RU2788131C2 - Therapy methods based on car t-cells with increased efficiency - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to an immune effector cell modified by engineering methods for expression of a chimeric antigen receptor (hereinafter – CAR), containing Tet2 inhibitor and Tet2 gene disorder, to its production method, as well as to a composition containing it, and a population. A method for increasing therapeutic efficiency of an immune effector cell is also disclosed, including a stage of bringing the specified immune effector cell into contact with Tet2 inhibitor. The invention also relates to a vector containing a sequence encoding CAR and a sequence encoding Tet2 inhibitor.

EFFECT: invention is effective for the treatment of a disease associated with the expression of a tumor antigen in an individual.

44 cl, 32 dwg, 6 tbl

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application Serial No. 62/220196, filed September 17, 2015, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LIST

This application contains a sequence listing, which is provided in electronic form in ASCII format and is hereby incorporated by reference in its entirety. The specified ASCII copy, created on September 14, 2016, is named N2067-7098WO_SL.txt and has a size of 507996 bytes.

FIELD OF THE INVENTION

The present invention relates primarily to the use of immune effector cells (eg, T cells, NK cells) engineered to express a chimeric antigen receptor (CAR) for the treatment of a disease associated with tumor antigen expression.

STATE OF THE ART TO which THE INVENTION RELATES

Adoptive cell transfer (ACT) therapy using autologous T cells, especially T cells transduced with chimeric antigen receptors (CARs), has shown promise in trials in hematological malignancies. There is a medical need for T cell based therapies, especially CAR T cell based therapies with improved efficacy.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods that disrupt methylcytosine dioxygenase (e.g., Tet1, Tet2, Tet3) genes and uses of such compositions and methods to enhance the functional activity of engineered cells (e.g., genetically modified antigen-specific T cells, such as CAR T cells). In particular, the present invention relates to methods and compositions for enhancing the therapeutic efficacy of chimeric antigen receptor (CAR) T cells. Without wishing to be bound by theory, disruption of a single allele in the Tet gene (eg, Tet1, Tet2, or Tet3) results in a decrease in total 5-hydroxymethylcytosine levels, together with increased proliferation, regulation of effector cytokine production, and degranulation, and thereby enhances proliferation and/or CAR function. T cells.

Thus, the present invention provides a cell (e.g., a population of cells, such as a population of immune effector cells) engineered to express a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein the expression and /or the function of Tet1, Tet2 and/or Tet3 in the specified cell is reduced or eliminated. In one embodiment, expression and/or function of Tet2 in said cell is reduced or abolished. In some embodiments, the antigen binding domain that binds to the tumor antigen is selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA -4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, 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-1a, MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, protein sperm 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, protein, survivin and telomerase, PCTA-1/ha lectin 8, MelanA/MART1, Ras mutant, hTERT, breakpoints in sarcoma translocation, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2 , CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1. In one embodiment, the tumor antigen is CD19. In some embodiments, the antigen binding domain is an antibody or antibody fragment as described in, for example, WO2012/079000 or WO2014/153270.

In one aspect, the present invention relates to a cell (e.g., a population of cells, such as a population of immune effector cells) engineered to express CAR, and where the expression and/or function of Tet1, Tet2 and/or Tet3 in said cell is reduced or abolished. In one embodiment, expression and/or function of Tet2 in said cell is reduced or abolished. In some embodiments, the transmembrane domain of said CAR comprises: (i) an amino acid sequence having at least one, two, or three modifications, but no more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NO: 12, or a sequence having 95- 99% identity with the amino acid sequence of SEQ ID NO: 12; or (ii) the sequence of SEQ ID NO: 12.

In one aspect, the present invention relates to a cell (e.g., a population of cells, such as a population of immune effector cells) engineered to express CAR, and where the expression and/or function of Tet1, Tet2 and/or Tet3 in said cell is reduced or abolished. In one embodiment, the antigen-binding domain of said CAR is linked to a transmembrane domain by a hinge region, wherein said hinge region contains SEQ ID NO: 2 or SEQ ID NO: 6, or a sequence with 95-99% identity thereto. In some embodiments, the intracellular signaling domain of said CAR comprises a primary signaling domain and/or a costimulatory signaling domain, wherein the primary signaling domain comprises a functional signaling domain of a protein selected from CD3-zeta, CD3-gamma, CD3-delta, CD3, total FcR-gamma (FCER1G), FcR-beta (Fc-epsilon R1b), CD79a, CD79b, Fc-gamma RIIa, DAP10 or DAP12.

In some embodiments, the primary signaling domain of said CAR comprises: (i) an amino acid sequence having at least one, two, or three, but no more than 20, 10, or 5 amino acid sequence modifications of SEQ ID NO: 18 or SEQ ID NO: 20 , or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20; or (ii) the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, the intracellular signaling domain of said CAR comprises a co-stimulatory signal domain or a primary signal domain and a co-stimulatory signal domain, wherein the co-stimulatory signal domain comprises a functional protein signal domain, selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytic function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8-alpha, CD8-beta, IL2R- beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, L y9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR , LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46 and NKG2D.

In some embodiments, the co-stimulatory signaling domain of said CAR comprises an amino acid sequence having at least one, two, or three modifications, but no more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16, or the sequence , having 95-99% identity with the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16. In some embodiments, the intracellular domain of the specified CAR contains the sequence of SEQ ID NO: 14 or SEQ ID NO: 16, and the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, where sequences containing an intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain. In some embodiments, the CAR of the present invention further comprises the leader sequence of SEQ ID NO: 2.

In some embodiments, the implementation of the immune effector cell of the present invention is a T cell or NK cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof. In one aspect, the cells of the present invention are human cells. In one aspect, the cells (eg, engineered immune effector cells, eg, CAR T cells) of the present invention comprise a Tet1, Tet2, and/or Tet3 inhibitor. In some embodiments, cells of the present invention comprise a CAR and an inhibitor of Tet1, Tet2, and/or Tet3, wherein said inhibitor is (1) a gene editing system targeting one or more regions in a gene encoding Tet1, Tet2, and/or Tet3, or in their regulatory elements, for example, in Tet2 or its regulatory elements; (2) a nucleic acid encoding one or more components of said gene editing system; or (3) combinations of them.

In some embodiments, the cells of the present invention comprise a CAR and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system that targets one or more regions in the gene encoding Tet1, Tet2 and/or Tet3, or in their regulatory elements, for example, in Tet2 or its regulatory elements, and where the gene editing system is selected from the group consisting of: CRISPR/Cas9 system, zinc finger nuclease system, TALEN system and meganuclease system.

In some embodiments, the cells of the present invention comprise a CAR and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system that targets one or more regions in a gene encoding Tet1, Tet2 and/or Tet3, or in their regulatory elements, eg in Tet2 or its regulatory elements, and where the gene editing system binds to a target sequence in an early exon or intron of a gene encoding Tet1, Tet2 and/or Tet3, eg Tet2.

In some embodiments, the cells of the present invention comprise a CAR and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system that targets one or more regions in the gene encoding Tet1, Tet2 and/or Tet3, or in their regulatory elements, for example, in Tet2 or its regulatory elements, and where the gene editing system links the target sequence of the gene encoding tet2, and the target sequence is upstream of exon 4, for example, in exon 1, exon 2, or exon 3 e.g. in exon 3.

In some embodiments, the cells of the present invention comprise a CAR and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system that targets one or more regions in a gene encoding Tet1, Tet2 and/or Tet3, or in their regulatory elements, eg in Tet2 or its regulatory elements, and where the gene editing system binds to a target sequence in the late exon or intron of a gene encoding Tet1, Tet2 and/or Tet3, eg Tet2.

In some embodiments, the cells of the present invention comprise a CAR and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system that targets one or more regions in a gene encoding Tet1, Tet2 and/or Tet3, or in their regulatory elements, e.g., in Tet2 or its regulatory elements, and where the gene editing system links the target sequence of the gene encoding tet2 and the target sequence is downstream of exon 8, e.g., in exon 9, exon 10, or exon 11, for example, is found in exon 9.

In some embodiments, the cells of the present invention comprise a CAR and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system that targets one or more regions in a gene encoding Tet1, Tet2 and/or Tet3, or in their regulatory elements, for example, in Tet2 or its regulatory elements, and where the gene editing system is a CRISPR/Cas system containing a gRNA molecule containing a targeting sequence that hybridizes to the target sequence of the Tet2 gene. In some embodiments, the targeting sequence is the targeting sequence shown in Table 3. In some embodiments, the targeting sequence is the targeting sequence shown in Table 5.

In some embodiments, cells of the present invention comprise a CAR and a Tet1, Tet2, and/or Tet3 inhibitor, wherein said inhibitor is an siRNA or shRNA (siRNA or shRNA) specific for Tet1, Tet2, Tet3, or a nucleic acid encoding said siRNA or shRNA. In some embodiments, the siRNA or shRNA contains a sequence that is complementary to the Tet2 mRNA sequence, for example, contains the shRNA target sequence shown in Table 4.

In some embodiments, the cells of the present invention comprise a CAR and a Tet1, Tet2 and/or Tet3 inhibitor, wherein said inhibitor is a small molecule compound.

In some embodiments, the cells of the present invention comprise a CAR and an inhibitor of Tet1, Tet2, and/or Tet3, where the inhibitor is a protein, e.g., a dominant negative binding partner for Tet1, Tet2, and/or Tet3 (e.g., histone deacetylase ( HDAC) that interacts with Tet1, Tet2 and/or Tet3), or a nucleic acid encoding said dominant negative binding partner for Tet1, Tet2 and Tet3.

In some embodiments, cells of the present invention comprise a CAR and an inhibitor of Tet1, Tet2, and/or Tet3, wherein the inhibitor is a protein, e.g., dominant negative (e.g., catalytically inactive) Tet1, Tet2, or Tet3, or a nucleic acid encoding the specified dominant-negative Tet1, Tet2 or Tet3.

In one aspect, the present invention relates to a method for increasing the therapeutic efficacy of a CAR expressing cell, e.g., a cell according to any one of the preceding provisions, e.g., a CAR19 expressing cell (e.g., CTL019), comprising the step of reducing the level of 5-hydroxymethylcytosine in said cell. In some embodiments, said step comprises contacting said cell with a Tet inhibitor (eg, Tet1, Tet2, and/or Tet3). In some embodiments, said Tet inhibitor is a Tet2 inhibitor. In some embodiments, the Tet inhibitor (e.g., Tet1, Tet2, and/or Tet3) of the present invention is selected from the group consisting of: (1) a gene editing system targeting one or more regions in a gene encoding Tet1, Tet2, or Tet3, or in their respective regulatory elements; (2) a nucleic acid (eg, siRNA or shRNA) that inhibits the expression of Tet1, Tet2, or Tet3; (3) a protein (eg, dominant negative, eg, catalytically inactive) Tet1, Tet2, or Tet3, or a binding partner for Tet1, Tet2, or Tet3; (4) a small molecule compound that inhibits the expression and/or function of Tet1, Tet2 or Tet3; (5) a nucleic acid encoding any of (1)-(3); and (6) any combination of (1)-(5). In some embodiments, the Tet inhibitor of the present invention is a Tet2 inhibitor.

In one aspect, the present invention relates to a method for increasing the therapeutic efficacy of a CAR expressing cell, e.g., a cell according to any one of the preceding provisions, e.g., a CAR19 expressing cell (e.g., CTL019), comprising the step of reducing the level of 5-hydroxymethylcytosine in said cell. In some embodiments, said step comprises contacting said cell with a Tet inhibitor (eg, Tet1, Tet2, and/or Tet3). In some embodiments, said contacting occurs ex vivo . In some embodiments, said contacting occurs in vivo . In some embodiments, said contacting occurs in vivo prior to delivery of the CAR-encoding nucleic acid into the cell. In some embodiments, said contacting occurs in vivo after the cells are administered to an individual in need thereof.

In one aspect, the present invention provides a method for enhancing the therapeutic efficacy of a CAR expressing cell, e.g., a cell according to any one of the preceding, e.g., a CAR19 expressing cell (e.g., CTL019), comprising the step of contacting said cell with a Tet inhibitor, e.g., a Tet1 inhibitor. , Tet2 and/or Tet3. In some embodiments, said Tet inhibitor is a Tet2 inhibitor. In some embodiments, the Tet inhibitor (e.g., Tet1, Tet2, and/or Tet3) of the present invention is selected from the group consisting of: (1) a gene editing system targeting one or more regions in a gene encoding Tet1, Tet2, or Tet3, or in their respective regulatory elements; (2) a nucleic acid (eg, siRNA or shRNA) that inhibits the expression of Tet1, Tet2, or Tet3; (3) a protein (eg, dominant negative, eg, catalytically inactive) Tet1, Tet2, or Tet3, or a binding partner for Tet1, Tet2, or Tet3; (4) a small molecule compound that inhibits the expression and/or function of Tet1, Tet2 or Tet3; (5) a nucleic acid encoding any of (1)-(3); and (6) any combination of (1)-(5). In some embodiments, the Tet inhibitor of the present invention is a Tet2 inhibitor.

In one aspect, the present invention provides a method for enhancing the therapeutic efficacy of a CAR expressing cell, e.g., a cell according to any one of the preceding, e.g., a CAR19 expressing cell (e.g., CTL019), comprising the step of contacting said cell with a Tet inhibitor, e.g., a Tet1 inhibitor. , Tet2 and/or Tet3. In some embodiments, said step comprises bringing said cells into contact with a Tet inhibitor (eg, Tet1, Tet2, and/or Tet3). In some embodiments, said contacting occurs ex vivo . In some embodiments, said contacting occurs in vivo . In some embodiments, said contacting occurs in vivo prior to delivery of the CAR-encoding nucleic acid into the cell. In some embodiments, said contacting occurs in vivo after the cells are administered to an individual in need thereof.

In one aspect, the present invention relates to a method of treating an individual in need thereof, comprising administering to said individual an effective amount of cells as described herein, for example, immune effector cells (eg, T cells or NK cells) containing CAR, and optionally administering to said individual a Tet1, Tet2 and/or Tet3 inhibitor. In some embodiments, the subject is pre-treated with a Tet1, Tet2, and/or Tet3 inhibitor prior to initiation of therapy with CAR-expressing cells. In some embodiments, the individual is given concomitant treatment with a Tet1, Tet2 and/or Tet3 inhibitor and therapy with CAR expressing cells. In some embodiments, the individual is treated with a Tet1, Tet2, and/or Tet3 inhibitor following therapy with CAR-expressing cells. In some embodiments, the individual has a disease associated with tumor antigen expression, eg, a proliferative disease, a premalignant condition, cancer, and a non-cancer associated disease associated with tumor antigen expression. In some embodiments, the individual has a hematologic malignancy selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia leukemia (T-ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphomas, mantle cell lymphomas, marginal zone cell lymphomas, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström's macroglobulinemia or preleukemia.

The present invention relates to the use of the compositions and/or methods described herein for the treatment of cancer, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, renal cell cancer, liver cancer, non-small cell lung carcinoma, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, endometrial cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer , endometrial cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer canal, penile cancer, childhood solid tumors, urinary tract cancer o bladder, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphomas, malignant tumors induced by environmental factors, combinations of said malignant tumors and metastatic foci of said tumors.

The present invention relates to inhibitors of Tet1, Tet2 and/or Tet3 for use in the treatment of an individual, where the specified individual was, is being treated or intends to be treated with a CAR expressing cell.

In addition, the present invention relates to a method for obtaining a CAR-expressing cell, which includes introducing a CAR-encoding nucleic acid into the cell, so that the specified nucleic acid (or its CAR-encoding part) is integrated into the genome of the cell in the Tet1, Tet2 and/or Tet3 gene ( for example, in the intron or exon of the Tet1, Tet2 and/or Tet3 gene), so that the expression and/or function of Tet1, Tet2 and/or Tet3 is reduced or abolished.

In addition, the present invention relates to a method for obtaining a CAR-expressing cell, comprising contacting said CAR-expressing cell with an inhibitor of Tet1, Tet2 and/or Tet3 ex vivo . In some embodiments, the inhibitor is a Tet2 inhibitor.

In addition, the present invention relates to a vector containing a sequence encoding a CAR and a sequence encoding a Tet inhibitor, for example, a Tet1, Tet2 and/or Tet3 inhibitor. In some embodiments, the Tet inhibitor is (1) a gene editing system that targets one or more regions in a gene encoding Tet1, Tet2, or Tet3, or their respective regulatory elements; (2) a nucleic acid (eg, siRNA or shRNA) that inhibits the expression of Tet1, Tet2, or Tet3; (3) a protein (eg, dominant negative, eg, catalytically inactive) Tet1, Tet2, or Tet3, or a binding partner for Tet1, Tet2, or Tet3; and (4) a nucleic acid encoding any of (1)-(3), or combinations thereof. In some embodiments, the CAR coding sequence and the Tet inhibitor coding sequence are separated by region 2A.

In addition, the present invention relates to a gene editing system that is specific to the sequence of the Tet gene or its regulatory elements, for example, the Tet1, Tet2 or Tet3 genes or their regulatory elements. In some embodiments, the gene editing system is sequence specific for the Tet2 gene. In some embodiments, the gene editing system is (1) a CRISPR/Cas gene editing system, (2) a zinc finger nuclease system, a TALEN system, and a meganuclease system. In some embodiments, the gene editing system is a CRISPR/Cas gene editing system. In some embodiments, the gene editing system comprises: a gRNA molecule containing a targeting sequence specific to the sequence of the Tet2 gene or its regulatory elements, and a Cas9 protein; a gRNA molecule containing a targeting sequence specific to the sequence of the Tet2 gene or its regulatory elements, and a nucleic acid encoding the Cas9 protein; a nucleic acid encoding an gRNA molecule containing a targeting sequence specific to the sequence of the Tet2 gene or its regulatory elements, and the Cas9 protein; or a nucleic acid encoding an gRNA molecule containing a targeting sequence specific to the sequence of the Tet2 gene or its regulatory elements, and a nucleic acid encoding a Cas9 protein. In some embodiments, the gene editing system further comprises template DNA. In some embodiments, the messenger DNA contains a nucleic acid sequence encoding a CAR, such as CAR as described herein.

Furthermore, the present invention relates to a composition for the production of an ex vivo CAR expressing cell comprising a Tet inhibitor, eg a Tet1, Tet2 and/or Tet3 inhibitor, eg a Tet2 inhibitor. In some embodiments, the Tet inhibitor is selected from N-[3-[7-(2,5-dimethyl-2H-pyrazol-3-ylamino)-1-methyl-2-oxo-1,4-dihydro-2H-pyrimido[ 4,5-d]pyrimidin-3-yl]-4-methylphenyl]-3-trifluoromethylbenzamide, 2-[(2,6-dichloro-3-methylphenyl)amino]benzoic acid and 2-hydroxyglutarate.

In addition, the present invention relates to a cell population containing one or more of the cells described in the present description, where the cell population contains a higher percentage of Tscm cells (for example, CD45RA+CD62L+CCR7+CD27+CD95+ T cells) than the cell population , which does not contain one or more cells in which the expression and/or function of Tet1, Tet2 and/or Tet3 is reduced or eliminated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : CD19 expressing CART cells were administered to a patient (UPCC04409-10) for the treatment of CLL. Monitoring of CART cells in patient UPCC04409-10 over time was performed by taking blood samples. The expression level of BBZ in cells was determined (red). The number of copies of the TCR family sequence Vbeta5.1 (blue) was determined. Both measurements were performed on samples obtained on the indicated day after the second infusion of CART cells.

FIG. 2A and 2B: The UPCC04409-10 patient's T cell receptor repertoire was determined in a sample obtained 28 days (FIG. 2A) or 51 days (FIG. 2B) after CART infusion. This demonstrates a large number of T-cell receptors of the TCRBV05-01 family at day 51, indicating clonal expansion over time.

FIG. 3: Isolated T cells from patient UPCC04409-10 were analyzed for co-expression of CAR19 and 2 different TCR family genes over time (day 50 and day 51) and compared to the original dosed material (product): the top panel represents the TCR family Vb13.1; the bottom panel shows the TCR Vb5.1 family. The data demonstrate that CAR19 positive cells contain one gene of the TCR family (Vb5.1), which increases rapidly between days 50 and 51.

FIG. 4: T cell receptor repertoire for CD8 positive cells from patient UPCC04409-10 was determined in a sample obtained 51 days after CART infusion. This demonstrates a large number of T-cell receptors of the TCRBV05-01 family at day 51, indicating a clonal expansion of CD8-positive cells over time.

FIG. 5: Patient T cell receptor UPCC04409-10 was sequenced and the sequence of alpha and beta chains is shown (amino acid sequences shown as SEQ ID NO: 1297-1298 and nucleotide sequences shown as SEQ ID NO: 1299 -1301, all, respectively, in order of appearance).

FIG. 6: Sonicated DNA was obtained from patient UPCC04409-10 T cells. This material is used to amplify genomic sequences adjacent to the CAR19 insertion region. These genes adjacent to CAR19 in the genome were identified as having increased expression relative to the infused product (D0). At different time points after said CART infusion (d=day; m=month), different relative increases in adjacent genes were observed, with increases in Tet2 expression peaking in both peripheral blood (PBMC) and CAR+CD8+ T cell samples. for 51 days.

FIG. 7: The CAR19 gene insertion site has been mapped to the Tet2 gene. More specifically, insertion occurred between exons 9 and 10 of the Tet2 gene. The catalytic domain for Tet2 is located in exon 11. Insertion at this position may result in expression of aberrant mRNA transcripts or reduced expression of functional Tet2 (wild type).

FIG. 8: Tet2 gene transcripts from isolated UPCC04409-10 patient mRNA were evaluated by RT-PCR using primers spanning the indicated regions of Tet2 or CAR19, or both, as shown in the figure on the right. Rxn 3 contains primers designed to amplify the region of the Tet2 transcript spanning exons 9 and 10. Rxn, 6, 7, 8, 9 and 10 are primers designed to amplify the indicated portions of the CAR19 lentivirus. Rxn 12-16 are primer pairs that contain the sequence of exon 9 of the Tet2 transcript as well as the sequence from the CAR19 lentiviral construct. These data show that the transcripts are derived from the Tet2 locus, which contains both the Tet2 sequence and the CAR19 sequence.

