a leader sequence encoding an N-terminal signal peptide.

[0470] Embodiment 38 provides the nucleic acid of embodiment 37, wherein the leader sequence encodes a CD8u signal peptide.

[0471] Embodiment 39 provides the nucleic acid of any one of embodiments 2-38, wherein the fourth polynucleotide further encodes a second transgene encoding a second protein of interest.

[0472] Embodiment 40 provides the nucleic acid of embodiment 39, wherein the first and second transgenes are operatively linked to the TetOR.

[0473] Embodiment 41 provides the nucleic acid of embodiments 39 or 40, wherein the second protein of interest is a second CAR, a checkpoint inhibitor, a cytokine, a chemokine, a cytokine inhibitor, or a switch receptor.

[0474] Embodiment 42 provides the nucleic acid of embodiment 41, wherein the second protein of interest is a checkpoint inhibitor antagonizing the activity of a checkpoint protein selected from the group consisting of PD1, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG 3, TIGIT, TIM-3, and VISTA.

[0475] Embodiment 43 provides the nucleic acid of embodiment 41, wherein the second protein of interest is a cytokine selected from the group consisting of IL -2, IL-7, IL-9, IL-12, IL-15, IL- 18, IL-21 IL-22, IL-23, CXCL9, and CCL19.

[0476] Embodiment 44 provides the nucleic acid of embodiment 41, wherein the second protein of interest is a chemokine, and wherein the chemokine is CXCL9 or CCL19.

[0477] Embodiment 45 provides the nucleic acid of embodiment 41, wherein the second protein of interest is a cytokine inhibitor selected from the group consisting of anti-IL-6, anti-IL-1, anti- IFN-y, anti-TNFa, anti-IL-8, anti-ILlO, anti-GM-SCM, including scFvs and / or nanobodies thereof.

[0478] Embodiment 46 provides the nucleic acid of embodiment 41, wherein the second protein of interest is a switch receptor selected from the group consisting of PD1-CD28, PD1A132L- Atorney Docket No. 046483 -7474WO 1(04010)

[0479] CD28, TIM3-CD28, PD1 -4-1BB, PD1 A132L-4-1BB, TGFpRI-IL-12R.pi, and TGFpRILIL- 12RP2.

[0480] Embodiment 47 provides the nucleic acid of any one of embodiments 39-46, wherein the nucleic acid encodes a self-cleaving 2A peptide domain between the first and second protein of interest.

[0481] Embodiment 48 provides the nucleic acid of embodiment 47, wherein the self-cleaving peptide domain is selected from the group consisting of T2A, P2A, E2A and F2A.

[0482] Embodiment 49 provides the nucleic acid of embodiment 48 or 49, wherein the nucleic acid further encodes a furin cleavage site between the C-terminal end of the protein of interest and the self-cleaving 2A peptide domain.

[0483] Embodiment 50 provides the nucleic acid of embodiment 1, wherein the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 127-132, 139, 141, 144, and 157-160.

[0484] Embodiment 51 provides an expression vector comprising the nucleic acid of any one of embodiments 1-50.

[0485] Embodiment 52 provides the expression vector of embodiment 51, wherein the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0486] Embodiment 53 provides the expression vector of embodiment 52, wherein the expression vector is a lentiviral vector.

[0487] Embodiment 54 provides the expression vector of embodiment 53, wherein the lentiviral vector is a self-inactivating HIV vector comprising an LTR comprising a U3 deletion.

[0488] Embodiment 55 provides the expression vector of embodiment 54, wherein the HIV vector comprises a partial gag sequence, optionally wherein the partial gag sequence comprises the nucleotide sequence of SEQ ID NO: 77.

[0489] Embodiment 56 provides the expression vector of embodiment 55, wherein the HIV vector further comprises a central polypurine tract (cPPT), optionally wherein the cPPT comprises the nucleotide sequence of SEQ ID NO: 78.