FIG. 9: Shows a schematic representation of transcripts derived from the Tet2 locus found according to FIGS. 10A and 10B. This figure indicates the splicing variants of this Tet2/CAR19 fusion construct that were found in the patient sample. This analysis showed that insertion of CAR19 into Tet2 resulted in transcripts containing stop codons upstream of exon 11. Exon 11 was demonstrated to be important for Tet2 function. This indicates that Tet2 function is impaired by insertion of CAR19. This also indicates that disruption of Tet2 function resulted in favorable expansion of these individual CART clones.

FIG. 10A and 10B: Schematic representation of Tet2 enzymatic activity (FIG. 10A). The Tet family protein converts 5-methylcytosine (5-mc) to 5-hydroxymethylcytosine (5-hmc) and then to 5-formylcytosine (5-fmc), resulting in demethylated cytosine. Methylated DNA is an epigenetic state known to affect the transcription profile. The methylation status of T cells from patient UPCC04409-10 was assessed (FIG. 10B). Patient T cells were stained for TCRVb5.1 (which contains the CAR19 insertion in Tet2) and 5-hmc and 5-fmc were evaluated in TCRVb5.1 positive (red) and TCRVb5.1 negative (blue) populations by flow cytometry. These data indicate that cells containing the CAR19 insertion in the Tet2 gene have a demethylation defect.

FIG. 11: Anti-TET2 shRNAs reduce 5-hmc levels in normal human T cells. TET2 and shRNA control mCherry-expressing shRNA constructs were introduced into normal human T cells. 5-hmc levels were determined by intracellular staining with FACS on day 6 post-expansion using anti-CD3/CD28 antibody beads. TET2 knockdown reduced overall 5-hmc levels.

FIG. 12: Anti-TET2 shRNA increased in Tscm T cells. TET2 and shRNA control mCherry-expressing shRNA constructs were introduced into normal human T cells. CD45RA+CD62L+CCR7+CD27+CD95+ Tscm T cells were detected by FACS staining on day 11 post-expansion with anti-CD3/CD28 antibody beads. Knockdown of TET2 stimulated the expansion of T cells with the Tscm phenotype.

FIG. 13A: Gating strategy for quantification of CAR+ cells.

FIG. 13B: CAR expression levels in cells electroporated with CRISPR/Cas systems targeting Tet2 compared to non-transfected cells.

FIG. 14: Quantification of CD4+ and CD8+ cells after CAR transduction and Tet2 editing.

FIG. 15: Effect of Tet2 inhibition on CAR T cell expansion using anti-CD3/CD28 antibody beads.

FIG. 16: Effect of Tet2 inhibition on antigen-dependent production of interleukin-2 (IL-2) by CAR T cells.

FIG. 17: Effect of Tet2 inhibition on antigen dependent interferon-gamma production by CAR T cells.

FIG. 18: Effect of Tet2 inhibition on antigen-driven proliferation of CAR+ T cells.

FIG. 19: Effect of Tet2 inhibition on antigen-driven T cell proliferation.

FIG. 20: Effect of Tet2 inhibition on antigen-driven CD4+ T cell proliferation.

FIG. 21: Effect of Tet2 inhibition on antigen-driven proliferation of CAR+ CD4+ T cells.

FIG. 22: Effect of Tet2 inhibition on antigen-driven CD8+ T cell proliferation.

FIG. 23: Effect of Tet2 inhibition on antigen-driven proliferation of CAR+ CD8+ T cells.

FIG. 24: % edit and % frameshift edit by CRISPR/Cas systems targeting Tet2 when determined using NGS.

FIG. 25: The 5 most common insertion-deletions observed in T cells following the addition of RNP that included the indicated gRNAs targeted at exon 3 of TET2 (SEQ ID NOs: 1302-1326, respectively, in order of appearance). Changes relative to the unmodified wt sequence are shown, with insertions indicated by lower case letters ("a", "t", "g", and "c") and deletions indicated by a dash ("-"). The insertion-deletion frequency is shown in the rightmost column.

FIG. 26: The 5 most common insertion-deletions observed in T cells following the addition of an RNP that included the indicated gRNAs targeted at exon 9 of TET2 (SEQ ID NOs: 1327-1356, respectively, in order of appearance). Changes relative to the unmodified wt sequence are shown, with insertions indicated by lower case letters ("a", "t", "g", and "c") and deletions indicated by a dash ("-"). Insertion-deletion rates are shown in the rightmost column.

FIG. 27: Schematic protocol of an experiment to determine TET2 knockdown in Jurkat cells in response to a lentivirus encoding shRNA inhibitors of TET2.

FIG. 28: Expression of RFP in shRNA-infected Jurkat cells. Determination of RFP expression was performed using FACS on the 6th day after treatment with puromycin. Based on the expression of RFP by treatment with puromycin, more than 99% of shRNA-introduced jurkat cells were selected.

FIG. 29: Efficacy of tet2 knockdown in anti-TET2 shRNA-infected Jurkat cells. An experiment was carried out using qRT-PCR. The expression levels of tet1 and tet3 were also measured. β-actin served as an internal control to quantify relative gene expression among the tested samples. To improve the reliability of qRT-PCR, two primers for β-actin and one primer for RPLP1 were used in this experiment.

FIG.30: TET2 protein knockdown in response to shRNA in Jurkat cells. An experiment was carried out using Western blotting.

FIG. 31A: Venn diagrams of ATAC peaks in CAR+CD8+ T cells from a Tet2 impaired patient compared to CAR-CD8+ T cells from the same patient at the corresponding time point without Tet2 impaired. Boxplots show differences in ATAC increase between the two cell populations.

FIG. 31B : GO terms associated with ATAC peaks most occluded in a Tet2 deficient cell population compared to its counterpart.

FIG. 32A: Tet2 silencing by shRNA in primary CD8+ T cells from healthy donors as determined by qPCR. Expression (mean, SEM) normalized to GAPDH is presented as fold change relative to untargeted shRNA control.

FIGS. 32B and 32C: Relative frequencies of central memory T cells (FIG. 32B) and effector CD8+ T cells (FIG. 32C) at day 14 after increasing in numbers by stimulation via CD3/CD28 in the same healthy donors as shown on A.

DETAILED DESCRIPTION

Definitions

Unless otherwise defined, all technical and scientific terms used in the present description have the same meaning, which usually means a specialist in the field to which the invention relates.

The singular form refers to one or more than one (i.e., at least one) grammatical entity in the singular form. By way of example, "element" means one element or more than one element.

The term "approximately" when referring to a measurable quantity such as an amount, a period of time, etc., covers deviations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases cases ± 1%, or in some cases ± 0.1% of the indicated value, since such deviations are suitable for performing the described methods.

The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, typically two in the simplest embodiments, which, when present in an immune effector cell, provides specificity for the cell to a target cell, typically a cancerous cell, and induction of an intracellular signal. In some embodiments, a CAR comprises at least an intracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") containing a functional signaling domain derived from a stimulatory molecule and/or a co-stimulatory molecule, as defined below. In some aspects, the polypeptides in the set are adjacent to each other. In some embodiments, the set of polypeptides includes a dimerization switch that, in the presence of a dimerization molecule, can bind polypeptides to each other, for example, can bind an antigen-binding domain to an intracellular signaling domain. In one aspect, the stimulatory molecule is a zeta chain associated with a T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In one aspect, the co-stimulatory molecule is selected from the co-stimulatory molecules described herein, eg 4-1BB (ie CD137), CD27 and/or CD28. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain containing a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. . In one aspect, the CAR contains an optional leader sequence at the N-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, where the leader sequence is optionally cleaved from the antigen-binding domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane.

A CAR that contains an antigen binding domain (eg, scFv or TCR) that targets a specific tumor marker X, such as the tumor markers described herein, is also referred to as XCAR. For example, a CAR that contains an antigen binding domain that targets CD19 is referred to as CD19CAR.

The term "signaling domain" refers to a functional portion of a protein that acts to communicate information in a cell to regulate cellular activity through certain signal transduction pathways by forming second messengers or acting as effectors by responding to such mediators.

The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. The antibodies may be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and may be from natural sources or from recombinant sources. The antibodies may be tetramers of immunoglobulin molecules.

The term "antibody fragment" refers to at least one portion of an antibody that retains the ability to specifically interact (eg, by binding, spatial obstruction, stabilization/destabilization, spatial distribution) with an antigen epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv fragments, Fv disulfide-linked scFv antibody fragments (sdFv), Fd fragment consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, multispecific antibodies formed by antibody fragments such as a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region, and isolated CDRs or other epitope-binding antibody fragments. The antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diantibodies, tribodies, tetrabodies, v-NARs and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005 ). Antigen-binding fragments can also be grafted into scaffolds based on polypeptides such as type III fibronectin (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide mini-antibodies).

The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are linked to each other, e.g. , via a synthetic linker, such as a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless indicated as used within the scope of the invention, the scFv may have the VL and VH variable regions in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may contain a VL linker-VH or may contain a VH linker-VL.

The antibody or antibody fragment portion of a CAR of the invention may exist in a variety of forms wherein the antigen binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or a bispecific antibody. (Harlow et al., 1999: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989: 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). In one aspect, the antigen-binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR contains an antibody fragment that contains the scFv. The precise boundaries of the amino acid sequences of a given CDR can be determined using any of a number of well known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme), or combinations thereof.

As used herein, the term "binding domain" or "antibody molecule" refers to a protein, such as an immunoglobulin chain or fragment thereof, containing at least one immunoglobulin variable domain sequence. The terms "binding domain" or "antibody molecule" encompass antibodies and antibody fragments. In one embodiment, the antibody molecule is a multispecific antibody molecule, for example, it contains a plurality of immunoglobulin variable domain sequences, where the first immunoglobulin variable domain sequence of the plurality has binding specificity for the first epitope, and the second immunoglobulin variable domain sequence of the plurality has binding specificity for regarding the second epitope. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

The portion of a CAR of the invention containing an antibody or antibody fragment can take a variety of forms where the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody ( Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, 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). In one aspect, the antigen-binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR contains an antibody fragment that contains the scFv.

The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and generally defining the class to which the antibody belongs.

The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains are the two main isotypes of antibody light chains.

The term "recombinant antibody" refers to an antibody that is produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or a yeast expression system. The term should also be construed as an antibody obtained by synthesizing a DNA molecule encoding an antibody, which DNA molecule expresses an antibody protein or an amino acid sequence characterizing the antibody, where the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technology that is available and well known in this area.

The term "antigen" or "Ag" refers to a molecule that induces an immune response. The immune response may involve either the production of antibodies or the activation of specific immunocompetent cells, or both. The skilled artisan will appreciate that any macromolecule, including virtually all proteins or peptides, can act as an antigen. Moreover, antigens can be derived from recombinant or genomic DNA. The skilled artisan will appreciate that any DNA that contains nucleotide sequences or a partial nucleotide sequence encoding a protein that induces an immune response thus encodes an "antigen" as the term is used herein. Moreover, one skilled in the art will appreciate that an antigen need not be encoded solely by the full nucleotide sequence of a gene. It is well understood that the present invention includes, but is not limited to, the use of partial nucleotide sequences from more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that induce the desired immune response. Moreover, one skilled in the art will recognize that an antigen need not be encoded by a "gene" at all. It is well understood that the antigen can be obtained by synthesis, or it can be derived from a biological sample, or it can be a macromolecule other than a polypeptide. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell, or a fluid with other biological components.

The term "anti-cancer effect" refers to a biological effect that can be manifested in a variety of ways, including, but not limited to, for example, a decrease in tumor volume, a decrease in the number of malignant cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in the proliferation of malignant cells, a decrease in the survival of malignant cells or alleviate various physiological symptoms associated with the malignant condition. "Anti-cancer effect" can also be manifested by the ability of peptides, polynucleotides, cells and antibodies to prevent the onset of a malignant tumor in the first place. The term "antitumor effect" refers to a biological effect that can be manifested in various ways, including, but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

The term "autologous" refers to any material originating from the same individual to whom it is subsequently reintroduced.

The term "allogeneic" refers to any material derived from a different animal of the same species relative to the individual to whom the material is administered. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be genetically different enough for antigenic interaction to occur.

The term "xenogeneic" refers to a transplant derived from an animal of another species.

The term "malignant tumor" refers to a disease characterized by uncontrolled growth of aberrant cells. Malignant cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, malignant brain tumor, lymphoma, leukemia, lung cancer, and the like. The terms "tumor" and "malignant tumor" are used interchangeably herein, eg, both terms encompass solid and fluid, eg, diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes premalignant as well as malignant neoplasms and tumors.

The term "derived from" as used herein indicates the relationship between the first and second molecule. It generally refers to a structural similarity between a first molecule and a second molecule and does not mean or include a process or source limitation on the first molecule that is derived from the second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3-zeta molecule, the intracellular signaling domain retains sufficient CD3-zeta structure for it to have the desired function, namely the ability to generate a signal under appropriate conditions. It does not mean or include limitation to a particular process for obtaining an intracellular signaling domain, for example, it does not mean that in order to provide an intracellular signaling domain, it is necessary to start with a CD3-zeta sequence and delete an unwanted sequence or introduce mutations to obtain an intracellular signaling domain.

The expression "a disease associated with the expression of a tumor antigen as described herein" includes, but is not limited to, a disease associated with the expression of a tumor antigen as described herein, or a condition associated with cells that express a tumor antigen, as described herein, including, for example, proliferative diseases such as cancer or malignancy, or a premalignant condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or a non-cancerous condition associated with cells that express a tumor antigen as described herein. In one aspect, the cancer associated with the expression of a tumor antigen as described herein is a hematologic cancer. In one aspect, the cancer associated with tumor antigen expression as described herein is a solid cancer. The following diseases associated with the expression of a tumor antigen described herein include, but are not limited to, for example, atypical and/or non-classical cancer, malignancies, premalignant conditions, or proliferative diseases associated with the expression of a tumor antigen as described herein. . Non-malignant conditions associated with tumor antigen expression as described herein include, but are not limited to, for example, autoimmune disease (eg, lupus), inflammatory disorders (allergy and asthma), and transplantation. In some embodiments, cells expressing a tumor antigen express or have at any time expressed an mRNA encoding a tumor antigen. In one embodiment, cells expressing a tumor antigen produce a tumor antigen protein (eg, wild-type or mutant) and the tumor antigen protein may be present at normal levels or reduced levels. In one embodiment, tumor antigen-expressing cells produce detectable levels of tumor antigen protein at one point in time and then substantially no detectable tumor antigen protein.

The term "conservative sequence modifications" refers to amino acid modifications that do not affect or significantly alter the binding characteristics of an antibody or an antibody fragment containing an amino acid sequence. Such conservative modifications include amino acid substitutions, insertions or deletions. Modifications can be made to an antibody or antibody fragment of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are 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), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine). Thus, one or more amino acid residues in a CAR of the invention may be replaced with other amino acid residues from the same side chain family, and the altered CAR may be examined using the functional assays described herein.

The term "stimulation" refers to the primary response induced by the binding of a stimulatory molecule (e.g., TCR/CD3 or CAR complex) to its own ligand (or tumor antigen in the case of CAR), thereby mediating a signaling event such as, but not limited to, signaling through the TCR/CD3 complex, or signaling through the appropriate NK receptor or CAR signaling domains. Stimulation can mediate altered expression of certain molecules.

The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides a signal sequence(s) that regulates immune cell activation in a stimulatory manner for at least some aspect of the cascade signaling by immune cells. In one aspect, the signal is a primary signal that is initiated, for example, by binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, and that results in the mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. .P. The primary cytoplasmic signaling sequence (also referred to as "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif, which is known as an immunoreceptor tyrosine activating motif or ITAM. Examples of ITAM-containing cytoplasmic signal sequences that are particularly useful within the scope of the invention include, but are not limited to, sequences derived from CD3-zeta, total FcR-gamma (FCER1G), Fc-gamma RIIa, FcR-beta (Fc-epsilon R1b), CD3-gamma, CD3-delta, CD3-epsilon, CD79a, CD79b, DAP10 and DAP12. In a particular CAR of the invention, the intracellular signaling domain in any one or more CARs of the invention contains an intracellular signal sequence, for example, the primary CD3-zeta signal sequence. In a particular CAR of the invention, the primary CD3-zeta signal sequence is the sequence shown in SEQ ID NO: 17, or equivalent residues from a non-human species, e.g., mouse, rodent, marmoset, great ape, and the like. In a particular CAR of the invention, the primary CD3-zeta signal sequence is the sequence shown in SEQ ID NO: 43, or equivalent residues from a non-human species, e.g., mouse, rodent, marmoset, great ape, and the like.

The term "antigen presenting cell" or "APC" refers to a cell of the immune system, such as a helper cell (e.g., B cell, dendritic cell, etc.), which displays a foreign antigen in complex with the major histocompatibility complex (MHC) on its surfaces. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

"Intracellular signaling domain", as used in the present description, refers to the intracellular part of the molecule. The intracellular signaling domain generates a signal that stimulates the immune effector function of a CAR-containing cell, such as a CART cell. Examples of immune effector function, for example in a CART cell, include cytolytic activity and helper activity, including cytokine secretion.

In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include domains derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a cost-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include domains derived from molecules responsible for cost-stimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may contain the cytoplasmic sequence of the T cell receptor, and the costimulatory intracellular signaling domain may contain the cytoplasmic sequence of the coreceptor or costimulatory molecule.

The primary intracellular signaling domain may contain a signaling motif, which is known as an immunoreceptor tyrosine activating motif or ITAM. Examples of ITAM-containing primary cytoplasmic signal sequences include, but are not limited to, sequences derived from CD3-zeta, total FcR-gamma (FCER1G), Fc-gamma RIIa, FcR-beta (Fc-epsilon R1b), CD3-gamma, CD3 -delta, CD3-epsilon, CD79a, CD79b, DAP10 and DAP12.

The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as a protein represented under GenBan Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g. mouse, rodent, monkey, etc., and "Zeta-stimulatory domain" or alternatively "CD3-Zeta stimulation domain" or "TCR-Zeta stimulation domain" is defined as amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient for functional transmission of the initial signal required for T-cell activation. In one aspect, the cytoplasmic domain of the zeta chain contains residues 52 to 164 of GenBank Accession No. BAG36664.1 or equivalent residues from a non-human species, e.g., mouse, rodent, marmoset, great ape, etc., which are their functional orthologs. In one aspect, "zeta-stimulating domain" or "CD3-zeta stimulating domain" is the sequence provided as SEQ ID NO: 18. In one aspect, "zeta-stimulating domain" or "CD3-zeta stimulating domain" is the sequence provided as SEQ ID NO: 20.

The term "costimulatory molecule" refers to a recognition binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an effective immune response. Costimulatory molecules include, but are not limited to, the MHC class I molecule, BTLA, and the Toll Ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4 -1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8-alpha, CD8-beta, IL2R- beta, IL2R-gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX , CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, 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, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR , LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds to CD83.

The costimulatory intracellular signaling domain may be an intracellular portion of a costimulatory molecule. The costimulatory molecule may belong to one of the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CDS, CD7 , CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds to CD83, and the like.

An intracellular signaling domain may comprise the entire intracellular portion or the entire native intracellular signaling domain of the molecule from which it is derived, or a functional fragment or derivative thereof.

The term "4-1BB" refers to a member of the TNFR superfamily with an amino acid sequence shown as GenBank Accession No. AAA62478.2, or equivalent residues from a non-human species, e.g., mouse, rodent, marmoset, great ape, etc. P.; and "costimulatory domain 4-1BB" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or equivalent residues from a non-human species, e.g., mouse, rodent, marmoset, great ape, and the like. In one aspect, the "4-1BB costimulatory domain" is the sequence shown as SEQ ID NO: 14, or equivalent residues from a non-human species, e.g., mouse, rodent, marmoset, great ape, and the like.

"Immune effector cell", as this term is used in the present description, refers to a cell that is involved in the immune response, for example, in stimulating the response of immune effector cells. Examples of immune effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK), natural killer T (NKT) cells, mast cells, and phagocytes of myeloid origin. .

"Immune effector function or immune effector response", as the term is used herein, refers to a function or response, for example, of an immune effector cell that enhances or stimulates an immune attack on a target cell. For example, immune effector function or response refers to a property of T cells or NK cells that stimulates the destruction or inhibition of the growth or proliferation of a target cell. In the case of a T cell, primary stimulation and costimulation are examples of an immune effector function or response.

The term "coding" refers to the property inherent in specific nucleotide sequences in a polynucleotide, such as a gene, cDNA or mRNA, to act as a template for the synthesis of other polymers and macromolecules in biological processes having either a specific nucleotide sequence (for example, rRNA, tRNA and mRNA ), or a certain sequence of amino acids, and to the biological properties that are a consequence of this property. Thus, a gene, cDNA or RNA, codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both a coding strand whose nucleotide sequence is identical to an mRNA sequence and is commonly provided in sequence listings, and a non-coding strand used as a template for transcription of a gene or cDNA may be referred to as encoding a protein or other product of that gene or cDNA.

Unless otherwise indicated, "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 expression nucleotide sequence that codes for a protein or RNA may also include introns, since a nucleotide sequence that codes for a protein may, in some version, contain intron(s).

The terms "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.

The term "endogenous" refers to any material from or produced within an organism, cell, tissue, or system.

The term "exogenous" refers to any material introduced from or produced outside of an organism, cell, tissue, or system.

The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

The term "transfer vector" refers to a composition that contains an isolated nucleic acid and which can be used to deliver the isolated nucleic acid into 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 "transfer vector" includes an autonomously replicating plasmid or virus. The term should also be construed as further including non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, a liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

The term "expression vector" refers to a vector containing a recombinant polynucleotide containing expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-regulatory elements for expression; other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include vectors known in the art, including cosmids, plasmids (eg, naked or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that include a recombinant polynucleotide.

The term "lentivirus" refers to a genus in the family Retroviridae. Lentiviruses are unique among retroviruses in that they are able to infect non-dividing cells; they can deliver a significant amount of genetic information to the DNA of the host cell, so they are one of the most efficient ways to deliver a vector with a gene. Examples of lentiviruses are HIV, SIV and FIV.

The term "lentiviral vector" refers to a vector derived from at least a portion of the lentivirus genome, including but not limited to the self-inactivating lentiviral vector presented in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used in the clinic include, but are not limited to, for example, LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX ^™ vector system from Lentigen, and the like. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

The term "homologous" or "identity" refers to the subunit sequence identity between two polymeric molecules, for example between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If the position of the subunit in both of the two molecules is occupied by the same monomeric subunit, for example, if the position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half (eg, five positions in a ten subunit long polymer) of the positions in two sequences are homologous, then the two sequences are 50% homologous; if 90% of the positions (eg, 9 out of 10) are the same or homologous, then the two sequences are 90% homologous.