[0490] Embodiment 57 provides the expression vector of any one of embodiments 51-56, comprising a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE), optionally wherein the WPRE comprising the nucleotide sequence of SEQ ID NO: 83. Atorney Docket No. 046483 -7474WO 1(04010)

[0491] Embodiment 58 provides a cell comprising the nucleic acid of any one of embodiments 1-50, or the expression construct of any one of embodiments 51-57.

[0492] Embodiment 59 provides the cell of embodiment 58, wherein the cell is an immune cell.

[0493] Embodiment 60 provides the cell of embodiment 58, wherein the immune cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell (Treg)

[0494] Embodiment 61 provides the cell of embodiment 60, wherein the cell is a T cell.

[0495] Embodiment 62 provides the cell of any one of embodiments 58-61, wherein the cell is an autologous cell.

[0496] Embodiment 63 provides the cell of embodiment 62, wherein the autologous cell obtained from a human subject.

[0497] Embodiment 64 provides a pharmaceutical composition comprising a therapeutically effective amount of the cell of any one of embodiments 58-63.

[0498] Embodiment 65 provides a method of treating a disease or disorder in a subject in need thereof, comprising administering the cell of any one of embodiments 58-63, or the pharmaceutical composition of embodiment 67 to the subject, and further administering doxycycline in an amount sufficient to induce expression of the transgene.

[0499] Embodiment 66 provides the method of embodiment 65, wherein the doxycycline is administered after at least 12 hours have elapsed since administering the modified cell or pharmaceutical composition.

[0500] Embodiment 67 provides the method of embodiment 65, wherein the doxycycline is administered after at least 24 hours have elapsed since administering the modified cell or pharmaceutical composition.

[0501] Embodiment 68 provides the method of embodiment 65, wherein the doxycycline is administered after at least 36 hours have elapsed since administering the modified cell or pharmaceutical composition.

[0502] Embodiment 69 provides the method of embodiment 65, wherein the doxycycline is administered after at least 48 hours have elapsed since administering the modified cell or pharmaceutical composition.

[0503] Embodiment 70 provides the method of any one of embodiments 65-69, wherein administration of doxycycline is stopped when the subject demonstrates a side effect. Atorney Docket No. 046483 -7474WO 1(04010)

[0504] Embodiment 71 provides the method of any one of embodiments 65-70, wherein the disease or disorder is a cancer.

[0505] Embodiment 72 provides the method of embodiment 71, wherein the cancer is a liquid tumor.

[0506] Embodiment 73 provides the method of embodiment 72, wherein the cancer is a hematological malignancy.

[0507] Embodiment 74 provides the method of embodiment 71, wherein the cancer is a solid tumor.

[0508] Embodiment 75 provides the method of embodiment 74, wherein the solid tumor is selected from the group consisting of the solid tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, melanoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, hepatic cancer and brain cancer.

[0509] Embodiment 76 provides the method of any one of embodiments 65-75, wherein the subject is human.

[0510] Embodiment 77 provides a method for generating the cell of any one of embodiments 58- 63, comprising: introducing into the cell the nucleic acid of nucleic acid of any one of embodiments 1-50 or the expression construct of any one of embodiments 51-57.

[0511] Embodiment 78 provides the method of embodiment 77, wherein the cell is cultured in the presence of IL-2, IL-7, IL- 15, IL-21, or a combination thereof.

[0512] Other Embodiments

[0513] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0514] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure can be devised by others skilled in the art without departing from the Attorney Docket No. 046483 -7474WO 1(04010) true spirit and scope of the present disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attorney Docket No. 046483 -7474WO 1(04010)CLAIMSWhat is claimed is:

1. A nucl ei c aci d compri si ng : a first polynucleotide encoding an NF-KB p65-rTetO fusion protein comprising one or more transactivation domains (TAD) of human NF-KB p65 fused to a reverse Tet operator (rTetO) binding domain; a second polynucleotide comprising a Tet operator region (TetOR) for providing inducible expression of one or more transgene(s) operatively linked thereto; and a third polynucleotide comprising a promoter directing transcription of p65-rTetO from a first strand of DNA, wherein p65-rTetO is configured to induce transcription of a transgene of interest operatively linked to the TetOR in the presence of tetracycline or doxycycline (Dox) from an opposite strand of DNA.

2. The nucleic acid of claim 1, comprising a spacer between the TetOR and the promoter directing transcription of p65-rTetO.

3. The nucleic acid of claim 2, wherein the spacer comprises the nucleotide sequence of SEQ ID NO: 4.

4. The nucleic acid of any one of claims 1-3, further comprising a fourth polynucleotide comprising a transgene encoding a protein of interest, which is operatively linked to the TetOR.

5. The nucleic acid of any one of claims 1-4, wherein a portion of the first polynucleotide encoding p65-rTetO encodes p65 TAD2.

6. The nucleic acid of claim 5, wherein the portion of the first polynucleotide encoding p65 TAD2 comprises the nucleic acid sequence of SEQ ID NO: 1 and / or the amino acid sequence of SEQ ID NO: 2.Attorney Docket No. 046483 -7474WO 1(04010)7. The nucleic acid of any one of claims 1-3, wherein a portion of the first polynucleotide encoding p65-rTetO encodes p65 TAD1 and p65 TAD2.

8. The nucleic acid of claim 7, wherein the portion of the first polynucleotide encoding p65 TAD1 and p65 TAD2 comprises the nucleic acid sequence of SEQ ID NO: 21 or 23 and / or the amino acid sequence of SEQ ID NO: 22 or 24.

9. The nucleic acid of any one of claims 1-8, wherein a portion of the first polynucleotide encoding p65-rTetO encodes an rTetO, wherein the portion of the first polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 6 and / or encodes an amino acid sequence of SEQ ID NO: 7.

10. The nucleic acid of claim 9, wherein a portion of the first polynucleotide encoding rTetO is codon-optimized to enhance expression of p65-rTetO.

11. The nucleic acid of any one of claims 1-10, wherein the second polynucleotide comprises a Tet operator region (TetOR) comprising one or more copies of a nucleotide sequence of SEQ ID NO: 1.

12. The nucleic acid of claim 11, wherein the second polynucleotide comprising the TetOR comprises the nucleotide sequence of SEQ ID NO: 2.

13. The nucleic acid of any one of claims 1-10, comprising a nucleotide sequence between the transgene of interest and the p65-rTetO, wherein the nucleotide sequence is set forth in SEQ ID NO: 3.

14. The nucleic acid of any one of claims 1-13, wherein the third polynucleotide comprises a constitutive promoter.

15. The nucleic acid of claim 14, wherein the constitutive promoter is selected from the group consisting of an EF-1 alpha promoter, a phosphoglycerate kinase- 1 (PGK)Attorney Docket No. 046483 -7474WO 1(04010) promoter, a Rous sarcoma virus (RSV) promoter, a cytomegalovirus (CMV) immediate- early promoter (CMV), a human Ubiquitin C promoter (UBC), a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV), a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus (EBV) immediate early promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.

16. The nucleic acid of claim 15, wherein the constitutive promoter is an EF-1 alpha promoter, optionally wherein the EF-1 alpha promoter comprises the nucleotide sequence of SEQ ID NO: 82.

17. The nucleic acid of any one of claims 1-16, wherein the third polynucleotide comprises a cell type-specific promoter.

18. The nucleic acid of claim 17, wherein the cell type-specific promoter is selected from the group consisting of light and / or heavy chain immunoglobulin gene promoters, a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, an NK cell-specific promoter.

19. The nucleic acid of any one of claims 2-18, wherein the protein of interest is a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain.

20. The nucleic acid of claim 19, wherein the CAR comprises an antigen binding domain selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a nanobody.

21. The nucleic acid of claim 19 or 20, wherein the CAR comprises an antigen binding domain that specifically binds a B cell protein.Attorney Docket No. 046483 -7474WO 1(04010)22. The nucleic acid of claim 21, wherein the B cell protein is selected from the group consisting of CD10, CD19, CD20, CD22, CD79b, CD34, CD52, CD123, FLT-3, R0R1, CD 179b, and CD79a.