"Humanized" forms of non-human antibodies (eg, mice) are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding antibody subsequences) that contain minimal a sequence derived from a non-human immunoglobulin. In general, humanized antibodies and fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as a 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. Moreover, the humanized antibody/antibody fragment may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Typically, a humanized antibody or fragment thereof will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDRs correspond to the CDRs of a non-human immunoglobulin and all or a significant portion of the regions FRs are FR regions from a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. For a more detailed description, 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.

"Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the entire molecule is human-derived and consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

The term "isolated" means altered or extracted from a natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from coexisting natural state materials is "isolated". The isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell.

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

The term "operably linked" or "transcriptional control" refers to a functional relationship between a regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is operably linked to a second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Functionally linked DNA sequences can be adjacent to each other and, for example, when two protein-coding regions are to be linked, are in the same reading frame.

The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (SC), intravenous (IV), intramuscular (IM), or intralesional injection, intratumoral administration, or infusion methods.

The terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof, either in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of naturally occurring nucleotides that have similar binding properties to a reference nucleic acid and are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also includes, by implication, its conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences, as well as the sequence expressly specified. In particular, degenerate codon substitutions can be made by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed residues and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol Chem 260:2605-2608 (1985) and Rossolini et al., Mol Cell Probes 8:91-98 (1994)).

The terms "peptide," "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be contained in a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art, such as peptides, oligopeptides, and oligomers, and longer chains, which are commonly 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, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

The term "promoter" refers to a DNA sequence recognized by the synthetic apparatus of the cell or introduced by the synthetic apparatus, which is required to initiate transcription of the polynucleotide sequence.

The term "promoter/regulatory sequence" refers to a nucleic acid sequence that is required for the expression of a gene product operably linked to a promoter/regulatory sequence. In some cases, this sequence may be the main promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements that are required for expression of the gene product. The promoter/regulatory sequence, for example, may be a sequence that expresses the gene product in a tissue-specific manner.

The term "constitutive" promoter refers to a nucleotide sequence which, when operably linked to a polynucleotide that encodes or defines a gene product, causes the production of the gene product in a cell under most or all physiological conditions in the cell.

The term "inducible" promoter refers to a nucleotide sequence which, when operably linked to a polynucleotide that encodes or defines a gene product, causes the gene product to be produced in a cell substantially only when an inducer that corresponds to the promoter is present in the cell.

The term "tissue-specific" promoter refers to a nucleotide sequence which, when operably linked to a polynucleotide that encodes or defines a gene product, 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.

The terms "cancer-associated antigen" or "tumor antigen" interchangeably refer to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of a cancer cell, either in its entirety or as a fragment (e.g., MHC/peptide), and which is suitable for preferentially targeting the pharmacological agent to the cancer cell. In some embodiments, the tumor antigen is a marker expressed by both normal cells and malignant cells, such as a lineage marker, such as CD19 on B cells. In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in a cancer cell compared to a normal cell, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to a normal cell. In some embodiments, the tumor antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for example, a molecule that contains deletions, insertions, or mutations compared to a molecule expressed by a normal cell. In some embodiments, the tumor antigen is exclusively expressed on the cell surface of the cancer cell, in whole or as a fragment (eg, MHC/peptide), and is not synthesized or expressed on the surface of a normal cell. In some embodiments, the CARs of the present invention include CARs containing an antigen-binding domain (eg, an antibody or antibody fragment) that binds to a peptide presented by the MHC. Typically, peptides derived from endogenous proteins fill pockets of class I major histocompatibility complex (MHC) molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and/or tumor-specific peptide/MHC complexes correspond to a unique class of cell surface targets for immunotherapy. TCR-like antibodies have been described targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 (see, for example, Sastry et al., J Virol. 2011 85(5) :1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21 ):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, a TCR-like antibody can be identified by screening a library, such as a human scFv phage display library.

The terms "tumor-supporting antigen" or "cancer-supporting antigen" interchangeably refer to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of a cell that is not itself cancerous but supports the growth of cancerous cells, for example, by stimulating their growth or survival, such as immune cell resistance. Exemplary cells of this type include stromal cells and myeloid-derived suppressor cells (MDSC). The tumor-supporting antigen itself should not play a role in the maintenance of tumor cells if the antigen is present on a cell that supports malignant cells.

The terms "flexible polypeptide linker" or "linker" as used in the context of scFv refers to a peptide linker that consists of amino acids, such as glycine and/or serine residues, used alone or in combination, to link the heavy chain variable region and the variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and it contains the amino acid sequence (Gly-Gly-Gly-Ser) _n , where n is a positive integer equal to or greater than 1. For example, n=1, n =2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10 (SEQ ID NO: 28). In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly ₄ Ser) ₄ (SEQ ID NO: 29) or (Gly ₄ Ser) ₃ (SEQ ID NO: 30). In another embodiment, the linkers include multiple repeats of (Gly ₂ Ser), (GlySer) or (Gly ₃ Ser) (SEQ ID NO: 31). Also within the scope of the invention are the linkers described in WO2012/138475, incorporated herein by reference).

As used herein, the 5' cap (also referred to as the RNA cap, 7-methylguanosine RNA cap, or m ⁷ G-cap RNA) is a modified guanine nucleotide that is added "in front" or at the 5' end of the eukaryotic messenger RNA shortly after the start of transcription. The 5' cap consists of an end group that is linked to the first nucleotide to be transcribed. Its presence is important for ribosome recognition and protection against RNases. Cap attachment is transcriptionally coupled and occurs co-transcriptionally, so that each of these events affects the other. Shortly after the start of transcription, the 5' end of the synthesized mRNA is bound by the synthesizing cap complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-stage biochemical reaction. The capping portion can be modified to modulate the functionality of the mRNA, such as its stability or translation efficiency.

As used herein, " in vitro transcribed RNA" refers to RNA, preferably mRNA, synthesized in vitro . Typically, in vitro transcribed RNA is obtained using an in vitro transcription vector. An in vitro transcription vector contains a template that is used to generate transcribed RNA in vitro .

As used herein, "poly(A)" is a sequence of adenosine residues attached by polyadenylation of an mRNA. In a preferred embodiment, the poly-A transient expression constructs have 50 to 5000 (SEQ ID NO: 34), preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400 bases. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability, or translation efficiency.

As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety, or a modified variant thereof, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3'-poly(A) tail is a long sequence of adenine nucleotides (often several hundred) attached to the pre-mRNA by the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is attached to transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein that binds to it contribute to the protection of mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after the transcription of DNA into RNA, but in addition it also occurs later in the cytoplasm. After completion of transcription, the mRNA chain is cleaved off by the action of the endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the AAUAAA base sequence near the cleavage site. After mRNA cleavage, adenosine residues are linked to the free 3' end of the cleavage site.

As used herein, "transient" refers to the expression of a non-integrated transgene for a period of hours, days, or weeks, where the expression period is less than the expression period of the gene if it is inserted into the genome or contained in a stable plasmid replicon in the cell. -owner.

As used herein, the terms "treat", "treatment", and "administering treatment" refer to reducing or alleviating the progression, severity and/or duration of a proliferative disorder, or alleviating one or more symptoms (preferably one or more noticeable symptoms) a proliferative disorder resulting from the administration of one or more therapeutic agents (eg, one or more drugs such as the CARs of the invention). In specific embodiments, the terms "treat", "treating", and "administering treatment" refer to alleviating at least one quantifiable physical parameter of a proliferative disorder, such as tumor growth, not necessarily perceptible to a patient. In other embodiments, the terms "treat", "treating", and "administering treatment" refer to inhibiting the progression of a proliferative disorder, either physically, such as by stabilizing a noticeable symptom, or physiologically, such as by stabilizing a physical parameter, or both. In other embodiments, the terms "treat", "treatment", and "treatment" refer to reducing or stabilizing the size of a tumor or the number of malignant cells.

The term "signaling cascade" refers to the biochemical relationship between various signaling molecules that play a role in signaling from one part of a cell to another part of the cell. The expression "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and transmitting a signal across the cell membrane.

The term "individual" includes living organisms in which an immune response can be induced (eg, mammals, humans).

The term "substantially clear" cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that is separated from other cell types with which it is normally associated in its natural state. In some instances, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to a cell that is separate from the cells with which it is associated in its natural state. In some aspects, the cells are cultured in vitro . In other aspects, the cells are not cultured in vitro .

The term "therapeutic", as used in the framework of the invention, means treatment. The therapeutic effect is achieved by reducing, suppressing, providing remission or eliminating the disease state.

The term "prophylaxis" as used herein means the prevention or protective treatment of a disease or disease state.

In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with hyperproliferative disorder" refers to antigens that are common to specific hyperproliferative disorders. In some aspects, the hyperproliferative disorder antigens of the present invention are derived from malignancies, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias, endometrial cancer, cancer cervix, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

The terms "transfected" or "transformed" or "transduced" refer to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. A cell includes an individual's primary cell and its progeny.

The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner protein (e.g., a tumor antigen) present in a sample, but that antibody or ligand does not substantially recognize or bind other molecules in the sample. .

"Regulated chimeric antigen receptor (RCAR)", as the term is used herein, refers to a set of polypeptides, typically two in the simplest embodiments, which, when present in an RCARX cell, provides specificity to the RCARX cell for cell- target, typically a malignant cell, and regulated intracellular signal generation or proliferation, which can optimize the immune effector property of the RCARX cell. The RCARX cell is at least partially based on an antigen binding domain to provide specificity for a target cell that contains an antigen bound by the antigen binding domain. In one embodiment, the RCAR includes a dimerization switch that, in the presence of a dimerization molecule, can bind an intracellular signaling domain to an antigen-binding domain.

A "membrane anchor" or "membrane-binding domain" as used herein refers to a polypeptide or moiety, such as a myristoyl group, sufficient to anchor an extracellular or intracellular domain to a plasma membrane.

A "switch domain" as used herein, for example when referring to an RCAR, refers to a structure, typically a polypeptide-based structure, that associates with another switch domain in the presence of a dimerization molecule. This association results in the operative linking of a first structure associated, eg fused, with the first switch domain, and a second structure associated, eg fused, with the second switching domain. The first and second switch domains are collectively referred to herein as dimerization switches. In embodiments, the first and second switch domains are the same, eg, they are polypeptides having the same primary amino acid sequence, and are collectively referred to as homodimerization switches. In embodiments, the first and second switch domains are different from each other, for example, they are polypeptides having different primary amino acid sequences and are collectively referred to as heterodimerization switches. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based structure, such as FKBP or FRB, and the dimerization molecule is a small molecule, such as rapalogue. In embodiments, the switch domain is a polypeptide-based structure, such as scFv, that binds the myc peptide, and the dimerization molecule is a polypeptide, a fragment thereof, or a polypeptide multimer, such as a myc ligand or myc ligand multimers, that bind to one or more scFv to myc. In embodiments, the switch domain is a polypeptide-based structure, such as a myc receptor, and the dimerization molecule is an antibody or fragments thereof, such as an anti-myc antibody.

"Dimerization molecule" as used herein, for example when referring to RCAR, refers to a molecule that allows the first switch domain to be associated with the second switch domain. In embodiments, the dimerization molecule is not naturally occurring in the individual or does not occur at concentrations that would result in significant dimerization. In embodiments, the dimerization molecule is a small molecular weight compound, such as rapamycin or rapalogue, such as RAD001.

The term "bioequivalent" refers to the amount of an agent other than a reference compound (eg, RAD001) required to provide an effect equivalent to that provided by a reference dose or reference amount of a reference compound (eg, RAD001). In one embodiment, the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as measured in an in vivo or in vitro assay , e.g., as measured using an assay described herein, e.g., the Boulay assay . In one embodiment, the effect is a change in the ratio of PD-1 positive/PD-1 negative T cells as measured by cell sorting. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as a reference dose or reference amount of a reference compound. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of change in the ratio of PD-1 positive/PD-1 negative T cells as a reference dose or reference amount of a reference compound.

The term "low immune-boosting dose" when used in relation to an mTOR inhibitor, e.g. an allosteric mTOR inhibitor, e.g. , as measured by inhibition of P70 S6 kinase activity. Methods for assessing mTOR activity, for example, by inhibition of P70 S6 kinase, are described herein. The dose is insufficient to ensure complete suppression of the immune response, but is sufficient to enhance the immune response. In one embodiment, a low immune-enhancing dose of an mTOR inhibitor results in a decrease in the number of PD-1 positive T cells and/or an increase in the number of PD-1 negative T cells, or an increase in the ratio of PD-1 negative T cells/positive by PD-1 T cells. In one embodiment, a low immune-boosting dose of an mTOR inhibitor results in an increase in naïve T cells. In one embodiment, a low immune-boosting dose of an mTOR inhibitor results in one or more of the following:

increased expression of one or more of the following markers: CD62L ^high , CD127 ^high , CD27 ⁺ and BCL2, eg on memory T cells, eg memory T cell precursors;

downregulation of KLRG1, eg on memory T cells, eg memory T cell progenitors; and

an increase in memory T cell precursors, eg, cells with any or a combination of the following: increased CD62L ^high , increased CD127 ^high , increased CD27 ⁺ , decreased KLRG1, and increased BCL2;

where any of the changes described above occurs, for example, at least temporarily, for example, compared with an individual without treatment.

"Refractory", as used herein, refers to a disease, such as cancer, that does not respond to treatment. In embodiments, the refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. Refractory cancer is also called resistant cancer.

"Relapsing", as used herein, refers to the return of a disease (eg, cancer) or signs and symptoms of a disease, such as cancer, after a period of improvement, such as after a previous treatment, such as cancer therapy.

Ranges: Throughout the present description, various aspects of the invention may be presented in the format of ranges. It should be understood that the description in range format is provided for convenience and brevity only and should not be construed as a strict limitation on the scope of the invention. Thus, a description of a range should be considered to have specifically described all possible numerical subranges, as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be construed as a range having specifically described subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers in this range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as the identity 95- 99%, includes anything with 95%, 96%, 97%, 98% or 99% identity and includes sub-ranges such as 96-99% identity, 96-98% identity, 96-97% identity, 97-99% identity, 97-98% and 98-99%. This applies regardless of the range width.

"Tet" as used herein refers to the family of genes and to the methylcytosine dioxygenase family proteins encoded by said genes with translocation ten to eleven. Tet includes, for example, Tet1, Tet2 and Tet3.

"Tet2" as used herein refers to the methylcytosine dioxygenase 2 tet gene and the methylcytosine dioxygenase tet2 protein encoded by said gene, which catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. It is sometimes also referred to as "KIAA1546", "FLJ20032", and "tet oncogene family member 2". The encoded protein is involved in myelopoiesis and defects in this gene have been associated with several myeloproliferative disorders. In the human genome, TET2 is located on chromosome 4q24. Currently, six TET2 isoforms have been described and their Genebank numbers are: NM_001127208.2; XM_005263082.1; XM_006714242.2; NM_017628.4; XM_011532044.1 and XM_011532043.1.

An example of the human Tet2 protein sequence is given under UniProt accession number Q6N021:

The tet2 gene is located on chromosome 4, region GRCh38.p2 (GCF_000001405.28) (NC_000004.12 (from 105145875 to 105279803); gene ID 54790.

Examples of nucleic acid sequences encoding Tet2 are shown below. Six human Tet2 isoforms have been identified. The mRNA sequences are shown below (In embodiments, T may be replaced by U in each sequence). In embodiments, Tet2 includes proteins encoded by each of the following sequences:

Name Reference sequence at NCBI Sequence Homo sapiens methylcytosine dioxygenase 2 tet (TET2), transcript variant 1, mRNA
[SEQ ID NO: 1358] NNM_001127208.2 GGCAGTGGCAGCGGCGAGAGCTTGGGCGGCCGCCGCCGCCTCCTCGCGAGCGCCGCGCGCCCGGGTCCCGCTCGCATGCAAGTCACGTCCGCCCCCTCGGCGCGGCCGCCCCGAGACGCCGGCCCCGCTGAGTGATGAGAACAGACGTCAAACTGCCTTATGAATATTGATGCGGAGGCTAGGCTGCTTTCGTAGAGAAGCAGAAGGAAGCAAGATGGCTGCCCTTTAGGATTTGTTAGAAAGGAGACCCGACTGCAACTGCTGGATTGCTGCAAGGCTGAGGGACGAGAACGAGGCTGGCAAACATTCAGCAGCACACCCTCTCAAGATTGTTTACTTGCCTTTGCTCCTGTTGAGTTACAACGCTTGGAAGCAGGAGATGGGCTCAGCAGCAGCCAATAGGACATGATCCAGGAAGAGCAGTAAGGGACTGAGCTGCTGAATTCAACTAGAGGGCAGCCTTGTGGATGGCCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAACCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATACCATCACCTCCCATTTGCCAGACAGAACCTCTGGCTACAAAGCTCCAGAATGGAAGCCCACTGCCTGAGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTATTATGGAATACCCTGTATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTACACAAGAAAGTAGAGGGTATTCCAAGTGTTTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTCTCTCTCTGGGCTCCTTCAGATCAAGAAATTGAAACAAGACCAAAAGGCTAATGGAGAAAGACGTAACTTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAATGTCTCCGATTTGAGTGATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCAGTTAAAGATTTCACCAGTTTTTCAACACATAACTGC AGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAATGAGCAGGAGGGGAAAAGTGCTAATTACCATGACAAGAACATTGTATTACTTAAAAACAAGGCAGTGCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACTCCTGGAAAAAACACTGTCTCAATATTATCCAGATTGTGTTTCCATTGCGGTGCAGAAAACCACATCTCACATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCATACCTCAGGGCAGATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAGCTGCAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCCAGTAAACTAGCTGCAATGCTAAATACCTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATCAGTTTTTGAGATATGCCCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAATTCTGTTCAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAACAAAATGAAATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTCTGCCACTACCACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCACAGGTTCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAACACCACCACTACCCCAACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACCTGAGGCACCACCTTCCCAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGCTTTCTGAAAGGCCTCAGAATAATTGTGTGAACAGGAATGACATACAGACTGCAGGGACAATGACTGTTCCATTGTGTTCTGAGAAAA CAAGACCAATGTCAGAACACCTCAAGCATAACCCACCAATTTTTGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTGATGAGAAACAAAGAGCAAGAGATTCTGAAGGGTCGAGACAAGGAGCAAACACGAGATCTTGTGCCCCCAACACAGCACTATCTGAAACCAGGATGGATTGAATTGAAGGCCCCTCGTTTTCACCAAGCGGAATCCCATCTAAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTATCAACCCAATCTCTCCAATCAAATGACCTCCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCTTACACCCAGAAAACAACACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATGAATCAAGGGCAGTCCCAAGGTACAGTGGACCAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACTTCTCCAAAACAGACCATTTACCAAAAGCTCATGTGCAGTCACTGTGTGGCACTAGATTTCATTTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTCCCCAGTGTTGAAACAGCACTTGAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAACTCACACCTTTTGCAACATAAGCCTCATAAACAGGCAGCACAAACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGCAACAGCAGCAAAAATTACAAATAAAGAATAAAGAGGAAATACTCCAGACTTTTCCTCACCCCCAAAGCAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAAGTGGAAGAATGTTTTCATGGTGAAAATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACTGGAGGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCAAGTGCATGCAAAATACAGGTTTCTTGTTCAAACAATAC ACACCTAGTTTCAGAGAATAAAGAACAGACTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAAAACTTGCATCACATGCAATATTTTCCAAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGAGCAGAAGTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATAGAAACCAAGATATGTCTGGTCAACAAGCTGCGCAACTTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTGTGCCTGACCAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGCTCTAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAGTCTTGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGTATGCACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAGCTGTGATAATGTGCAGCAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAAGCAGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCTTACTCTCAAATCACAGAAGCAAGTAAAAGTTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGAACTTGATAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAGAACAGCTGCTTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATAAAAAATTTATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGAGCAAATTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGGCAGCTATTAGAGAAATCATGGAAGAAAGGTTTGGACAGAAG GGTAAAGCTATTAGGATTGAAAGAGTCATCTATACTGGTAAAGAAGGCAAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGTGGTTCGCAGAAGCAGCAGTGAAGAGAAGCTACTGTGTTTGGTGCGGGAGCGAGCTGGCCACACCTGTGAGGCTGCAGTGATTGTGATTCTCATCCTGGTGTGGGAAGGAATCCCGCTGTCTCTGGCTGACAAACTCTACTCGGAGCTTACCGAGACGCTGAGGAAATACGGCACGCTCACCAATCGCCGGTGTGCCTTGAATGAAGAGAGAACTTGCGCCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCCTCCTTCTCTTTTGGTTGTTCATGGAGCATGTACTACAATGGATGTAAGTTTGCCAGAAGCAAGATCCCAAGGAAGTTTAAGCTGCTTGGGGATGACCCAAAAGAGGAAGAGAAACTGGAGTCTCATTTGCAAAACCTGTCCACTCTTATGGCACCAACATATAAGAAACTTGCACCTGATGCATATAATAATCAGATTGAATATGAACACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCCATTCTCAGGGGTCACTGCATGTTTGGACTTCTGTGCTCATGCCCACAGAGACTTGCACAACATGCAGAATGGCAGCACATTGGTATGCACTCTCACTAGAGAAGACAATCGAGAATTTGGAGGAAAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTATACAAAGTCTCTGACGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGAGGAGAAAAAACGGAGTGGTGCCATTCAGGTACTGAGTTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGACTTGCCGACAAAGGAAACTAGAAGCCAAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGCTCAAATAAAAATGAAAAGGAAAAGTCAGCCCCATCACGTACAAAACAAACTGAAAACG CAAGCCAGGCTAAACAGTTGGCAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTCCCAGCAGCCCCAGCCTCTACAGAAGCAGCCACCACAGCCCCAGCAGCAGCAGAGACCCCAGCAGCAGCAGCCACATCACCCTCAGACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACATGAGACGGCCCAATCCAGTTAGTCCTTATCCAAACTCTTCACACACTTCAGATATCTATGGAAGCACCAGCCCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCTAATCCCATGAACCCTTACCCTGGGCTTTTGAATCAGAATACCCAATATCCATCATATCAATGCAATGGAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCTATTCTCCCCAGTCTCAGCCGATGGATCTGTATAGGTATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTCTACCACCCATCCATACACTTTACCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGGTTATGGAAACCAAAATATGCAGGGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTACATCATGTAGGGAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCCACCTCTAGATTACCACCCAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCATTCACCTTCTCACATAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAGCTCTCTTCATGCCCTGCATCTCCAAAACAAGGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCAAAGATGCTTCCAGCTCTTAACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGTGATGCTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTC TGGTGCAGAGGACAACGATGAGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGGGGAGTGGCCGTGGCTCCAACTCATGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATGCCACAACCCCTTTAAAGAATCCCAATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTTACCAGCATAAGAGCATGAATGAGCCAAAACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCGTGAGAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGACTATGTGCCTCAGAAATCCCATGGCAAAAAAGTGAAACGGGAGCCTGCTGAGCCACATGAAACTTCAGAGCCCACTTACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGACCATGTCCGTGACCACAGACTCCACAGTAACTACATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACAACAGATATATATGATATCACCCCCTTTTGTTGGTTACCTCACTTGAAAAGACCACAACCAACCTGTCAGTAGTATAGTTCTCATGACGTGGGCAGTGGGGAAAGGTCACAGTATTCATGACAAATGTGGTGGGAAAAACCTCAGCTCACCAGCAACAAAAGAGGTTATCTTACCATAGCACTTAATTTTCACTGGCTCCCAAGTGGTCACAGATGGCATCTAGGAAAAGACCAAAGCATTCTATGCAAAAAGAAGGTGGGGAAGAAAGTGTTCCGCAATTTACATTTTTAAACACTGGTTCTATTATTGGACGAGATGATATGTAAATGTGATCCCCCCCCCCCGCTTACAACTCTACACATCTGTGACCACTTTTAATAATATCAAGTTTGCATAGTCATGGAACACAAATCAAACAAGTACTGTAGTATTACAGTGACAGGAATCTTAAAATACCATCTGGTGCTGAATATATGATGTACTGAAATACTGGAATTATGGCTTTTTGAAATGCAGTT TTTACTGTAATCTTAACTTTTATTTATCAAAATAGCTACAGGAAACATGAATAGCAGGAAAACACTGAATTTGTTTGGATGTTCTAAGAAATGGTGCTAAGAAAATGGTGTCTTTAATAGCTAAAAATTTAATGCCTTTATATCATCAAGATGCTATCAGTGTACTCCAGTGCCCTTGAATAATAGGGGTACCTTTTCATTCAAGTTTTTATCATAATTACCTATTCTTACACAAGCTTAGTTTTTAAAATGTGGACATTTTAAAGGCCTCTGGATTTTGCTCATCCAGTGAAGTCCTTGTAGGACAATAAACGTATATATGTACATATATACACAAACATGTATATGTGCACACACATGTATATGTATAAATATTTTAAATGGTGTTTTAGAAGCACTTTGTCTACCTAAGCTTTGACAACTTGAACAATGCTAAGGTACTGAGATGTTTAAAAAACAAGTTTACTTTCATTTTAGAATGCAAAGTTGATTTTTTTAAGGAAACAAAGAAAGCTTTTAAAATATTTTTGCTTTTAGCCATGCATCTGCTGATGAGCAATTGTGTCCATTTTTAACACAGCCAGTTAAATCCACCATGGGGCTTACTGGATTCAAGGGAATACGTTAGTCCACAAAACATGTTTTCTGGTGCTCATCTCACATGCTATACTGTAAAACAGTTTTATACAAAATTGTATGACAAGTTCATTGCTCAAAAATGTACAGTTTTAAGAATTTTCTATTAACTGCAGGTAATAATTAGCTGCATGCTGCAGACTCAACAAAGCTAGTTCACTGAAGCCTATGCTATTTTATGGATCATAGGCTCTTCAGAGAACTGAATGGCAGTCTGCCTTTGTGTTGATAATTATGTACATTGTGACGTTGTCATTTCTTAGCTTAAGTGTCCTCTTTAACAAGAGGATTGAGCAGACTGATGCCTGCATAAGATGAATAAACAGGGTTAGTTCCATGTGAATCTGTCAGTTAAAAAGAAACA AAAACAGGCAGCTGGTTTGCTGTGGTGGTTTTAAATCATTAATTTGTATAAAGAAGTGAAAGAGTTGTATAGTAAATTAAATTGTAAACAAAACTTTTTTAATGCAATGCTTTAGTATTTTAGTACTGTAAAAAAATTAAATATATACATATATATATATATATATATATATATATATATGAGTTTGAAGCAGAATTCACATCATGATGGTGCTACTCAGCCTGCTACAAATATATCATAATGTGAGCTAAGAATTCATTAAATGTTTGAGTGATGTTCCTACTTGTCATATACCTCAACACTAGTTTGGCAATAGGATATTGAACTGAGAGTGAAAGCATTGTGTACCATCATTTTTTTCCAAGTCCTTTTTTTTATTGTTAAAAAAAAAAGCATACCTTTTTTCAATACTTGATTTCTTAGCAAGTATAACTTGAACTTCAACCTTTTTGTTCTAAAAATTCAGGGATATTTCAGCTCATGCTCTCCCTATGCCAACATGTCACCTGTGTTTATGTAAAATTGTTGTAGGTTAATAAATATATTCTTTGTCAGGGATTTAACCCTTTTATTTTGAATCCCTTCTATTTTACTTGTACATGTGCTGATGTAACTAAAACTAATTTTGTAAATCTGTTGGCTCTTTTTATTGTAAAGAAAAGCATTTTAAAAGTTTGAGGAATCTTTTGACTGTTTCAAGCAGGAAAAAAAAATTACATGAAAATAGAATGCACTGAGTTGATAAAGGGAAAAATTGTAAGGCAGGAGTTTGGCAAGTGGCTGTTGGCCAGAGACTTACTTGTAACTCTCTAAATGAAGTTTTTTTGATCCTGTAATCACTGAAGGTACATACTCCATGTGGACTTCCCTTAAACAGGCAAACACCTACAGGTATGGTGTGCAACAGATTGTACAATTACATTTTGGCCTAAATACATTTTTGCTTACTAGTATTTAAAATAAATTCTTAATCAGAGGAGGCCTTTGGGTTTTATTGGTC AAATCTTTGTAAGCTGGCTTTTGTCTTTTTAAAAAATTTCTTGAATTTGTGGTTGTGTCCAATTTGCAAACATTTCCAAAAATGTTTGCTTTGCTTACAAACCACATGATTTTAATGTTTTTTGTATACCATAATATCTAGCCCCAAACATTTGATTACTACATGTGCATTGGTGATTTTGATCATCCATTCTTAATATTTGATTTCTGTGTCACCTACTGTCATTTGTTAAACTGCTGGCCAACAAGAACAGGAAGTATAGTTTGGGGGGTTGGGGAGAGTTTACATAAGGAAGAGAAGAAATTGAGTGGCATATTGTAAATATCAGATCTATAATTGTAAATATAAAACCTGCCTCAGTTAGAATGAATGGAAAGCAGATCTACAATTTGCTAATATAGGAATATCAGGTTGACTATATAGCCATACTTGAAAATGCTTCTGAGTGGTGTCAACTTTACTTGAATGAATTTTTCATCTTGATTGACGCACAGTGATGTACAGTTCACTTCTGAAGCTAGTGGTTAACTTGTGTAGGAAACTTTTGCAGTTTGACACTAAGATAACTTCTGTGTGCATTTTTCTATGCTTTTTTAAAAACTAGTTTCATTTCATTTTCATGAGATGTTTGGTTTATAAGATCTGAGGATGGTTATAAATACTGTAAGTATTGTAATGTTATGAATGCAGGTTATTTGAAAGCTGTTTATTATTATATCATTCCTGATAATGCTATGTGAGTGTTTTTAATAAAATTTATATTTATTTAATGCACTCTAAAAAAAAAAAAAAAAAA Predicted: Homo sapiens methylcytosine dioxygenase 2 tet (TET2), transcript variant X1, mRNA
[SEQ ID NO: 1359] XXM_005263082.1 AAGCAGAAGGAAGCAAGATGGCTGCCCTTTAGGATTTGTTAGAAAGGAGACCCGACTGCAACTGCTGGATTGCTGCAAGGCTGAGGGACGAGAACGAGAATTCAACTAGAGGGCAGCCTTGTGGATGGCCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAACCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATACCATCACCTCCCATTTGCCAGACAGAACCTCTGGCTACAAAGCTCCAGAATGGAAGCCCACTGCCTGAGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTATTATGGAATACCCTGTATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTACACAAGAAAGTAGAGGGTATTCCAAGTGTTTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTCTCTCTCTGGGCTCCTTCAGATCAAGAAATTGAAACAAGACCAAAAGGCTAATGGAGAAAGACGTAACTTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAATGTCTCCGATTTGAGTGATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCAGTTAAAGATTTCACCAGTTTTTCAACACATAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAATGAGCAGGAGGGGAAAAGTGCTAATTACCATGACAAGAACATTGTATTACTTAAAAACAAGGCAGTGCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACTCCTGGAAAAAACACTGTCTCAATATTATCCAGATTGTGTTTCCATTGCGGTGCAGAAAACCACATCTCACATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCATACCTCAGGGCAGATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTC CAAAGCCAGCTGCAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCCAGTAAACTAGCTGCAATGCTAAATACCTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATCAGTTTTTGAGATATGCCCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAATTCTGTTCAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAACAAAATGAAATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTCTGCCACTACCACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCACAGGTTCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAACACCACCACTACCCCAACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACCTGAGGCACCACCTTCCCAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGCTTTCTGAAAGGCCTCAGAATAATTGTGTGAACAGGAATGACATACAGACTGCAGGGACAATGACTGTTCCATTGTGTTCTGAGAAAACAAGACCAATGTCAGAACACCTCAAGCATAACCCACCAATTTTTGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTGATGAGAAACAAAGAGCAAGAGATTCTGAAGGGTCGAGACAAGGAGCAAACACGAGATCTTGTGCCCCCAACACAGCACTATCTGAAACCAGGATGGATTGAATTGAAGGCCCCTCGTTTTCACCAAGCGGAATCCCATCTAAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTATCAACCCAATCTCTCCAATCAAATGACCTCCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGC TTACACCCAGAAAACAACACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATGAATCAAGGGCAGTCCCAAGGTACAGTGGACCAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACTTCTCCAAAACAGACCATTTACCAAAAGCTCATGTGCAGTCACTGTGTGGCACTAGATTTCATTTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTCCCCAGTGTTGAAACAGCACTTGAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAACTCACACCTTTTGCAACATAAGCCTCATAAACAGGCAGCACAAACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGCAACAGCAGCAAAAATTACAAATAAAGAATAAAGAGGAAATACTCCAGACTTTTCCTCACCCCCAAAGCAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAAGTGGAAGAATGTTTTCATGGTGAAAATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACTGGAGGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCAAGTGCATGCAAAATACAGGTTTCTTGTTCAAACAATACACACCTAGTTTCAGAGAATAAAGAACAGACTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAAAACTTGCATCACATGCAATATTTTCCAAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGAGCAGAAGTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATAGAAACCAAGATATGTCTGGTCAACAAGCTGCGCAACTTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTGTGCCTGACCAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGCTCTA AGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAGTCTTGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGTATGCACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAGCTGTGATAATGTGCAGCAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAAGCAGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCTTACTCTCAAATCACAGAAGCAAGTAAAAGTTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGAACTTGATAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAGAACAGCTGCTTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATAAAAAATTTATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGAGCAAATTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGGCAGCTATTAGAGAAATCATGGAAGAAAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAAAGAGTCATCTATACTGGTAAAGAAGGCAAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGTGGTTCGCAGAAGCAGCAGTGAAGAGAAGCTACTGTGTTTGGTGCGGGAGCGAGCTGGCCACACCTGTGAGGCTGCAGTGATTGTGATTCTCATCCTGGTGTGGGAAGGAATCCCGCTGTCTCTGGCTGACAAACTCTACTCGGAGCTTACCGAGACGCTGAGGAAATACGGCACGCTCACCAATCGCCGGTGTGCCTTGAATGAAGAGAGAACTTGCGCCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCCTCCTTCT CTTTTGGTTGTTCATGGAGCATGTACTACAATGGATGTAAGTTTGCCAGAAGCAAGATCCCAAGGAAGTTTAAGCTGCTTGGGGATGACCCAAAAGAGGAAGAGAAACTGGAGTCTCATTTGCAAAACCTGTCCACTCTTATGGCACCAACATATAAGAAACTTGCACCTGATGCATATAATAATCAGATTGAATATGAACACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCCATTCTCAGGGGTCACTGCATGTTTGGACTTCTGTGCTCATGCCCACAGAGACTTGCACAACATGCAGAATGGCAGCACATTGGTATGCACTCTCACTAGAGAAGACAATCGAGAATTTGGAGGAAAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTATACAAAGTCTCTGACGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGAGGAGAAAAAACGGAGTGGTGCCATTCAGGTACTGAGTTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGACTTGCCGACAAAGGAAACTAGAAGCCAAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGCTCAAATAAAAATGAAAAGGAAAAGTCAGCCCCATCACGTACAAAACAAACTGAAAACGCAAGCCAGGCTAAACAGTTGGCAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTCCCAGCAGCCCCAGCCTCTACAGAAGCAGCCACCACAGCCCCAGCAGCAGCAGAGACCCCAGCAGCAGCAGCCACATCACCCTCAGACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACATGAGACGGCCCAATCCAGTTAGTCCTTATCCAAACTCTTCACACACTTCAGATATCTATGGAAGCACCAGCCCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCTAATCCCATGAACCCTTACCC TGGGCTTTTGAATCAGAATACCCAATATCCATCATATCAATGCAATGGAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCTATTCTCCCCAGTCTCAGCCGATGGATCTGTATAGGTATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTCTACCACCCATCCATACACTTTACCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGGTTATGGAAACCAAAATATGCAGGGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTACATCATGTAGGGAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCCACCTCTAGATTACCACCCAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCATTCACCTTCTCACATAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAGCTCTCTTCATGCCCTGCATCTCCAAAACAAGGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCAAAGATGCTTCCAGCTCTTAACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGTGATGCTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTCTGGTGCAGAGGACAACGATGAGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGGGGAGTGGCCGTGGCTCCAACTCATGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATGCCACAACCCCTTTAAAGAATCCCAATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTTACCAGCATAAGAGCATGAATGAGCCAAAACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCGTGAGAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGACTATGTGCCTCAGAAATCCCATGGCAAAAAAGTGAAACGGGAG CCTGCTGAGCCACATGAAACTTCAGAGCCCACTTACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGACCATGTCCGTGACCACAGACTCCACAGTAACTACATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACAACAGATATATATGATATCACCCCCTTTTGTTGGTTACCTCACTTGAAAAGACCACAACCAACCTGTCAGTAGTATAGTTCTCATGACGTGGGCAGTGGGGAAAGGTCACAGTATTCATGACAAATGTGGTGGGAAAAACCTCAGCTCACCAGCAACAAAAGAGGTTATCTTACCATAGCACTTAATTTTCACTGGCTCCCAAGTGGTCACAGATGGCATCTAGGAAAAGACCAAAGCATTCTATGCAAAAAGAAGGTGGGGAAGAAAGTGTTCCGCAATTTACATTTTTAAACACTGGTTCTATTATTGGACGAGATGATATGTAAATGTGATCCCCCCCCCCCGCTTACAACTCTACACATCTGTGACCACTTTTAATAATATCAAGTTTGCATAGTCATGGAACACAAATCAAACAAGTACTGTAGTATTACAGTGACAGGAATCTTAAAATACCATCTGGTGCTGAATATATGATGTACTGAAATACTGGAATTATGGCTTTTTGAAATGCAGTTTTTACTGTAATCTTAACTTTTATTTATCAAAATAGCTACAGGAAACATGAATAGCAGGAAAACACTGAATTTGTTTGGATGTTCTAAGAAATGGTGCTAAGAAAATGGTGTCTTTAATAGCTAAAAATTTAATGCCTTTATATCATCAAGATGCTATCAGTGTACTCCAGTGCCCTTGAATAATAGGGGTACCTTTTCATTCAAGTTTTTATCATAATTACCTATTCTTACACAAGCTTAGTTTTTAAAATGTGGACATTTTAAAGGCCTCTGGATTTTGCTCATCCAGTGAAGTCCTTGTAGGACAATAAACGTATATATGTACATATATACACAAACATGT ATATGTGCACACACATGTATATGTATAAATATTTTAAATGGTGTTTTAGAAGCACTTTGTCTACCTAAGCTTTGACAACTTGAACAATGCTAAGGTACTGAGATGTTTAAAAAACAAGTTTACTTTCATTTTAGAATGCAAAGTTGATTTTTTTAAGGAAACAAAGAAAGCTTTTAAAATATTTTTGCTTTTAGCCATGCATCTGCTGATGAGCAATTGTGTCCATTTTTAACACAGCCAGTTAAATCCACCATGGGGCTTACTGGATTCAAGGGAATACGTTAGTCCACAAAACATGTTTTCTGGTGCTCATCTCACATGCTATACTGTAAAACAGTTTTATACAAAATTGTATGACAAGTTCATTGCTCAAAAATGTACAGTTTTAAGAATTTTCTATTAACTGCAGGTAATAATTAGCTGCATGCTGCAGACTCAACAAAGCTAGTTCACTGAAGCCTATGCTATTTTATGGATCATAGGCTCTTCAGAGAACTGAATGGCAGTCTGCCTTTGTGTTGATAATTATGTACATTGTGACGTTGTCATTTCTTAGCTTAAGTGTCCTCTTTAACAAGAGGATTGAGCAGACTGATGCCTGCATAAGATGAATAAACAGGGTTAGTTCCATGTGAATCTGTCAGTTAAAAAGAAACAAAAACAGGCAGCTGGTTTGCTGTGGTGGTTTTAAATCATTAATTTGTATAAAGAAGTGAAAGAGTTGTATAGTAAATTAAATTGTAAACAAAACTTTTTTAATGCAATGCTTTAGTATTTTAGTACTGTAAAAAAATTAAATATATACATATATATATATATATATATATATATATATATGAGTTTGAAGCAGAATTCACATCATGATGGTGCTACTCAGCCTGCTACAAATATATCATAATGTGAGCTAAGAATTCATTAAATGTTTGAGTGATGTTCCTACTTGTCATATACCTCAACACTAGTTTGGCAATAGGATATTGAACTGAGAGTGAAAGCATTG TGTACCATCATTTTTTTCCAAGTCCTTTTTTTTATTGTTAAAAAAAAAAGCATACCTTTTTTCAATACTTGATTTCTTAGCAAGTATAACTTGAACTTCAACCTTTTTGTTCTAAAAATTCAGGGATATTTCAGCTCATGCTCTCCCTATGCCAACATGTCACCTGTGTTTATGTAAAATTGTTGTAGGTTAATAAATATATTCTTTGTCAGGGATTTAACCCTTTTATTTTGAATCCCTTCTATTTTACTTGTACATGTGCTGATGTAACTAAAACTAATTTTGTAAATCTGTTGGCTCTTTTTATTGTAAAGAAAAGCATTTTAAAAGTTTGAGGAATCTTTTGACTGTTTCAAGCAGGAAAAAAAAATTACATGAAAATAGAATGCACTGAGTTGATAAAGGGAAAAATTGTAAGGCAGGAGTTTGGCAAGTGGCTGTTGGCCAGAGACTTACTTGTAACTCTCTAAATGAAGTTTTTTTGATCCTGTAATCACTGAAGGTACATACTCCATGTGGACTTCCCTTAAACAGGCAAACACCTACAGGTATGGTGTGCAACAGATTGTACAATTACATTTTGGCCTAAATACATTTTTGCTTACTAGTATTTAAAATAAATTCTTAATCAGAGGAGGCCTTTGGGTTTTATTGGTCAAATCTTTGTAAGCTGGCTTTTGTCTTTTTAAAAAATTTCTTGAATTTGTGGTTGTGTCCAATTTGCAAACATTTCCAAAAATGTTTGCTTTGCTTACAAACCACATGATTTTAATGTTTTTTGTATACCATAATATCTAGCCCCAAACATTTGATTACTACATGTGCATTGGTGATTTTGATCATCCATTCTTAATATTTGATTTCTGTGTCACCTACTGTCATTTGTTAAACTGCTGGCCAACAAGAACAGGAAGTATAGTTTGGGGGGTTGGGGAGAGTTTACATAAGGAAGAGAAGAAATTGAGTGGCATATTGTAAATATCAGATCTATAATTGTAAA TATAAAACCTGCCTCAGTTAGAATGAATGGAAAGCAGATCTACAATTTGCTAATATAGGAATATCAGGTTGACTATATAGCCATACTTGAAAATGCTTCTGAGTGGTGTCAACTTTACTTGAATGAATTTTTCATCTTGATTGACGCACAGTGATGTACAGTTCACTTCTGAAGCTAGTGGTTAACTTGTGTAGGAAACTTTTGCAGTTTGACACTAAGATAACTTCTGTGTGCATTTTTCTATGCTTTTTTAAAAACTAGTTTCATTTCATTTTCATGAGATGTTTGGTTTATAAGATCTGAGGATGGTTATAAATACTGTAAGTATTGTAATGTTATGAATGCAGGTTATTTGAAAGCTGTTTATTATTATATCATTCCTGATAATGCTATGTGAGTGTTTTTAATAAAATTTATATTTATTTAATGCACTCTAA Predicted: Homo sapiens methylcytosine dioxygenase 2 tet (TET2), transcript variant X2, mRNA
[SEQ ID NO: 1360] XXM_006714242.2 GTAGAGAAGCAGAAGGAAGCAAGATGGCTGCCCTTTAGGATTTGTTAGAAAGGAGACCCGACTGCAACTGCTGGATTGCTGCAAGGCTGAGGGACGAGAACGAGGCTGGCAAACATTCAGCAGCACACCCTCTCAAGATTGTTTACTTGCCTTTGCTCCTGTTGAGTTACAACGCTTGGAAGCAGGAGATGGGCTCAGCAGCAGCCAATAGGACATGATCCAGGAAGAGCAGTAAGGGACTGAGCTGCTGAATTCAACTAGAGGGCAGCCTTGTGGATGGCCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAACCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATACCATCACCTCCCATTTGCCAGACAGAACCTCTGGCTACAAAGCTCCAGAATGGAAGCCCACTGCCTGAGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTATTATGGAATACCCTGTATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTACACAAGAAAGTAGAGGGTATTCCAAGTGTTTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTCTCTCTCTGGGCTCCTTCAGATCAAGAAATTGAAACAAGACCAAAAGGCTAATGGAGAAAGACGTAACTTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAATGTCTCCGATTTGAGTGATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCAGTTAAAGATTTCACCAGTTTTTCAACACATAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAATGAGCAGGAGGGGAAAAGTGCTAATTACCATGACAAGAACATTGTATTACTTAAAAACAAGGCAGTGCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACTCCTGGAAAAAACACTGTCTCAATATTATCC AGATTGTGTTTCCATTGCGGTGCAGAAAACCACATCTCACATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCATACCTCAGGGCAGATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAGCTGCAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCCAGTAAACTAGCTGCAATGCTAAATACCTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATCAGTTTTTGAGATATGCCCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAATTCTGTTCAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAACAAAATGAAATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTCTGCCACTACCACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCACAGGTTCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAACACCACCACTACCCCAACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACCTGAGGCACCACCTTCCCAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGCTTTCTGAAAGGCCTCAGAATAATTGTGTGAACAGGAATGACATACAGACTGCAGGGACAATGACTGTTCCATTGTGTTCTGAGAAAACAAGACCAATGTCAGAACACCTCAAGCATAACCCACCAATTTTTGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTGATGAGAAACAAAGAGCAAGAGATTCTGAAGGGTCGAGACAAGGAGCAAACACGAGATCTTGTGCCCCCAACACAGCACTATCTGAAACCAGGATGGATTGAATTGAAG GCCCCTCGTTTTCACCAAGCGGAATCCCATCTAAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTATCAACCCAATCTCTCCAATCAAATGACCTCCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCTTACACCCAGAAAACAACACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATGAATCAAGGGCAGTCCCAAGGTACAGTGGACCAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACTTCTCCAAAACAGACCATTTACCAAAAGCTCATGTGCAGTCACTGTGTGGCACTAGATTTCATTTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTCCCCAGTGTTGAAACAGCACTTGAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAACTCACACCTTTTGCAACATAAGCCTCATAAACAGGCAGCACAAACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGCAACAGCAGCAAAAATTACAAATAAAGAATAAAGAGGAAATACTCCAGACTTTTCCTCACCCCCAAAGCAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAAGTGGAAGAATGTTTTCATGGTGAAAATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACTGGAGGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCAAGTGCATGCAAAATACAGGTTTCTTGTTCAAACAATACACACCTAGTTTCAGAGAATAAAGAACAGACTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAAAACTTGCATCACATGCAATATTTTCCAAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGAGCAGAAGTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATA GAAACCAAGATATGTCTGGTCAACAAGCTGCGCAACTTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTGTGCCTGACCAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGCTCTAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAGTCTTGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGTATGCACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAGCTGTGATAATGTGCAGCAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAAGCAGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCTTACTCTCAAATCACAGAAGCAAGTAAAAGTTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGAACTTGATAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAGAACAGCTGCTTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATAAAAAATTTATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAAAGAGTCATCTATACTGGTAAAGAAGGCAAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGAGAACTTGCGCCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCCTCCTTCTCTTTTGGTTGTTCATGGAGCATGTACTACAATGGATGTAAGTTTGCCAGAAGCAAGATCCCAAGGAAGTTTAAGCTGCTTGGGGATGACCCAAAAGAGGAAGAGAAACTGGAGTCTCATTTGCAAAACCTGTCCACTCTTATGGCACC AACATATAAGAAACTTGCACCTGATGCATATAATAATCAGATTGAATATGAACACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCCATTCTCAGGGGTCACTGCATGTTTGGACTTCTGTGCTCATGCCCACAGAGACTTGCACAACATGCAGAATGGCAGCACATTGGTATGCACTCTCACTAGAGAAGACAATCGAGAATTTGGAGGAAAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTATACAAAGTCTCTGACGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGAGGAGAAAAAACGGAGTGGTGCCATTCAGGTACTGAGTTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGACTTGCCGACAAAGGAAACTAGAAGCCAAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGCTCAAATAAAAATGAAAAGGAAAAGTCAGCCCCATCACGTACAAAACAAACTGAAAACGCAAGCCAGGCTAAACAGTTGGCAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTCCCAGCAGCCCCAGCCTCTACAGAAGCAGCCACCACAGCCCCAGCAGCAGCAGAGACCCCAGCAGCAGCAGCCACATCACCCTCAGACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACATGAGACGGCCCAATCCAGTTAGTCCTTATCCAAACTCTTCACACACTTCAGATATCTATGGAAGCACCAGCCCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCTAATCCCATGAACCCTTACCCTGGGCTTTTGAATCAGAATACCCAATATCCATCATATCAATGCAATGGAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCTATTCTCCCCAGTCTCAGCCGATGGATCTGTATAGGTATCCAAGCCAAGACCCTCTGTCT