23. The nucleic acid of claim 22, wherein the CAR comprises an anti-CD19 antigen binding domain.

24. The nucleic acid of claim 19 or 20, wherein the CAR comprises an antigen binding domain that specifically binding a tumor associated antigen.

25. The nucleic acid of claim 24, wherein the tumor associated antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, B7H4, BCMA, CA-IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, CEACAM5, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D- Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, Pl 6, PD-L1 , PSCA, PSMA, R0R1, R0R2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

26. The nucleic acid of claim 25, where the tumor associated antigen is mesothelin.

27. The nucleic acid of any one of claims 19-26, wherein the CAR comprises a transmembrane domain from a protein selected from the group consisting of alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

28. The nucleic acid of claim 27, wherein the CAR comprises a transmembrane domain selected from the group consisting of CD8, CD28, ICOS, and 0X40.Attorney Docket No. 046483 -7474WO 1(04010)29. The nucleic acid of any one of claims 19-28, wherein each intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

30. The nucleic acid of claim 29, wherein the costimulatory domain is from a protein selected from the group consisting of CD28, 4-1BB (CD137), ICOS (CD278), 0X40, CD5, CD27, LFA-1 (CDl la / CD18), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, GDI Id, ITGAE, CD 103, ITGAL, ITGAM, CD1 lb, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, BTLA, an MHC class I molecule, and a ligand that specifically binds with CD8.

31. The nucleic acid of any one of claims 19-30, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif.

32. The nucleic acid of claim 31 , wherein the intracellular signaling domain is from a protein selected from the group consisting of CD3 zeta, common FcR gamma, FcyRIII Fc gamma Rlla, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12.

33. The nucleic acid of claim 32, wherein the intracellular signaling domain is from CD3 zeta.

34. The nucleic acid of any one of claims 19-33, wherein each antigen-binding domain is connected to the transmembrane domain by a hinge region.

35. The nucleic acid of claim 34, wherein the hinge region is from a protein selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof.Attorney Docket No. 046483 -7474WO 1(04010)36. The nucleic acid of claim 35, wherein the hinge region is from CD8 or CD28.

37. The nucleic acid of any one of claims 19-36, wherein the CAR comprises a leader sequence encoding an N-terminal signal peptide.

38. The nucleic acid of claim 37, wherein the leader sequence encodes a CD8a signal peptide.

39. The nucleic acid of any one of claims 2-38, wherein the fourth polynucleotide further encodes a second transgene encoding a second protein of interest.

40. The nucleic acid of claim 39, wherein the first and second transgenes are operatively linked to the TetOR.

41. The nucleic acid of claims 39 or 40, wherein the second protein of interest is a second CAR, a checkpoint inhibitor, a cytokine, a chemokine, a cytokine inhibitor, or a switch receptor.

42. The nucleic acid of claim 41, wherein the second protein of interest is a checkpoint inhibitor antagonizing the activity of a checkpoint protein selected from the group consisting of PD1, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG 3, TIGIT, TIM-3, and VISTA.

43. The nucleic acid of claim 41, wherein the second protein of interest is a cytokine selected from the group consisting of IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, IL-21 IL-22, IL-23, CXCL9, and CCL19.

44. The nucleic acid of claim 41, wherein the second protein of interest is a chemokine, and wherein the chemokine is CXCL9 or CCL19.

45. The nucleic acid of claim 41, wherein the second protein of interest is a cytokine inhibitor selected from the group consisting of anti-IL-6, anti-IL-1, anti-IFN-y, anti-TNFa, anti-IL- 8, anti-ILlO, anti-GM-SCM, including scFvs and / or nanobodies thereof.Attorney Docket No. 046483 -7474WO 1(04010)46. The nucleic acid of claim 41 , wherein the second protein of interest is a switch receptor selected from the group consisting of PD1-CD28, PD1A132L-CD28, TIM3-CD28, PD1-4- 1BB, PD1A132L-4-1BB, TGFPRI-IL-12RP1, and TGFPRII-IL-12RP2.