AAGCTCAGTCTACCACCCATCCATACACTTTACCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGGTTATGGAAACCAAAATATGCAGGGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTACATCATGTAGGGAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCCACCTCTAGATTACCACCCAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCATTCACCTTCTCACATAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAGCTCTCTTCATGCCCTGCATCTCCAAAACAAGGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCAAAGATGCTTCCAGCTCTTAACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGTGATGCTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTCTGGTGCAGAGGACAACGATGAGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGGGGAGTGGCCGTGGCTCCAACTCATGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATGCCACAACCCCTTTAAAGAATCCCAATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTTACCAGCATAAGAGCATGAATGAGCCAAAACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCGTGAGAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGACTATGTGCCTCAGAAATCCCATGGCAAAAAAGTGAAACGGGAGCCTGCTGAGCCACATGAAACTTCAGAGCCCACTTACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGACCATGTCCGTGACCACAGACTCCACAGTAACTACATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACAACAGATATA TATGATATCACCCCCTTTTGTTGGTTACCTCACTTGAAAAGACCACAACCAACCTGTCAGTAGTATAGTTCTCATGACGTGGGCAGTGGGGAAAGGTCACAGTATTCATGACAAATGTGGTGGGAAAAACCTCAGCTCACCAGCAACAAAAGAGGTTATCTTACCATAGCACTTAATTTTCACTGGCTCCCAAGTGGTCACAGATGGCATCTAGGAAAAGACCAAAGCATTCTATGCAAAAAGAAGGTGGGGAAGAAAGTGTTCCGCAATTTACATTTTTAAACACTGGTTCTATTATTGGACGAGATGATATGTAAATGTGATCCCCCCCCCCCGCTTACAACTCTACACATCTGTGACCACTTTTAATAATATCAAGTTTGCATAGTCATGGAACACAAATCAAACAAGTACTGTAGTATTACAGTGACAGGAATCTTAAAATACCATCTGGTGCTGAATATATGATGTACTGAAATACTGGAATTATGGCTTTTTGAAATGCAGTTTTTACTGTAATCTTAACTTTTATTTATCAAAATAGCTACAGGAAACATGAATAGCAGGAAAACACTGAATTTGTTTGGATGTTCTAAGAAATGGTGCTAAGAAAATGGTGTCTTTAATAGCTAAAAATTTAATGCCTTTATATCATCAAGATGCTATCAGTGTACTCCAGTGCCCTTGAATAATAGGGGTACCTTTTCATTCAAGTTTTTATCATAATTACCTATTCTTACACAAGCTTAGTTTTTAAAATGTGGACATTTTAAAGGCCTCTGGATTTTGCTCATCCAGTGAAGTCCTTGTAGGACAATAAACGTATATATGTACATATATACACAAACATGTATATGTGCACACACATGTATATGTATAAATATTTTAAATGGTGTTTTAGAAGCACTTTGTCTACCTAAGCTTTGACAACTTGAACAATGCTAAGGTACTGAGATGTTTAAAAAACAAGTTTACTTTCATTTTAGAATGCAAAGTTGAT TTTTTTAAGGAAACAAAGAAAGCTTTTAAAATATTTTTGCTTTTAGCCATGCATCTGCTGATGAGCAATTGTGTCCATTTTTAACACAGCCAGTTAAATCCACCATGGGGCTTACTGGATTCAAGGGAATACGTTAGTCCACAAAACATGTTTTCTGGTGCTCATCTCACATGCTATACTGTAAAACAGTTTTATACAAAATTGTATGACAAGTTCATTGCTCAAAAATGTACAGTTTTAAGAATTTTCTATTAACTGCAGGTAATAATTAGCTGCATGCTGCAGACTCAACAAAGCTAGTTCACTGAAGCCTATGCTATTTTATGGATCATAGGCTCTTCAGAGAACTGAATGGCAGTCTGCCTTTGTGTTGATAATTATGTACATTGTGACGTTGTCATTTCTTAGCTTAAGTGTCCTCTTTAACAAGAGGATTGAGCAGACTGATGCCTGCATAAGATGAATAAACAGGGTTAGTTCCATGTGAATCTGTCAGTTAAAAAGAAACAAAAACAGGCAGCTGGTTTGCTGTGGTGGTTTTAAATCATTAATTTGTATAAAGAAGTGAAAGAGTTGTATAGTAAATTAAATTGTAAACAAAACTTTTTTAATGCAATGCTTTAGTATTTTAGTACTGTAAAAAAATTAAATATATACATATATATATATATATATATATATATATATATGAGTTTGAAGCAGAATTCACATCATGATGGTGCTACTCAGCCTGCTACAAATATATCATAATGTGAGCTAAGAATTCATTAAATGTTTGAGTGATGTTCCTACTTGTCATATACCTCAACACTAGTTTGGCAATAGGATATTGAACTGAGAGTGAAAGCATTGTGTACCATCATTTTTTTCCAAGTCCTTTTTTTTATTGTTAAAAAAAAAAGCATACCTTTTTTCAATACTTGATTTCTTAGCAAGTATAACTTGAACTTCAACCTTTTTGTTCTAAAAATTCAGGGATATTTCAGCTCATGCTCTCCCT ATGCCAACATGTCACCTGTGTTTATGTAAAATTGTTGTAGGTTAATAAATATATTCTTTGTCAGGGATTTAACCCTTTTATTTTGAATCCCTTCTATTTTACTTGTACATGTGCTGATGTAACTAAAACTAATTTTGTAAATCTGTTGGCTCTTTTTATTGTAAAGAAAAGCATTTTAAAAGTTTGAGGAATCTTTTGACTGTTTCAAGCAGGAAAAAAAAATTACATGAAAATAGAATGCACTGAGTTGATAAAGGGAAAAATTGTAAGGCAGGAGTTTGGCAAGTGGCTGTTGGCCAGAGACTTACTTGTAACTCTCTAAATGAAGTTTTTTTGATCCTGTAATCACTGAAGGTACATACTCCATGTGGACTTCCCTTAAACAGGCAAACACCTACAGGTATGGTGTGCAACAGATTGTACAATTACATTTTGGCCTAAATACATTTTTGCTTACTAGTATTTAAAATAAATTCTTAATCAGAGGAGGCCTTTGGGTTTTATTGGTCAAATCTTTGTAAGCTGGCTTTTGTCTTTTTAAAAAATTTCTTGAATTTGTGGTTGTGTCCAATTTGCAAACATTTCCAAAAATGTTTGCTTTGCTTACAAACCACATGATTTTAATGTTTTTTGTATACCATAATATCTAGCCCCAAACATTTGATTACTACATGTGCATTGGTGATTTTGATCATCCATTCTTAATATTTGATTTCTGTGTCACCTACTGTCATTTGTTAAACTGCTGGCCAACAAGAACAGGAAGTATAGTTTGGGGGGTTGGGGAGAGTTTACATAAGGAAGAGAAGAAATTGAGTGGCATATTGTAAATATCAGATCTATAATTGTAAATATAAAACCTGCCTCAGTTAGAATGAATGGAAAGCAGATCTACAATTTGCTAATATAGGAATATCAGGTTGACTATATAGCCATACTTGAAAATGCTTCTGAGTGGTGTCAACTTTACTTGAATGAATTTTTCATCTTGATTGACGCA CAGTGATGTACAGTTCACTTCTGAAGCTAGTGGTTAACTTGTGTAGGAAACTTTTGCAGTTTGACACTAAGATAACTTCTGTGTGTGCATTTTTCTATGCTTTTTTAAAAACTAGTTTCATTTCATTTTCATGAGATGTTTGGTTTATAAGATCTGAGGATGGTTATAAATACTGTAAGTATTGTAATGTTATGAATGCAGGTTATTTGAAAGCTGTTTTATTATTATATTCTTATTAGATATGCATTTATATGATTTATATGATTTATATG Methylcytosine dioxygenase 2 tet (TET2) Homo sapiens, transcript variant 2, mRNA
[SEQ ID NO: 1361] NM_017628.4 AAACAGAAGGTGGGCCGGGGCGGGGAGAAACAGAACTCGGTCAATTTCCCAGTTTGTCGGGTCTTTAAAAATACAGGCCCCTAAAGCACTAAGGGCATGCCCTCGGTGAAACAGGGGAGCGCTTCTGCTGAATGAGATTAAAGCGACAGAAAAGGGAAAGGAGAGCGCGGGCAACGGGATCTAAAGGGAGATAGAGACGCGGGCCTCTGAGGGCTGGCAAACATTCAGCAGCACACCCTCTCAAGATTGTTTACTTGCCTTTGCTCCTGTTGAGTTACAACGCTTGGAAGCAGGAGATGGGCTCAGCAGCAGCCAATAGGACATGATCCAGGAAGAGCAGTAAGGGACTGAGCTGCTGAATTCAACTAGAGGGCAGCCTTGTGGATGGCCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAACCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATACCATCACCTCCCATTTGCCAGACAGAACCTCTGGCTACAAAGCTCCAGAATGGAAGCCCACTGCCTGAGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTATTATGGAATACCCTGTATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTACACAAGAAAGTAGAGGGTATTCCAAGTGTTTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTCTCTCTCTGGGCTCCTTCAGATCAAGAAATTGAAACAAGACCAAAAGGCTAATGGAGAAAGACGTAACTTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAATGTCTCCGATTTGAGTGATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCAGTTAAAGATTTCACCAGTTTTTCAACACATAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAATGAGCAGGAGGGGAAAAGTGCTAATTACCATGACAAGAACATTGTATT ACTTAAAAACAAGGCAGTGCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACTCCTGGAAAAAACACTGTCTCAATATTATCCAGATTGTGTTTCCATTGCGGTGCAGAAAACCACATCTCACATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCATACCTCAGGGCAGATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAGCTGCAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCCAGTAAACTAGCTGCAATGCTAAATACCTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATCAGTTTTTGAGATATGCCCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAATTCTGTTCAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAACAAAATGAAATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTCTGCCACTACCACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCACAGGTTCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAACACCACCACTACCCCAACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACCTGAGGCACCACCTTCCCAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGCTTTCTGAAAGGCCTCAGAATAATTGTGTGAACAGGAATGACATACAGACTGCAGGGACAATGACTGTTCCATTGTGTTCTGAGAAAACAAGACCAATGTCAGAACACCTCAAGCATAACCCACCAATTTTTGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTG ATGAGAAACAAAGAGCAAGAGATTCTGAAGGGTCGAGACAAGGAGCAAACACGAGATCTTGTGCCCCCAACACAGCACTATCTGAAACCAGGATGGATTGAATTGAAGGCCCCTCGTTTTCACCAAGCGGAATCCCATCTAAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTATCAACCCAATCTCTCCAATCAAATGACCTCCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCTTACACCCAGAAAACAACACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATGAATCAAGGGCAGTCCCAAGGTACAGTGGACCAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACTTCTCCAAAACAGACCATTTACCAAAAGCTCATGTGCAGTCACTGTGTGGCACTAGATTTCATTTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTCCCCAGTGTTGAAACAGCACTTGAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAACTCACACCTTTTGCAACATAAGCCTCATAAACAGGCAGCACAAACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGCAACAGCAGCAAAAATTACAAATAAAGAATAAAGAGGAAATACTCCAGACTTTTCCTCACCCCCAAAGCAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAAGTGGAAGAATGTTTTCATGGTGAAAATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACTGGAGGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCAAGTGCATGCAAAATACAGGTTTCTTGTTCAAACAATACACACCTAGTTTCAGAGAATAAAGAACAGACTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAAAACTTGCATCACATGC AATATTTTCCAAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGAGCAGAAGTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATAGAAACCAAGATATGTCTGGTCAACAAGCTGCGCAACTTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTGTGCCTGACCAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGCTCTAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAGTCTTGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGTATGCACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAGCTGTGATAATGTGCAGCAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAAGCAGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCTTACTCTCAAATCACAGAAGCAAGTAAAAGTTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGAACTTGATAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAGAACAGCTGCTTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATAAAAAATTTATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGGTAAGTGCCAGAAATGTACTGAGACACATGGCGTTTATCCAGAATTAGCAAATTTATCTTCAGATATGGGATTTTCCTTCTTTTTTTAAATCTTGAGTCTGGCAGCAATTTGTAAAGGCTCATAAAAATCTGAAGCTTACATTTTTTGTCAAGTTACCGATGCTTGTGTCTTGTGAAAGAGAACTTC ACTTACATGCAGTTTTTCCAAAAGAATTAAATAATCGTGCATGTTTATTTTTCCCTCTCTTCAGATCCTGTAAAATTTGAATGTATCTGTTTTAGATCAATTCGCCTATTTAGCTCTTTGTATATTATCTCCTGGAGAGACAGCTAGGCAGCAAAAAAACAATCTATTAAAATGAGAAAATAACGACCATAGGCAGTCTAATGTACGAACTTTAAATATTTTTTAATTCAAGGTAAAATATATTAGTTTCACAAGATTTCTGGCTAATAGGGAAATTATTATCTTCAGTCTTCATGAGTTGGGGGAAATGATAATGCTGACACTCTTAGTGCTCCTAAAGTTTCCTTTTCTCCATTTATACATTTGGAATGTTGTGATTTATATTCATTTTGATTCCCTTTTCTCTAAAATTTCATCTTTTTGATTAAAAAATATGATACAGGCATACCTCAGAGATATTGTGGGTTTGGCTCCATACCACAATAAAATGAATATTACAATAAAGCAAGTTGTAAGGACTTTTTGGTTTCTCACTGTATGTAAAAGTTATTTATATACTATACTGTAACATACTAAGTGTGCAATAGCATTGTGTCTAAAAAATATATACTTTAAAAATAATTTATTGTTAAAAAAATGCCAACAATTATCTGGGCCTTTAGTGAGTGCTAATCTTTTTGCTGGTGGAGGGTCGTGCTTCAGTATTGATCGCTGTGGACTGATCATGGTGGTAGTTGCTGAAGGTTGCTGGGATGGCTGTGTGTGTGGCAATTTCTTAAAATAAGACAACAGTGAAGTGCTGTATCAATTGATTTTTCCATTCACAAAAGATTTCTCTGTAGCATGCAATGCTGTTTGATAGCATTTAACCCACAGCAGAATTTCTTTGAAAATTGGACTCAGTCCTCTCAAACTGTGCTGCTGCTTTATCAACTAAGTTTTTGTAATTTTCTGAATCCTTTGTTGTCATTTCAGCAGTTTACAGCATCTTCATTGGAAG TATATTCCATCTCAAACATTCTTTGTTCATCCATAAGAAGCAACTTCTTATCAAGTTTTTTCATGACATTGCAGTAACTCAGCCCCATCTTCAGGCTCTACTTCTAATTCTGGTTCTCTTGCTACATCTCCCTCATCTGCAGTGACCTCTCCACGGAAGTCTTGAACTCCTCAAAGTAATCCATGAGGGTTGGAATCAACTTCTAAACTCCTGTTAATGTTGATATATTGACCCCCTCCCATGAATTATGAATGTTCTTAATAACTTCTAAATGGTGATACCTTTCCAGAAGGCTTTCAATGTACTTTGCCCGGATCCATCAGAAGACTATCTTGGCAGCTGTAGACTAACAATATATTTCTTAAATGATAAGACTTGAAAGTCAAAAGTACTCCTTAATCCATAGGCTGCAGAATCAATGTTGTATTAACAGGCACGAAAACAGCATTAATCTTGTGCATCTCCATCGGAGCTCTTGGGTGACTAGGTGCCTTGAGCAGTAATATTTTGAAAGGAGGTTTTGGTTTTGTTTTTTGTTTTTTTTTTTTGTTTTTTAGCAGTAAGTCTCAACACTGGGCTTAAAATATTCAGTAAACTATGTTGTAAAAAGATGTGTTATCATCCAGACTTTGTTGTTCCATTACTCTACACAAGCAGGGTACACTTAGCATAATTCTTAAGGGCCTTGGAATTTTCAGAATGGTAAATGAGTATGGGCTTCAACTTAAAATCATCAACTGCATTAGCCTGTAACAAGAGAGTCAGCCTGTCCTTTGAAGCAAGGCATTGACTTCTATCTATGAAAGTCTTAGATGGCACCTTGTTTCAATAGTAGGCTGTTTAGTACAGCCACCTTCATCAGTGATCTTAGCTAGATCTTCTGCATAACTTGCTGCAGCTTCTACATCAGCACTTGCTGCCTCACCTTGTCCTTTTATGTTATAGAGACAGCTGCGCTTCTTAAACTTTATAAACCAACTTCTGCTAGCTTCCAACTTCT CTTCTGCAGCTTCCTCATTCTCTTCATAGAACTGAAGGGAGTCAAGGCCTTGCTCTGGATTAAGCTTTGGCTTAAGGAATGTTGTGGCTGACGTGATCTTCTATCCAGACCACTAAAGCGCTCTCCATATCAGCAATAAGGCCGTTTTGCTTTCTTACCTTTCATGTGTTCACTGGAGTAATTTCCTTCAAGAATTTTTCCTTTACATTCACAACTTGGCTAACTGGCATGCAAGGCCTAGCTTTCAGCCTGTCTTGGCTTTTGACATGCCTTCCTCACTTAGCTCGTCATATCTAGCTTTTGATTTAAAGTGGCAGGCATACAACTCTTCCTTTCACTTGAACACTTAGAGGCCACTGTAGGGTTATTAATTGGCCTAATTTCAATATTGTTGTGTTTTAGGGAATAGAGAGGCCCAGGGAGAGGGAGAGAGCCCAAACGGCTGGTTGATAGAGCAGGCAGAATGCACACAACATTTATCAGATTATGTTTGCACCATTTACCAGATTATGGGTACGGTTTGTGGCACCCCCCAAAAATTAGAATAGTAACATCAAAGATCACTGATCACAGATCGCCATAACATAAATAATAATAAACTTTAAAATACTGTGAGAATTACCAAAATGTGATACAGAGACATGAAGTGAGCACATGCTGTTGAAAAAAATGACACTGATAGACATACTTAACACGTGGGATTGCCACAAACCTTCAGTTTGTAAAAGTCACAGTAACTGTGACTCACAAAAGAACAAAGCACAATAAAACGAGGTATGCCTGTATTTTTAAAAAAAGCTTTTTGTTAAAATTCAGGATATGTAATAGGTCTGTAGGAATAGTGAAATATTTTTGCTGATGGATGTAGATATATACGTGGATAGAGATGAAGATCTTAATTATAGCTATGCAGCATAGATTTAGTCAAAGACATTTGAAAAGACAAATGTTAAATTAGTGTGGCTAATGACCTACCCGTGCCATGTTTTCCCTCTTGCAA TGAGATACCCCACACTGTGTAGAAGGATGGAGGGAGGACTCCTACTGTCCCTCTTTGCGTGTGGTTATTAAGTTGCCTCACTGGGCTAAAACACCACACATCTCATAGATAATATTTGGTAAGTTGTAATCGTCTTCACTCTTCTCTTATCACCCACCCCTATCTTCCCACTTTTCCATCTTTGTTGGTTTGCAACAGCCCCTTCTTTTTGCCTGACTCTCCAGGATTTTCTCTCATCATAAATTGTTCTAAAGTACATACTAATATGGGTCTGGATTGACTATTCTTATTTGCAAAACAGCAATTAAATGTTATAGGGAAGTAGGAAGAAAAAGGGGTATCCTTGACAATAAACCAAGCAATATTCTGGGGGTGGGATAGAGCAGGAAATTTTATTTTTAATCTTTTAAAATCCAAGTAATAGGTAGGCTTCCAGTTAGCTTTAAATGTTTTTTTTTTCCAGCTCAAAAAATTGGATTGTAGTTGATACTACATATAATACATTCTAATTCCCTCACTGTATTCTTTGTTTAGTTTCATTTATTTGGTTTAAAATAATTTTTTATCCCATATCTGAAATGTAATATATTTTTATCCAACAACCAGCATGTACATATACTTAATTATGTGGCACATTTTCTAATAGATCAGTCCATCAATCTACTCATTTTAAAGAAAAAAAAATTTTAAAGTCACTTTTAGAGCCCTTAATGTGTAGTTGGGGGTTAAGCTTTGTGGATGTAGCCTTTATATTTAGTATAATTGAGGTCTAAAATAATAATCTTCTATTATCTCAACAGAGCAAATTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGGCAGCTATTAGAGAAATCATGGAAGAAAGGTAATTAACGCAAAGGCACAGGGCAGATTAACGTTTATCCTTTTGTATATGTCAGAATTTTTCCAGCCTTCACACACAAAGCAGTAAACAATTGTAAATTGAGTAATTA TTAGTAGGCTTAGCTATTCTAGGGTTGCCAACACTACACACTGTGCTATTCACCAGAGAGTCACAATATTTGACAGGACTAATAGTCTGCTAGCTGGCACAGGCTGCCCACTTTGCGATGGATGCCAGAAAACCCAGGCATGAACAGGAATCGGCCAGCCAGGCTGCCAGCCACAAGGTACTGGCACAGGCTCCAACGAGAGGTCCCACTCTGGCTTTCCCACCTGATAATAAAGTGTCAAAGCAGAAAGACTGGTAAAGTGTGGTATAAGAAAAGAACCACTGAATTAAATTCACCTAGTGTTGCAAATGAGTACTTATCTCTAAGTTTTCTTTTACCATAAAAAGAGAGCAAGTGTGATATGTTGAATAGAAAGAGAAACATACTATTTACAGCTGCCTTTTTTTTTTTTTTTCGCTATCAATCACAGGTATACAAGTACTTGCCTTTACTCCTGCATGTAGAAGACTCTTATGAGCGAGATAATGCAGAGAAGGCCTTTCATATAAATTTATACAGCTCTGAGCTGTTCTTCTTCTAGGGTGCCTTTTCATTAAGAGGTAGGCAGTATTATTATTAAAGTACTTAGGATACATTGGGGCAGCTAGGACATATTCAGTATCATTCTTGCTCCATTTCCAAATTATTCATTTCTAAATTAGCATGTAGAAGTTCACTAAATAATCATCTAGTGGCCTGGCAGAAATAGTGAATTTCCCTAAGTGCCTTTTTTTTGTTGTTTTTTTGTTTTGTTTTTTAAACAAGCAGTAGGTGGTGCTTTGGTCATAAGGGAAGATATAGTCTATTTCTAGGACTATTCCATATTTTCCATGTGGCTGGATACTAACTATTTGCCAGCCTCCTTTTCTAAATTGTGAGACATTCTTGGAGGAACAGTTCTAACTAAAATCTATTATGACTCCCCAAGTTTTAAAATAGCTAAATTTAGTAAGGGAAAAAATAGTTTATGTTTTAGAAGACTGAACTTAGCAAACTAACC TGAATTTTGTGCTTTGTGAAATTTTATATCGAAATGAGCTTTCCCATTTTCACCCACATGTAATTTACAAAATAGTTCATTACAATTATCTGTACATTTTGATATTGAGAAAAACAAGGCTTAAAAACCATTATCCAGTTTGCTTGGCGTAGACCTGTTTAAAAAATAATAAACCGTTCATTTCTCAGGATGTGGTCATAGAATAAAGTTATGCTCAAATGTTCAAATATTTAAA Predicted: Homo sapiens methylcytosine dioxygenase 2 tet (TET2), transcript variant X9, mRNA
[SEQ ID NO: 1362] XXM_011532044.1 TCAGGCTCTACTTCTAATTCTGGTTCTCTTGCTACATCTCCCTCATCTGCAGTGACCTCTCCACGGAAGTCTTGAACTCCTCAAAAGCAAATTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGGCAGCTATTAGAGAAATCATGGAAGAAAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAAAGAGTCATCTATACTGGTAAAGAAGGCAAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGTGGTTCGCAGAAGCAGCAGTGAAGAGAAGCTACTGTGTTTGGTGCGGGAGCGAGCTGGCCACACCTGTGAGGCTGCAGTGATTGTGATTCTCATCCTGGTGTGGGAAGGAATCCCGCTGTCTCTGGCTGACAAACTCTACTCGGAGCTTACCGAGACGCTGAGGAAATACGGCACGCTCACCAATCGCCGGTGTGCCTTGAATGAAGAGAGAACTTGCGCCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCCTCCTTCTCTTTTGGTTGTTCATGGAGCATGTACTACAATGGATGTAAGTTTGCCAGAAGCAAGATCCCAAGGAAGTTTAAGCTGCTTGGGGATGACCCAAAAGAGGAAGAGAAACTGGAGTCTCATTTGCAAAACCTGTCCACTCTTATGGCACCAACATATAAGAAACTTGCACCTGATGCATATAATAATCAGATTGAATATGAACACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCCATTCTCAGGGGTCACTGCATGTTTGGACTTCTGTGCTCATGCCCACAGAGACTTGCACAACATGCAGAATGGCAGCACATTGGTATGCACTCTCACTAGAGAAGACAATCGAGAATTTGGAGGAAAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTATACAAAGTCTCTGACGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGAGGAGAAAAAACGGAGTG GTGCCATTCAGGTACTGAGTTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGACTTGCCGACAAAGGAAACTAGAAGCCAAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGCTCAAATAAAAATGAAAAGGAAAAGTCAGCCCCATCACGTACAAAACAAACTGAAAACGCAAGCCAGGCTAAACAGTTGGCAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTCCCAGCAGCCCCAGCCTCTACAGAAGCAGCCACCACAGCCCCAGCAGCAGCAGAGACCCCAGCAGCAGCAGCCACATCACCCTCAGACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACATGAGACGGCCCAATCCAGTTAGTCCTTATCCAAACTCTTCACACACTTCAGATATCTATGGAAGCACCAGCCCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCTAATCCCATGAACCCTTACCCTGGGCTTTTGAATCAGAATACCCAATATCCATCATATCAATGCAATGGAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCTATTCTCCCCAGTCTCAGCCGATGGATCTGTATAGGTATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTCTACCACCCATCCATACACTTTACCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGGTTATGGAAACCAAAATATGCAGGGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTACATCATGTAGGGAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCCACCTCTAGATTACCACCCAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCATTCACCTTCTCACATAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAG CTCTCTTCATGCCCTGCATCTCCAAAACAAGGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCAAAGATGCTTCCAGCTCTTAACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGTGATGCTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTCTGGTGCAGAGGACAACGATGAGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGGGGAGTGGCCGTGGCTCCAACTCATGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATGCCACAACCCCTTTAAAGAATCCCAATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTTACCAGCATAAGAGCATGAATGAGCCAAAACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCGTGAGAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGACTATGTGCCTCAGAAATCCCATGGCAAAAAAGTGAAACGGGAGCCTGCTGAGCCACATGAAACTTCAGAGCCCACTTACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGACCATGTCCGTGACCACAGACTCCACAGTAACTACATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACAACAGATATATATGATATCACCCCCTTTTGTTGGTTACCTCACTTGAAAAGACCACAACCAACCTGTCAGTAGTATAGTTCTCATGACGTGGGCAGTGGGGAAAGGTCACAGTATTCATGACAAATGTGGTGGGAAAAACCTCAGCTCACCAGCAACAAAAGAGGTTATCTTACCATAGCACTTAATTTTCACTGGCTCCCAAGTGGTCACAGATGGCATCTAGGAAAAGACCAAAGCATTCTATGCAAAAAGAAGGTGGGGAAGAAAGTGTTCCGCAATTTACATTTTTAAACACTGGTTCTATTATTGGACGAGATGATATGTAAATG TGATCCCCCCCCCCCGCTTACAACTCTACACATCTGTGACCACTTTTAATAATATCAAGTTTGCATAGTCATGGAACACAAATCAAACAAGTACTGTAGTATTACAGTGACAGGAATCTTAAAATACCATCTGGTGCTGAATATATGATGTACTGAAATACTGGAATTATGGCTTTTTGAAATGCAGTTTTTACTGTAATCTTAACTTTTATTTATCAAAATAGCTACAGGAAACATGAATAGCAGGAAAACACTGAATTTGTTTGGATGTTCTAAGAAATGGTGCTAAGAAAATGGTGTCTTTAATAGCTAAAAATTTAATGCCTTTATATCATCAAGATGCTATCAGTGTACTCCAGTGCCCTTGAATAATAGGGGTACCTTTTCATTCAAGTTTTTATCATAATTACCTATTCTTACACAAGCTTAGTTTTTAAAATGTGGACATTTTAAAGGCCTCTGGATTTTGCTCATCCAGTGAAGTCCTTGTAGGACAATAAACGTATATATGTACATATATACACAAACATGTATATGTGCACACACATGTATATGTATAAATATTTTAAATGGTGTTTTAGAAGCACTTTGTCTACCTAAGCTTTGACAACTTGAACAATGCTAAGGTACTGAGATGTTTAAAAAACAAGTTTACTTTCATTTTAGAATGCAAAGTTGATTTTTTTAAGGAAACAAAGAAAGCTTTTAAAATATTTTTGCTTTTAGCCATGCATCTGCTGATGAGCAATTGTGTCCATTTTTAACACAGCCAGTTAAATCCACCATGGGGCTTACTGGATTCAAGGGAATACGTTAGTCCACAAAACATGTTTTCTGGTGCTCATCTCACATGCTATACTGTAAAACAGTTTTATACAAAATTGTATGACAAGTTCATTGCTCAAAAATGTACAGTTTTAAGAATTTTCTATTAACTGCAGGTAATAATTAGCTGCATGCTGCAGACTCAACAAAGCTAGTTCACTGAAGCCTATGCTAT TTTATGGATCATAGGCTCTTCAGAGAACTGAATGGCAGTCTGCCTTTGTGTTGATAATTATGTACATTGTGACGTTGTCATTTCTTAGCTTAAGTGTCCTCTTTAACAAGAGGATTGAGCAGACTGATGCCTGCATAAGATGAATAAACAGGGTTAGTTCCATGTGAATCTGTCAGTTAAAAAGAAACAAAAACAGGCAGCTGGTTTGCTGTGGTGGTTTTAAATCATTAATTTGTATAAAGAAGTGAAAGAGTTGTATAGTAAATTAAATTGTAAACAAAACTTTTTTAATGCAATGCTTTAGTATTTTAGTACTGTAAAAAAATTAAATATATACATATATATATATATATATATATATATATATATGAGTTTGAAGCAGAATTCACATCATGATGGTGCTACTCAGCCTGCTACAAATATATCATAATGTGAGCTAAGAATTCATTAAATGTTTGAGTGATGTTCCTACTTGTCATATACCTCAACACTAGTTTGGCAATAGGATATTGAACTGAGAGTGAAAGCATTGTGTACCATCATTTTTTTCCAAGTCCTTTTTTTTATTGTTAAAAAAAAAAGCATACCTTTTTTCAATACTTGATTTCTTAGCAAGTATAACTTGAACTTCAACCTTTTTGTTCTAAAAATTCAGGGATATTTCAGCTCATGCTCTCCCTATGCCAACATGTCACCTGTGTTTATGTAAAATTGTTGTAGGTTAATAAATATATTCTTTGTCAGGGATTTAACCCTTTTATTTTGAATCCCTTCTATTTTACTTGTACATGTGCTGATGTAACTAAAACTAATTTTGTAAATCTGTTGGCTCTTTTTATTGTAAAGAAAAGCATTTTAAAAGTTTGAGGAATCTTTTGACTGTTTCAAGCAGGAAAAAAAAATTACATGAAAATAGAATGCACTGAGTTGATAAAGGGAAAAATTGTAAGGCAGGAGTTTGGCAAGTGGCTGTTGGCCAGAGACTTACTTGTAACTCTCT AAATGAAGTTTTTTTGATCCTGTAATCACTGAAGGTACATACTCCATGTGGACTTCCCTTAAACAGGCAAACACCTACAGGTATGGTGTGCAACAGATTGTACAATTACATTTTGGCCTAAATACATTTTTGCTTACTAGTATTTAAAATAAATTCTTAATCAGAGGAGGCCTTTGGGTTTTATTGGTCAAATCTTTGTAAGCTGGCTTTTGTCTTTTTAAAAAATTTCTTGAATTTGTGGTTGTGTCCAATTTGCAAACATTTCCAAAAATGTTTGCTTTGCTTACAAACCACATGATTTTAATGTTTTTTGTATACCATAATATCTAGCCCCAAACATTTGATTACTACATGTGCATTGGTGATTTTGATCATCCATTCTTAATATTTGATTTCTGTGTCACCTACTGTCATTTGTTAAACTGCTGGCCAACAAGAACAGGAAGTATAGTTTGGGGGGTTGGGGAGAGTTTACATAAGGAAGAGAAGAAATTGAGTGGCATATTGTAAATATCAGATCTATAATTGTAAATATAAAACCTGCCTCAGTTAGAATGAATGGAAAGCAGATCTACAATTTGCTAATATAGGAATATCAGGTTGACTATATAGCCATACTTGAAAATGCTTCTGAGTGGTGTCAACTTTACTTGAATGAATTTTTCATCTTGATTGACGCACAGTGATGTACAGTTCACTTCTGAAGCTAGTGGTTAACTTGTGTAGGAAACTTTTGCAGTTTGACACTAAGATAACTTCTGTGTGCATTTTTCTATGCTTTTTTAAAAACTAGTTTCATTTCATTTTCATGAGATGTTTGGTTTATAAGATCTGAGGATGGTTATAAATACTGTAAGTATTGTAATGTTATGAATGCAGGTTATTTGAAAGCTGTTTATTATTATATCATTCCTGATAATGCTATGTGAGTGTTTTTAATAAAATTTATATTTATTTAATGCACTCTAA Predicted: Homo sapiens methylcytosine dioxygenase 2 tet (TET2), transcript variant X7, mRNA
[SEQ ID NO: 1363] XXM_011532043.1 GTAGAGAAGCAGAAGGAAGCAAGATGGCTGCCCTTTAGGATTTGTTAGAAAGGAGACCCGACTGCAACTGCTGGATTGCTGCAAGGCTGAGGGACGAGAACGAGGCTGGCAAACATTCAGCAGCACACCCTCTCAAGATTGTTTACTTGCCTTTGCTCCTGTTGAGTTACAACGCTTGGAAGCAGGAGATGGGCTCAGCAGCAGCCAATAGGACATGATCCAGGAAGAGCAGTAAGGGACTGAGCTGCTGAATTCAACTAGAGGGCAGCCTTGTGGATGGCCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAACCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATACCATCACCTCCCATTTGCCAGACAGAACCTCTGGCTACAAAGCTCCAGAATGGAAGCCCACTGCCTGAGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTATTATGGAATACCCTGTATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTACACAAGAAAGTAGAGGGTATTCCAAGTGTTTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTCTCTCTCTGGGCTCCTTCAGATCAAGAAATTGAAACAAGACCAAAAGGCTAATGGAGAAAGACGTAACTTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAATGTCTCCGATTTGAGTGATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCAGTTAAAGATTTCACCAGTTTTTCAACACATAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAATGAGCAGGAGGGGAAAAGTGCTAATTACCATGACAAGAACATTGTATTACTTAAAAACAAGGCAGTGCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACTCCTGGAAAAAACACTGTCTCAATATTATCC AGATTGTGTTTCCATTGCGGTGCAGAAAACCACATCTCACATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCATACCTCAGGGCAGATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAGCTGCAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCCAGTAAACTAGCTGCAATGCTAAATACCTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATCAGTTTTTGAGATATGCCCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAATTCTGTTCAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAACAAAATGAAATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTCTGCCACTACCACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCACAGGTTCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAACACCACCACTACCCCAACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACCTGAGGCACCACCTTCCCAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGCTTTCTGAAAGGCCTCAGAATAATTGTGTGAACAGGAATGACATACAGACTGCAGGGACAATGACTGTTCCATTGTGTTCTGAGAAAACAAGACCAATGTCAGAACACCTCAAGCATAACCCACCAATTTTTGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTGATGAGAAACAAAGAGCAAGAGATTCTGAAGGGTCGAGACAAGGAGCAAACACGAGATCTTGTGCCCCCAACACAGCACTATCTGAAACCAGGATGGATTGAATTGAAG GCCCCTCGTTTTCACCAAGCGGAATCCCATCTAAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTATCAACCCAATCTCTCCAATCAAATGACCTCCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCTTACACCCAGAAAACAACACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATGAATCAAGGGCAGTCCCAAGGTACAGTGGACCAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACTTCTCCAAAACAGACCATTTACCAAAAGCTCATGTGCAGTCACTGTGTGGCACTAGATTTCATTTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTCCCCAGTGTTGAAACAGCACTTGAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAACTCACACCTTTTGCAACATAAGCCTCATAAACAGGCAGCACAAACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGCAACAGCAGCAAAAATTACAAATAAAGAATAAAGAGGAAATACTCCAGACTTTTCCTCACCCCCAAAGCAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAAGTGGAAGAATGTTTTCATGGTGAAAATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACTGGAGGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCAAGTGCATGCAAAATACAGGTTTCTTGTTCAAACAATACACACCTAGTTTCAGAGAATAAAGAACAGACTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAAAACTTGCATCACATGCAATATTTTCCAAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGAGCAGAAGTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATA GAAACCAAGATATGTCTGGTCAACAAGCTGCGCAACTTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTGTGCCTGACCAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGCTCTAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAGTCTTGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGTATGCACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAGCTGTGATAATGTGCAGCAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAAGCAGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCTTACTCTCAAATCACAGAAGCAAGTAAAAGTTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGAACTTGATAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAGAACAGCTGCTTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATAAAAAATTTATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGAGCAAATTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGGCAGCTATTAGAGAAATCATGGAAGAAAGGTATACAAGTACTTGCCTTTACTCCTGCATGTAGAAGACTCTTATGAGCGAGATAATGCAGAGAAGGCCTTTCATATAAATTTATACAGCTCTGAGCTGTTCTTCTTCTAGGGTGCCTTTTCATTAAGAGGTAGGCAGTATTATTATTAAAGTACTTAGGATACATTGGGGCAGCTAGGACATATTCAGTATCATTCTTGCTCC ATTTCCAAATTATTCATTTCTAAATTAGCATGTAGAAGTTCACTAAATAATCATCTAGTGGCCTGGCAGAAATAGTGAATTTCCCTAAGTGCCTTTTTTTTGTTGTTTTTTTGTTTTGTTTTTTAAACAAGCAGTAGGTGGTGCTTTGGTCATAAGGGAAGATATAGTCTATTTCTAGGACTATTCCATATTTTCCATGTGGCTGGATACTAACTATTTGCCAGCCTCCTTTTCTAAATTGTGAGACATTCTTGGAGGAACAGTTCTAACTAAAATCTATTATGACTCCCCAAGTTTTAAAATAGCTAAATTTAGTAAGGGAAAAAATAGTTTATGTTTTAGAAGACTGAACTTAGCAAACTAACCTGAATTTTGTGCTTTGTGAAATTTTATATCGAAATGAGCTTTCCCATTTTCACCCACATGTAATTTACAAAATAGTTCATTACAATTATCTGTACATTTTGATATTGAGGAAAAACAAGGCTTAAAAACCATTATCCAGTTTGCTTGGCGTAGACCTGTTTAAAAAATAATAAACCGTTCATTTCTCAGGATGTGGTCATAGAATAAAGTTATGCTCAAATGTTCAAA

The terms "Tet inhibitor" or "Tet[x] inhibitor" (e.g., "Tet1 inhibitor", "Tet2 inhibitor", or "Tet3 inhibitor"), as used herein, refer to a molecule or group of molecules (e.g., a system) which reduce or abolish the function and/or expression of the corresponding Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2. In some embodiments, a Tet inhibitor, such as Tet1, Tet2, and/or Tet3, such as Tet2, is a molecule that inhibits the expression of Tet, such as Tet1, Tet2, and/or Tet3, such as Tet2, such as reducing or abolishing Tet expression, eg Tet1, Tet2 and/or Tet3, eg Tet2. In some embodiments, a Tet inhibitor, eg Tet1, Tet2 and/or Tet3, eg Tet2, is a molecule that inhibits Tet function, eg Tet1, Tet2 and/or Tet3, eg Tet2. An example of a Tet inhibitor, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, which inhibits the expression of Tet, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, is a gene editing system, e.g., as described herein, which targets to a nucleic acid in the Tet gene, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, or in its regulatory elements, such that modification of the nucleic acid at or near the binding site(s) of the gene editing system is altered to reduce or abolish Tet expression, eg Tet1, Tet2 and/or Tet3, eg Tet2. Another example of a Tet inhibitor, such as Tet1, Tet2 and/or Tet3, such as Tet2, which inhibits the expression of Tet, such as Tet1, Tet2 and/or Tet3, such as Tet2, is a nucleic acid molecule, such as an RNA molecule, such as short hairpin RNA (shRNA) or small interfering RNA (siRNA) capable of hybridizing to mRNA of Tet, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, and cause reduction or elimination of Tet translation, e.g. Tet2. Tet inhibitors, such as Tet1, Tet2 and/or Tet3, such as Tet2, also include nucleic acids encoding molecules that inhibit the expression of Tet, such as Tet1, Tet2 and/or Tet3, such as Tet2 (for example, a nucleic acid encoding shRNA or anti-Tet siRNA, eg Tet1, Tet2 and/or Tet3, eg Tet2, or a nucleic acid encoding one or more, eg, components of an anti-Tet gene editing system, eg Tet1, Tet2 and/or Tet3, eg Tet2) . An example of a molecule that inhibits Tet function, such as Tet1, Tet2 and/or Tet3, such as Tet2, is a molecule, such as a protein or small molecule, that inhibits one or more Tet activities, such as Tet1, Tet2 and/or Tet3, e.g. Tet2. An example is a small molecule inhibitor of Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2. Another example is a dominant negative Tet protein, eg Tet1, Tet2 and/or Tet3, eg Tet2. Another example is a dominant negative version of a Tet binding partner, eg Tet1, Tet2 and/or Tet3, eg Tet2, eg their associated histone deacetylase (HDAC). Another example is a molecule, e.g., a small molecule, that inhibits a Tet binding partner, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, e.g. a Tet-associated inhibitor, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2 HDAC . Tet inhibitors, eg Tet1, Tet2 and/or Tet3, eg Tet2, also include nucleic acids encoding inhibitors of Tet function, eg Tet1, Tet2 and/or Tet3, eg Tet2.