47. The nucleic acid of any one of claims 39-46, wherein the nucleic acid encodes a selfcleaving 2A peptide domain between the first and second protein of interest.

48. The nucleic acid of claim 47, wherein the self-cleaving peptide domain is selected from the group consisting of T2A, P2A, E2A and F2A.

49. The nucleic acid of claim 48 or 49, wherein the nucleic acid further encodes a furin cleavage site between the C-terminal end of the protein of interest and the self-cleaving 2A peptide domain.

50. The nucleic acid of claim 1, wherein the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 127-132, 139, 141, 144, and 157- 160.

51. An expression vector comprising the nucleic acid of any one of claims 1-50.

52. The expression vector of claim 51, wherein the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

53. The expression vector of claim 52, wherein the expression vector is a lentiviral vector.

54. The expression vector of claim 53, wherein the lentiviral vector is a self-inactivating HIV vector comprising an LTR comprising a U3 deletion.

55. The expression vector of claim 54, wherein the HIV vector comprises a partial gag sequence, optionally wherein the partial gag sequence comprises the nucleotide sequence of SEQ ID NO: 77.Attorney Docket No. 046483 -7474WO 1(04010)56. The expression vector of claim 55, wherein the HIV vector further comprises a central polypurine tract (cPPT), optionally wherein the cPPT comprises the nucleotide sequence of SEQ ID NO: 78.

57. The expression vector of any one of claims 51-56, comprising a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE), optionally wherein the WPRE comprising the nucleotide sequence of SEQ ID NO: 83.

58. A cell comprising the nucleic acid of any one of claims 1-50, or the expression construct of any one of claims 50-56.

59. The cell of claim 58, wherein the cell is an immune cell.

60. The cell of claim 59, wherein the immune cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell (Treg)61. The cell of claim 60, wherein the cell is a T cell.

62. The cell of any one of claims 58-61, wherein the cell is an autologous cell.

63. The cell of claim 62, wherein the autologous cell obtained from a human subject.

64. A pharmaceutical composition comprising a therapeutically effective amount of the cell of any one of claims 58-63.

65. A method of treating a disease or disorder in a subject in need thereof, comprising administering the cell of any one of claims 58-63, or the pharmaceutical composition of claim 67 to the subject, and further administering doxycycline in an amount sufficient to induce expression of the transgene.

66. The method of claim 65, wherein the doxycycline is administered after at least 12 hours have elapsed since administering the modified cell or pharmaceutical composition.Attorney Docket No. 046483 -7474WO 1(04010)67. The method of claim 65, wherein the doxycycline is administered after at least 24 hours have elapsed since administering the modified cell or pharmaceutical composition.

68. The method of claim 65, wherein the doxycycline is administered after at least 36 hours have elapsed since administering the modified cell or pharmaceutical composition.

69. The method of claim 65, wherein the doxycycline is administered after at least 48 hours have elapsed since administering the modified cell or pharmaceutical composition.

70. The method of any one of claims 65-69, wherein administration of doxycycline is stopped when the subject demonstrates a side effect.

71. The method of any one of claims 65-70, wherein the disease or disorder is a cancer.

72. The method of claim 71, wherein the cancer is a liquid tumor.

73. The method of claim 72, wherein the cancer is a hematological malignancy.

74. The method of claim 71, wherein the cancer is a solid tumor.

75. The method of claim 74, wherein the solid tumor is selected from the group consisting of the solid tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, melanoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, hepatic cancer and brain cancer.

76. The method of any one of claims 65-75, wherein the subject is human.

77. A method for generating the cell of any one of claims 58-63, comprising:Attorney Docket No. 046483 -7474WO 1(04010) introducing into the cell the nucleic acid of nucleic acid of any one of claims 1-50 or the expression construct of any one of claims 51-57.

78. The method of claim 77, wherein the cell is cultured in the presence of IL-2, IL-7, IL-15, IL-21, or a combination thereof.

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