"System" as used herein in relation to gene editing or inhibition of Tet, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, refers to a group of molecules, e.g. one or more molecules that act together to provide a desired function. .

"Gene editing system" as used herein refers to a system, e.g., one or more molecules, that provides for and effectuates a change, e.g., deletion, of one or more nucleic acids at or near the region of genomic DNA targeted by said system. Gene editing systems are known in the art and are described in more detail below.

"Binding partner", as used herein in the context of a Tet binding partner, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, refers to a molecule, e.g. a protein, that interacts, e.g. binds, with a Tet protein, eg Tet1, Tet2 and/or Tet3, eg Tet2. Without being bound by theory, it is believed that Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, binds to one or more HDAC proteins. Such HDAC proteins are considered examples of Tet binding partners, eg Tet1, Tet2 and/or Tet3, eg Tet2.

A "dominant negative" gene product or protein is a product or protein that interferes with the function of another gene product or protein. The other gene product being affected may be the same or different from the dominant negative protein. Dominant-negative gene products can take many forms, including truncations, full-length point mutation proteins or fragments thereof, or fusions of full-length wild-type or mutant proteins or fragments thereof with other proteins. The level of inhibition observed may be very low. For example, a large excess of the dominant negative protein over the functional protein or proteins involved in the process may be required to observe an effect. It can be difficult to observe effects under normal biological assay conditions. In one embodiment, the dominant negative Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, is a catalytically inactive Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2. In another embodiment, the dominant negative Tet binding partner, eg Tet1, Tet2 and/or Tet3, eg Tet2, is a catalytically inactive inhibitor of HDAC binding to Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2.

Description

The present invention relates to Tet inhibitors, eg Tet1, Tet2 and/or Tet3, eg Tet2, and methods of using them. In particular, the invention relates to CAR expressing T cells containing Tet inhibitors, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, and the use of Tet inhibitors, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, in conjunction with CART cells. The Tet inhibitors, eg Tet1, Tet2 and/or Tet3, eg Tet2, of the present invention, together with methods for their use, are described in more detail below. CAR, CART cells and methods of using them are further described below.

Tet inhibitors, eg Tet1, Tet2 and/or Tet3, eg Tet2

The present invention relates to compositions of, for example, Tet inhibitors, for example, Tet1, Tet2 and/or Tet3, for example Tet2, and to methods for enhancing the functions of immune effector cells, for example, the functions of CAR expressing cells, using such compositions and/or other means, as described in the present description. Any Tet inhibitors, eg Tet1, Tet2 and/or Tet3, eg Tet2, known in the art can be used as a Tet inhibitor, eg Tet1, Tet2 and/or Tet3, eg Tet2, in accordance with the present invention. Examples of Tet inhibitors, eg Tet1, Tet2 and/or Tet3, eg Tet2, are described below.

Gene editing systems

In accordance with the present invention, gene editing systems can be used as inhibitors of Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2. The present invention also contemplates the use of a nucleic acid encoding one or more components of the Tet gene editing system, eg Tet1, Tet2 and/or Tet3, eg Tet2.

CRISPR/Cas9 gene editing systems

Naturally occurring CRISPR/Cas systems are found in approximately 40% of sequenced eubacteria and 90% of sequenced archaeal genomes. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.

The CRISPR/Cas system has been modified for use in gene editing (silencing, amplification or modification of specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This was done by introducing into a eukaryotic cell a plasmid containing a specially designed CRISPR and one or more of the corresponding Cas.

The CRISPR sequence, sometimes referred to as the CRISPR locus, contains alternating repeats and spacers. In naturally occurring CRISPR, spacers typically contain sequences foreign to the bacterium, such as a plasmid or phage sequence; in an exemplary CRISPR/Cas system directed against Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, the spacers are derived from a Tet gene sequence, eg Tet1, Tet2 and/or Tet3, eg Tet2, or sequences of their regulatory elements.

RNA from the CRISPR locus is constitutively expressed and processed into small RNAs. They contain a spacer flanked by repeat sequences. RNA directs other Cas proteins to suppress exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1:7. Thus, spacers act as templates for RNA molecules, similar to miRNAs. Pennisi (2013) Science 341: 833-836.

Because they occur naturally in many different types of bacteria, the precise organization and structure of CRISPR, the function and number of Cas genes and their products differ to some extent from species to species. Haft et al. (2005) PLoS Comput. Biol. 1:e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mol. Evol. 60:174-182; Bolotin et al. (2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151:653-663; and Stern et al. (2010) Trends. Genet . 28:335-340. For example, Cse proteins (a subtype of Cas, E. coli ) (eg, CasA) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat elements that Cascade retains. Browns et al. (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes the CRISPR transcript. CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2. Cmr proteins (RAMP Cas module) in Pyrococcus furiosus and other prokaryotes form a functional complex with CRISPR small RNAs that recognize and cleave target RNAs. A simpler CRISPR system is based on the Cas9 protein, which is a nuclease with two active cleavage sites, one for each strand of the double helix. The combination of Cas9 and a modified CRISPR RNA locus can be used in a gene editing system. Pennisi (2013) Science 341: 833-836.

Thus, the CRISPR/Cas system can be used to modify, for example, delete, one or more Tet gene nucleic acids, for example, Tet1, Tet2 and/or Tet3, for example Tet2, or a Tet gene regulatory element, for example, Tet1, Tet2 and/ or Tet3, such as Tet2, or the introduction of a premature stop codon, thereby reducing the expression of functional Tet, such as Tet1, Tet2 and/or Tet3, such as Tet2. Alternatively, the CRISPR/Cas system can be used similar to RNA interference by turning off the Tet gene, eg Tet1, Tet2 and/or Tet3, eg Tet2, in a reversible manner. In a mammalian cell, for example, the RNA can direct the Cas protein to the Tet promoter, eg Tet1, Tet2 and/or Tet3, eg Tet2, spatially blocking RNA polymerases.

CRISPR/Cas systems for gene editing in eukaryotic cells typically involve (1) a guide RNA (gRNA) molecule containing a targeting sequence (which is capable of hybridizing to a genomic DNA target sequence) and a sequence that is capable of binding to the Cas enzyme , eg Cas9, and (2) a Cas protein, eg Cas9. The targeting sequence and the sequence that is capable of binding to a Cas enzyme, eg Cas9, may be on the same or on different molecules. If they are located on different molecules, each of them includes a hybridization domain, which allows the molecules to associate, for example, by hybridization.

An exemplary gRNA molecule of the present invention comprises, for example, consists of the first nucleic acid having the sequence (where "n" refers to residues of the targeting sequence (for example, as described herein, for example, in Table 3), and may consist of 15- 25 nucleotides, for example, consists of 20 nucleotides):

nnnnnnnnnnnnnnnnnnnnGUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 40);

and a second nucleic acid sequence having the sequence:

AACUUACCAAGGAACAGCAUAGCAAGUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC, optionally with 1, 2, 3, 4, 5, 6 or 7 (eg 4 or 7, eg 7) additional U nucleotides at the 3' end (SEQ ID NO: 41).

The second nucleic acid molecule may alternatively consist of a fragment of the sequence given above, wherein such fragment is capable of hybridizing to the first nucleic acid. An example of such a second nucleic acid molecule is:

AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC, optionally with 1, 2, 3, 4, 5, 6, or 7 (eg 4 or 7, eg 7) additional U nucleotides at the 3' end (SEQ ID NO: 42).

Another exemplary gRNA molecule of the present invention contains, for example, consists of the first nucleic acid having the sequence (where "n" refers to residues of the targeting sequence (for example, as described in the present description, for example, in table 3), and may consist of 15 -25 nucleotides, for example, consists of 20 nucleotides):

nnnnnnnnnnnnnnnnnnnn GUUUUAGAGCUA GAAA UAGCAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 43), optionally with 1, 2, 3, 4, 5, 6 or 7 (e.g. 4 or 7, e.g. 4) additional U nucleotides at the 3' end. It is possible to generate artificial CRISPR/Cas systems that inhibit Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, using technology known in the art, such as those described in US Publication No. 20140068797, WO2015/048577 and Cong ( 2013) Science 339: 819-823. Other artificial CRISPR/Cas systems, as known in the art, that inhibit Tet, such as Tet1, Tet2, and/or Tet3, such as Tet2, such as those described in Tsai (2014) Nature Biotechnol., 32:6, can also be made. 569-576, US Pat. No. 8,871,445; 8865406; 8795965; 8771945 and 8697359, the contents of which are incorporated into the present description by reference in full. Such systems that inhibit Tet, such as Tet1, Tet2 and/or Tet3, such as Tet2, can be obtained, for example, by modifying CRISPR/Cas system engineering to include a gRNA molecule containing a targeting sequence that hybridizes to the tet gene sequence, for example, Tet1, Tet2 and/or Tet3, such as Tet2. In some embodiments, the gRNA contains a targeting sequence that is fully complementary to 15-25 nucleotides, such as 20 nucleotides, of the tet gene, such as the Tet1, Tet2 and/or Tet3 gene, such as Tet2. In some embodiments, 15-25 nucleotides, e.g. 20 nucleotides, of the tet gene, e.g. Tet1, Tet2 and/or Tet3 gene, e.g. Tet2, are located immediately 5' to the protospacer motif (PAM) adjacent sequence recognized by the protein Cas of the CRISPR/Cas system (for example, when the system contains the S. pyogenes Cas9 protein, the PAM sequence contains NGG, where N can be any of A, T, G, or C). In some embodiments, the targeting sequence of the gRNA contains, for example, consists of an RNA sequence that is complementary to the sequence shown in Table 2. In some embodiments, the implementation of the gRNA contains the targeting sequence shown in Table 3.

In one embodiment, foreign DNA can be introduced into the cell along with the CRISPR/Cas system, eg DNA encoding CAR, eg as described herein; depending on the foreign DNA sequences and chromosomal sequence, this process can be used to insert DNA encoding a CAR, for example, as described herein, at or near the site targeted by the CRISPR/Cas system. As shown in the examples herein, but without being bound by theory, such insertion can result in CAR expression as well as disruption of the Tet gene, eg Tet1, Tet2 and/or Tet3, eg Tet2. Such a foreign DNA molecule is referred to herein as "messenger DNA." In some embodiments, the template DNA further comprises homology arms on the 5' side, 3' side, or both 5' and 3' sides of the nucleic acid of the template DNA that encodes the molecule or molecules of interest (e.g. , which encodes the CAR described in the present description), where the specified homology arms are complementary to the genomic DNA sequence flanking the target sequence.

In one embodiment, the CRISPR/Cas system of the present invention comprises Cas9, e.g., Cas9 S. pyogenes , and an gRNA containing a targeting sequence that hybridizes to a Tet gene sequence, e.g., Tet1, Tet2 and/or Tet3, e.g. Tet2. In one embodiment, the CRISPR/Cas system comprises a nucleic acid encoding a Tet gRNA, such as Tet1, Tet2, and/or Tet3, such as Tet2, and a nucleic acid encoding a Cas protein, such as Cas9, such as S. pyogenes Cas9. In one embodiment, the CRISPR/Cas system comprises a Tet gRNA, eg Tet1, Tet2 and/or Tet3, eg Tet2, and a nucleic acid encoding a Cas protein, eg Cas9, eg S. pyogenes Cas9.

Examples of Tet2 target genomic sequences for which gRNAs containing complementary targeting sequences can be generated for use in the present invention are shown in Table 2 below. In some embodiments, the gRNA contains an RNA complementary to the target sequence shown in the table below (eg, for sgTET2_1, the gRNA will contain CCUUGGACACCUUCUCCUCC (SEQ ID NO: 44)). In some embodiments, the gRNA contains an RNA analogue of the target sequence shown in Table 2 below (e.g., for sgTET2_1, the gRNA will contain GGAACCUGUGGAAGAGGAGG (SEQ ID NO: 45). In some embodiments, the Tet2 inhibitor is a nucleic acid encoding a gRNA molecule , specific to Tet2, where the nucleic acid contains the target sequence from table 2 below, for example, under the control of the U6 or H1 promoter:

table 2

gRNA ID Gene designation Chromosome Position Chain Target sequence in the Tet2 gene sequence sgTET2_1 TET2 chr4 106156327 - GGAACCTGTGGAAGAGGAGG (SEQ ID NO: 46) sgTET2_2 TET2 chr4 106156339 - GAAGGAAGCTGAGGAACCTG (SEQ ID NO: 47) sgTET2_3 TET2 chr4 106156897 + ATGACCTCCAAACAATACAC (SEQ ID NO: 48) sgTET2_4 TET2 chr4 106157189 - CAAGTGCTGTTTCACACTG (SEQ ID NO: 49) sgTET2_5 TET2 chr4 106157296 - GGGAGATGTGAACTCTGGGA (SEQ ID NO: 50) sgTET2_6 TET2 chr4 106155148 - GGAGGTGATGGTATCAGGAA (SEQ ID NO: 51) sgTET2_7 TET2 chr4 106155166 - GGTTCTGTCTGGCAAATGGG (SEQ ID NO: 52) sgTET2_8 TET2 chr4 106155217 - GGATGAGCTCTCTCAGGCAG (SEQ ID NO: 53) sgTET2_9 TET2 chr4 106155403 - TGAAGGAGCCCAGAGAGAGA (SEQ ID NO: 65) sgTET2_10 TET2 chr4 106155478 + GTAAGCCAAGAAAGAAATCC (SEQ ID NO: 66)

Examples of gRNA targeting sequences that are useful in various embodiments of the present invention for inhibiting Tet, such as Tet2, are shown in Table 3 below. Table 3. In some embodiments, the CRISPR/Cas system of the present invention comprises an gRNA molecule containing a targeting sequence that is the sequence shown in Table 3.

Table 3

ID Target target area Chain Position of target genomic sequence (hg38) gRNA targeting sequence SEQID NO: 54790_1_1 TET2 EXON + chr4:105145928-105145948 UGUCGGGUCUUUAAAAAAUAC 73 54790_1_3 TET2 EXON + chr4:105145945-105145965 UACAGGCCCCUAAAGCACUA 74 54790_1_4 TET2 EXON + chr4:105145946-105145966 ACAGGCCCCUAAAGCACUAA 75 54790_1_5 TET2 EXON + chr4:105145957-105145977 AAGCACUAAGGGCAUGCCCU 76 54790_1_8 TET2 EXON + chr4:105145966-105145986 GGGCAUGCCCUCGGUGAAAC 77 54790_1_10 TET2 EXON + chr4:105145967-105145987 GGCAUGCCCUCGGUGAAACA 78 54790_1_12 TET2 EXON + chr4:105145968-105145988 GCAUGCCCUCGGUGAAACAG 79 54790_1_20 TET2 EXON + chr4:105146006-105146026 UGAGAUUAAAGCGACAGAAA 80 54790_1_23 TET2 EXON + chr4:105146007-105146027 GAGAUUAAAGCGACAGAAAAA 81 54790_1_25 TET2 EXON + chr4:105146012-105146032 UAAAGCGACAGAAAAGGGAA 82 54790_1_30 TET2 EXON + chr4:105146021-105146041 AGAAAAGGGAAAGGAGAGCG 83 54790_1_31 TET2 EXON + chr4:105146022-105146042 GAAAAGGGAAAGGAGAGCGC 84 54790_1_33 TET2 EXON + chr4:105146028-105146048 GGAAAGGAGAGCGCGGGCAA 85 54790_1_35 TET2 EXON + chr4:105146029-105146049 GAAAGGAGAGCGCGGGCAAC 86 54790_1_38 TET2 EXON + chr4:105146038-105146058 GCGCGGGCAACGGGAUCUAA 87 54790_1_39 TET2 EXON + chr4:105146039-105146059 CGCGGGCAACGGGAUCUAAA 88 54790_1_43 TET2 EXON + chr4:105146053-105146073 UCUAAAGGGAGAUAGAGACG 89 54790_1_44 TET2 EXON + chr4:105146054-105146074 CUAAAGGGAGAAUAGAGACGC 90 54790_1_47 TET2 EXON + chr4:105146063-105146083 GAUAGAGACGCGGGCCUCUG 91 54790_1_48 TET2 EXON + chr4:105146064-105146084 AUAGAGACGCGGGCCUCUGA 92 54790_1_49 TET2 EXON + chr4:105146069-105146089 GACGCGGGCCUCUGAGGGUA 93 54790_1_51 TET2 EXON + chr4:105146072-105146092 GCGGGCCUCUGAGGGUAAGG 94 54790_1_52 TET2 EXON + chr4:105146073-105146093 CGGGCCUCUGAGGGUAAGGU 95 54790_1_54 TET2 EXON + chr4:105146082-105146102 GAGGGUAAGGUGGGCGCAAG 96 54790_1_61 TET2 EXON - chr4:105145954-105145974 GCAUGCCCUUAGUGCUUUAG 97 54790_1_62 TET2 EXON - chr4:105145955-105145975 GGCAUGCCCUUAGUGCUUUA 98 54790_1_64 TET2 EXON - chr4:105145956-105145976 GGGCAUGCCCUUAGUGCUUU 99 54790_1_68 TET2 EXON - chr4:105145976-105145996 GCGCUCCCCUGUUUCACGA one hundred 54790_1_69 TET2 EXON - chr4:105145977-105145997 AGCGCUCCCCUGUUUCACCG 101 54790_1_87 TET2 EXON - chr4:105146080-105146100 UGCGCCCACCUUACCCUCAG 102 54790_2_1 TET2 EXON + chr4:105146669-105146689 AGAGCCGGCGGUAGCGGCAG 103 54790_2_2 TET2 EXON + chr4:105146675-105146695 GGCGGUAGCGGCAGUGGCAG 104 54790_2_6 TET2 EXON + chr4:105146686-105146706 CAGUGGCAGCGGCGAGAGCU 105 54790_2_7 TET2 EXON + chr4:105146687-105146707 AGUGGCAGCGGCGAGAGCUU 106 54790_2_8 TET2 EXON + chr4:105146690-105146710 GGCAGCGGCGAGAGCUUGGG 107 54790_2_12 TET2 EXON + chr4:105146725-105146745 CCUCGCGAGCGCCGCGCGCC 108 54790_2_13 TET2 EXON + chr4:105146726-105146746 CUCGCGAGCGCCGCGCGCCC 109 54790_2_14 TET2 EXON + chr4:105146761-105146781 GCAAGUCACGUCCGCCCCCU 110 54790_2_15 TET2 EXON + chr4:105146766-105146786 UCACGUCCGCCCCCUCGGCG 111 54790_2_17 TET2 EXON + chr4:105146783-105146803 GCGCGGCCGCCCCGAGACGC 112 54790_2_24 TET2 EXON + chr4:105146836-105146856 CUGCCUUAUGAAUAUUGAUG 113 54790_2_25 TET2 EXON + chr4:105146839-105146859 CCUUAUGAAUAUUGAUGCGG 114 54790_2_27 TET2 EXON + chr4:105146844-105146864 UGAAUAUUGAUGCGGAGGCU 115 54790_2_34 TET2 EXON + chr4:105146868-105146888 UGCUUUCGUAGAGAGCAGA 116 54790_2_37 TET2 EXON + chr4:105146879-105146899 AGAAGCAGAAGGAAGCAAGA 117 54790_2_39 TET2 EXON + chr4:105146891-105146911 AAGCAAGAUGGCUGCCCUUU 118 54790_2_44 TET2 EXON + chr4:105146905-105146925 CCCUUUAGGAUUUGUUAGAA 119 54790_2_51 TET2 EXON + chr4:105146926-105146946 GGAGACCCGGACUGCAACUGC 120 54790_2_52 TET2 EXON + chr4:105146938-105146958 GCAACUGCUGGAUUGCUGCA 121 54790_2_56 TET2 EXON + chr4:105146944-105146964 GCUGGAUUGCUGCAAGGCUG 122 54790_2_57 TET2 EXON + chr4:105146945-105146965 CUGGAUUGCUGCAAGGCUGA 123 54790_2_62 TET2 EXON + chr4:105146957-105146977 AAGGCUGAGGGACGAGAACG 124 54790_2_64 TET2 EXON - chr4:105146676-105146696 GCUGCCACUGCCGCUACCGC 125 54790_2_65 TET2 EXON - chr4:105146716-105146736 CGCUCGCGAGGAGGCGGCGG 126 54790_2_66 TET2 EXON - chr4:105146719-105146739 CGGCGCUCGCGAGGAGGCGG 127 54790_2_67 TET2 EXON - chr4:105146722-105146742 GCGCGGCGCUCGCGAGGAGG 128 54790_2_68 TET2 EXON - chr4:105146725-105146745 GGCGCGCGGCGCUCGCGAGG 129 54790_2_69 TET2 EXON - chr4:105146728-105146748 CCGGGCGCGCGGCGCUCCGCG 130 54790_2_74 TET2 EXON - chr4:105146739-105146759 GCGAGCGGGACCCGGGCGCG 131 54790_2_75 TET2 EXON - chr4:105146746-105146766 CUUGCAUGCGAGCGGGACCCC 132 54790_2_76 TET2 EXON - chr4:105146747-105146767 ACUUGCAUGCGAGCGGGACC 133 54790_2_78 TET2 EXON - chr4:105146753-105146773 GACGUGACUUGCAUGCGAGC 134 54790_2_79 TET2 EXON - chr4:105146754-105146774 GGACGUGACUUGCAUGCGAG 135 54790_2_83 TET2 EXON - chr4:105146775-105146795 GGGCGGCCCGCGCCGAGGGGG 136 54790_2_85 TET2 EXON - chr4:105146778-105146798 UCGGGGCGGCCCGCGCCGAGG 137 54790_2_86 TET2 EXON - chr4:105146779-105146799 CUCGGGGCGGCCGCGCCGAG 138 54790_2_88 TET2 EXON - chr4:105146780-105146800 UCUCGGGGCGGCCGCGCCGA 139 54790_2_89 TET2 EXON - chr4:105146781-105146801 GUCUCGGGGCGGCCGCGCCG 140 54790_2_93 TET2 EXON - chr4:105146792-105146812 GCGGGGCCGGCGUCUCGGGG 141 54790_2_94 TET2 EXON - chr4:105146795-105146815 UCAGCGGGGCCGGCGUCUCG 142 54790_2_95 TET2 EXON - chr4:105146796-105146816 CUCAGCGGGGCCGGCGUCUC 143 54790_2_97 TET2 EXON - chr4:105146797-105146817 ACUCAGCGGGGCCGGCGUCU 144 54790_2_100 TET2 EXON - chr4:105146805-105146825 UUCUCAUCACUCAGCGGGGC 145 54790_2_101 TET2 EXON - chr4:105146809-105146829 UCUGUUCUCAUCACUCAGCG 146 54790_2_103 TET2 EXON - chr4:105146810-105146830 GUCUGUUCUCAUCACUCAGC 147 54790_2_106 TET2 EXON - chr4:105146811-105146831 CGUCUGUUCUCAUCACUCAG 148 54790_2_109 TET2 EXON - chr4:105146842-105146862 CCUCCGCAUCAAUAUUCAUA 149 54790_2_117 TET2 EXON - chr4:105146908-105146928 CCUUUCUAACAAAUCCUAAA 150 54790_2_118 TET2 EXON - chr4:105146909-105146929 UCCUUUCUAACAAAUCCUAA 151 54790_2_122 TET2 EXON - chr4:105146934-105146954 GCAAUCCAGCAGUUGCAGUC 152 54790_2_123 TET2 EXON - chr4:105146935-105146955 AGCAAUCCAGCAGUUGCAGU 153 54790_3_1 TET2 EXON + chr4:105190341-105190361 AAACUCUGUCUUCUCUAGGC 154 54790_3_13 TET2 EXON + chr4:105190411-105190431 UCCUGUUGAGUUACAACGCU 155 54790_3_16 TET2 EXON + chr4:105190418-105190438 GAGUUACAACGCUUGGAAGC 156 54790_3_19 TET2 EXON + chr4:105190424-105190444 CAACGCUUGGAAGCAGGAGA 157 54790_3_21 TET2 EXON + chr4:105190425-105190445 AACGCUUGGAAGCAGGAGAU 158 54790_3_24 TET2 EXON + chr4:105190444-105190464 UGGGCUCAGCAGCAGCCAAU 159 54790_3_26 TET2 EXON + chr4:105190456-105190476 CAGCCAAUAGGACAUGAUCC 160 54790_3_30 TET2 EXON + chr4:105190469-105190489 AUGAUCCAGGAAGAGCAGUA 161 54790_3_32 TET2 EXON + chr4:105190470-105190490 UGAUCCAGGAAGAGCAGUAA 162 54790_3_34 TET2 EXON + chr4:105190483-105190503 GCAGUAAGGGACUGAGCUGC 163 54790_3_37 TET2 EXON + chr4:105190494-105190514 CUGAGCUGCUGGUAAGACAG 164 54790_3_46 TET2 EXON - chr4:105190385-105190405 GCAAGUAAACAAUCUUGAGA 165 54790_3_47 TET2 EXON - chr4:105190386-105190406 GGCAAGUAAACAAUCUUGAG 166 54790_3_52 TET2 EXON - chr4:105190407-105190427 UUGUAACUCAACAGGAGCAA 167 54790_3_55 TET2 EXON - chr4:105190415-105190435 UCCAAGCGUUGUAACUCAAC 168 54790_3_60 TET2 EXON - chr4:105190462-105190482 CUUCCUGGAUCAUGUCCUAU 169 54790_3_62 TET2 EXON - chr4:105190477-105190497 CAGUCCCUUACUGCUCUUCC 170 54790_4_7 TET2 EXON + chr4:105233887-105233907 GCUCUUUAGAAUUCAACUAG 171 54790_4_8 TET2 EXON + chr4:105233888-105233908 CUCUUUAGAAUUCAACUAGA 172 54790_4_12 TET2 EXON + chr4:105233899-105233919 UCAACUAGAGGGCAGCCUUG 173 54790_4_14 TET2 EXON + chr4:105233903-105233923 CUAGAGGGCAGCCUUGUGGA 174 54790_4_19 TET2 EXON + chr4:105233923-105233943 UGGCCCCGAAGCAAGCCUGA 175 54790_4_21 TET2 EXON + chr4:105233929-105233949 CGAAGCAAGCCUGAUGGAAC 176 54790_4_25 TET2 EXON + chr4:105233950-105233970 GGAUAGAACCAACCAUGUUG 177 54790_4_26 TET2 EXON + chr4:105233951-105233971 GAUGAAACCAACCAUGUUGA 178 54790_4_30 TET2 EXON + chr4:105234010-105234030 CAUUUGCCAGACAGACCUC 179 54790_4_37 TET2 EXON + chr4:105234029-105234049 CUGGCUACAAAGCUCCAGAA 180 54790_4_44 TET2 EXON + chr4:105234068-105234088 AGAGCUCAUCCAGAAGUAAA 181 54790_4_45 TET2 EXON + chr4:105234081-105234101 AAGUAAAUGGAGACACCAAG 182 54790_4_47 TET2 EXON + chr4:105234104-105234124 CACUCUUUCAAAAGUUAUUA 183 54790_4_54 TET2 EXON + chr4:105234121-105234141 UUAUGGAAUACCCUGUAUGA 184 54790_4_57 TET2 EXON + chr4:105234122-105234142 UAUGGAAUACCCUGUAUGAA 185 54790_4_66 TET2 EXON + chr4:105234170-105234190 GACUUUACACAAAGAAAGUAG 186 54790_4_67 TET2 EXON + chr4:105234171-105234191 ACUUUACACAAGAAAGUAGA 187 54790_4_72 TET2 EXON + chr4:105234194-105234214 UAUUCCAAGGUUUGCAAAA 188 54790_4_74 TET2 EXON + chr4:105234197-105234217 UCCAAGUGUUUGCAAAAUGG 189 54790_4_81 TET2 EXON + chr4:105234233-105234253 GUUAGUGAACCUUCUCUCUC 190 54790_4_82 TET2 EXON + chr4:105234234-105234254 UUAGUGAACCUUCUCUCUCU 191 54790_4_89 TET2 EXON + chr4:105234271-105234291 GAAAUUGAAACAAGACCAAA 192 54790_4_93 TET2 EXON + chr4:105234278-105234298 AAACAAGACCAAAAGGCUAA 193 54790_4_97 TET2 EXON + chr4:105234296-105234316 AAUGGAGAAAAGACGUAACUU 194 54790_4_99 TET2 EXON + chr4:105234297-105234317 AUGGAGAAAAGACGUAACUUC 195 54790_4_100 TET2 EXON + chr4:105234298-105234318 UGGAGAAAAGACGUAACUUCG 196 54790_4_106 TET2 EXON + chr4:105234320-105234340 GUAAGCCAAGAAAGAAAUCC 197 54790_4_123 TET2 EXON + chr4:105234437-105234457 UUUUCAACACAUAACUGCAG 198 54790_4_124 TET2 EXON + chr4:105234438-105234458 UUUCAACACAUAACUGCAGU 199 54790_4_134 TET2 EXON + chr4:105234475-105234495 GCUUCAGAUUCUGAAUGAGC 200 54790_4_138 TET2 EXON + chr4:105234478-105234498 UCAGAUUCUGAAUGAGCAGG 201 54790_4_140 TET2 EXON + chr4:105234479-105234499 CAGAUUCUGAAUGAGCAGGA 202 54790_4_141 TET2 EXON + chr4:105234480-105234500 AGAUUCUGAAUGAGCAGGAG 203 54790_4_147 TET2 EXON + chr4:105234529-105234549 CAUUGUAUUACUUAAAAACA 204 54790_4_151 TET2 EXON + chr4:105234548-105234568 AAGGCAGUGCUAAUGCCUAA 205 54790_4_153 TET2 EXON + chr4:105234574-105234594 UACAGUUUCCUGCCUCUUCCG 206 54790_4_157 TET2 EXON + chr4:105234587-105234607 UCUUCCCGUGGAACACACCACA 207 54790_4_161 TET2 EXON + chr4:105234598-105234618 ACACACACAUGGUGAACCUCC 208 54790_4_163 TET2 EXON + chr4:105234643-105234663 UCCAGAUUGUGUUUCCAUUG 209 54790_4_171 TET2 EXON + chr4:105234685-105234705 CAUAAAUGCCAUUAACAGUC 210 54790_4_177 TET2 EXON + chr4:105234734-105234754 ACUCACCCAUCGCAUACCUC 211 54790_4_178 TET2 EXON + chr4:105234735-105234755 CUCACCCAUCGCAUACCUCA 212 54790_4_181 TET2 EXON + chr4:105234793-105234813 GCCUCCAAAGCCAGCUGCAG 213 54790_4_184 TET2 EXON + chr4:105234802-105234822 GCCAGCUGCAGUGGUGAGUG 214 54790_4_200 TET2 EXON + chr4:105234943-105234963 UCCUGCAGAAAAUAACAUCC 215 54790_4_201 TET2 EXON + chr4:105234944-105234964 CCUGCAGAAAAUAACAUCCA 216 54790_4_203 TET2 EXON + chr4:105234965-105234985 GGAACCACAAAAGCUAGCGUC 217 54790_4_207 TET2 EXON + chr4:105234983-105235003 UCUGGUGAAGAAUUCUGUUC 218 54790_4_211 TET2 EXON + chr4:105235010-105235030 AGCAGCAAUUUGCAAGCUCC 219 54790_4_212 TET2 EXON + chr4:105235013-105235033 AGCAAUUUGCAAGCUCCUGG 220 54790_4_216 TET2 EXON + chr4:105235026-105235046 CUCCUGGUGGCAGCUCUGAA 221 54790_4_219 TET2 EXON + chr4:105235052-105235072 UUAAAAACAAAAUGAAAUGAA 222 54790_4_225 TET2 EXON + chr4:105235087-105235107 GCAAAGCUCAGUGUUCACUA 223 54790_4_235 TET2 EXON + chr4:105235162-105235182 UCCCCCUCCUCCUCUUCCAC 224 54790_4_240 TET2 EXON + chr4:105235184-105235204 GUUCCUCAGACUUCCUUCAGA 225 54790_4_245 TET2 EXON + chr4:105235202-105235222 GAAGGAAAAAAGCACUCUGAA 226 54790_4_247 TET2 EXON + chr4:105235205-105235225 GGAAAAAAGCACUCUGAAUGG 227 54790_4_256 TET2 EXON + chr4:105235260-105235280 AAAGUAACACAACACUUUUA 228 54790_4_258 TET2 EXON + chr4:105235261-105235281 AAGUAACACAACACUUUUAA 229 54790_4_262 TET2 EXON + chr4:105235276-105235296 UUUAAGGGAAGUGAAAAUAG 230 54790_4_263 TET2 EXON + chr4:105235277-105235297 UUAAGGGAAGUGAAAAUGA 231 54790_4_268 TET2 EXON + chr4:105235288-105235308 GAAAAUAGAGGGUAAACCUG 232 54790_4_272 TET2 EXON + chr4:105235356-105235376 CUUCUCCGAUGCUUUCUGAA 233 54790_4_280 TET2 EXON + chr4:105235380-105235400 CUCAGAAUAAUUGUGUGAAC 234 54790_4_284 TET2 EXON + chr4:105235400-105235420 AGGAAUGACAUACAGACUGC 235 54790_4_286 TET2 EXON + chr4:105235401-105235421 GGAAUGACAUACAGACUGCA 236 54790_4_294 TET2 EXON + chr4:105235478-105235498 AAGCAUAACCCACCAAUUUU 237 54790_4_297 TET2 EXON + chr4:105235487-105235507 CCACCAAUUUUUGGUAGCAG 238 54790_4_302 TET2 EXON + chr4:105235498-105235518 UGGUAGCAGUGGAGAGCUAC 239 54790_4_313 TET2 EXON + chr4:105235546-105235566 CAAAGAGCAAGAGAUUCUGA 240 54790_4_314 TET2 EXON + chr4:105235547-105235567 AAAGAGCAAGAGAUUCUGAA 241 54790_4_317 TET2 EXON + chr4:105235558-105235578 GAUUCUGAAGGGUCGAGACA 242 54790_4_324 TET2 EXON + chr4:105235607-105235627 ACACAGCACUAUCUGAAACC 243 54790_4_326 TET2 EXON + chr4:105235611-105235631 AGCACUAUCUGAAACCAGGA 244 54790_4_329 TET2 EXON + chr4:105235624-105235644 ACCAGGAUGGAUUGAAUUGA 245 54790_4_333 TET2 EXON + chr4:105235645-105235665 GGCCCCUCGUUUUCACCAAG 246 54790_4_339 TET2 EXON + chr4:105235669-105235689 AUCCCAUCUAAAACGUAAUG 247 54790_4_343 TET2 EXON + chr4:105235739-105235759 AUGACCUCCAAACAAUACAC 248 54790_4_347 TET2 EXON + chr4:105235757-105235777 ACUGGAAAUUCCAACAUGCC 249 54790_4_349 TET2 EXON + chr4:105235758-105235778 CUGGAAAUUCCAACAUGCCU 250 54790_4_351 TET2 EXON + chr4:105235759-105235779 UGGAAAUUCCAACAUGCCUG 251 54790_4_352 TET2 EXON + chr4:105235760-105235780 GGAAAUUCCAACAUGCCUGG 252 54790_4_353 TET2 EXON + chr4:105235761-105235781 GAAAUUCCAACAUGCCUGGG 253 54790_4_355 TET2 EXON + chr4:105235770-105235790 ACAUGCCUGGGGGGCUCCCA 254 54790_4_360 TET2 EXON + chr4:105235801-105235821 CACCCAGAAAACAACACAGC 255 54790_4_365 TET2 EXON + chr4:105235841-105235861 UACCAAGUUGAAAUGAAUCA 256 54790_4_366 TET2 EXON + chr4:105235842-105235862 ACCAGUUGAAAUGAAUCAA 257 54790_4_368 TET2 EXON + chr4:105235853-105235873 AUGAAUCAAGGGCAGUCCCA 258 54790_4_370 TET2 EXON + chr4:105235861-105235881 AGGGCAGUCCCAAGGUACAG 259 54790_4_371 TET2 EXON + chr4:105235897-105235917 GUUCCAAAAACCCUCACACC 260 54790_4_376 TET2 EXON + chr4:105235952-105235972 GCUCAUGUGCAGUCACUGUG 261 54790_4_388 TET2 EXON + chr4:105236038-105236058 GAAACAGCACUUGAAUCAAC 262 54790_4_399 TET2 EXON + chr4:105236098-105236118 GCAACAUAAGCCUCAUAAAC 263 54790_4_407 TET2 EXON + chr4:105236182-105236202 AUUACAAAUAAAGAAUAAAG 264 54790_4_416 TET2 EXON + chr4:105236237-105236257 AACAAUGAUCAGCAAAGAGA 265 54790_4_417 TET2 EXON + chr4:105236249-105236269 CAAAGAGAAGGAUCAUUCUU 266 54790_4_419 TET2 EXON + chr4:105236263-105236283 AUUCUUUGGCCAGACUAAAG 267 54790_4_426 TET2 EXON + chr4:105236279-105236299 AAAGUGGAAGAAUGUUUUCA 268 54790_4_435 TET2 EXON + chr4:105236332-105236352 CGAGACUCAUAAUGUCCCAAA 269 54790_4_438 TET2 EXON + chr4:105236333-105236353 GAGACUCAUAAUGUCCAAAU 270 54790_4_440 TET2 EXON + chr4:105236338-105236358 UCAUAAUGUCCAAAUGGGAC 271 54790_4_444 TET2 EXON + chr4:105236341-105236361 UAAUGUCCAAAUGGGACUGG 272 54790_4_452 TET2 EXON + chr4:105236413-105236433 AUCAAGUGCAUGCAAAAUAC 273 54790_4_466 TET2 EXON + chr4:105236486-105236506 ACACAUCCUGAACUUUUUGC 274 54790_4_475 TET2 EXON + chr4:105236562-105236582 CAAAGCAAGAUCUUCUUCAC 275 54790_4_479 TET2 EXON + chr4:105236578-105236598 UCACAGGUGCUUUCAAGAAC 276 54790_4_486 TET2 EXON + chr4:105236611-105236631 ACAACAAGCUUCAGUUCUAC 277 54790_4_488 TET2 EXON + chr4:105236612-105236632 CAACAAGCUUCAGUUCUACA 278 54790_4_493 TET2 EXON + chr4:105236642-105236662 AAUGAAAACCAAGAUAUGUC 279 54790_4_494 TET2 EXON + chr4:105236673-105236693 CUGCGCAACUUGCUCAGCAA 280 54790_4_498 TET2 EXON + chr4:105236719-105236739 UGUUUUUCCUGUGCCUGACC 281 54790_4_501 TET2 EXON + chr4:105236720-105236740 GUUUUUCCUGUGCCUGACCA 282 54790_4_503 TET2 EXON + chr4:105236723-105236743 UUUCCUGUGCCUGACCAGGG 283 54790_4_511 TET2 EXON + chr4:105236752-105236772 CACUCAGACCCCUCCCCAGA 284 54790_4_512 TET2 EXON + chr4:105236778-105236798 CUCAAAAGCAUGCUGCUCUA 285 54790_4_513 TET2 EXON + chr4:105236781-105236801 AAAAGCAUGCUGCUCUAAGG 286 54790_4_518 TET2 EXON + chr4:105236856-105236876 CUUGCCAUAGUCAGAUGCAC 287 54790_4_520 TET2 EXON + chr4:105236866-105236886 UCAGAUGCACAGGCCAAUUA 288 54790_4_522 TET2 EXON + chr4:105236869-105236889 GAUGCACAGGCCAAUUAAGG 289 54790_4_525 TET2 EXON + chr4:105236876-105236896 AGGCCAAUUAAGGUGGAACC 290 54790_4_531 TET2 EXON + chr4:105236928-105236948 CACCACCAGAAAACAAAACA 291 54790_4_532 TET2 EXON + chr4:105236935-105236955 AGAAAACAAAACAUGGAAAAA 292 54790_4_540 TET2 EXON + chr4:105237004-105237024 AAAGAGCAUCAUUGAGACCA 293 54790_4_545 TET2 EXON + chr4:105237052-105237072 CAAGUCGUUAUUUGACCUA 294 54790_4_553 TET2 EXON + chr4:105237098-105237118 CAAGUAAAAGUUGAAAUGUC 295 54790_4_554 TET2 EXON + chr4:105237099-105237119 AAGUAAAAGUUGAAAUGUCA 296 54790_4_578 TET2 EXON + chr4:105237280-105237300 UACUCCUAUAAAAAAUUUAU 297 54790_4_582 TET2 EXON + chr4:105237329-105237349 UUCCCAUCUUGCAGAUGUGU 298 54790_4_589 TET2 EXON + chr4:105237359-105237379 CAGAAAUGUACUGAGACACA 299 54790_4_596 TET2 EXON + chr4:105237397-105237417 AGCAAUUUAUCUUCAGAUA 300 54790_4_597 TET2 EXON + chr4:105237398-105237418 GCAAAUUUAUCUUCAGAUAU 301 54790_4_606 TET2 EXON + chr4:105237430-105237450 CUUUUUUUAAAUCUUGAGUC 302 54790_4_614 TET2 EXON + chr4:105237446-105237466 AGUCUGGCAGCAAUUUGUAA 303 54790_4_657 TET2 EXON + chr4:105237650-105237670 GCUCUUUGUAUAUUAUCUCC 304 54790_4_662 TET2 EXON + chr4:105237663-105237683 UAUCUCCUGGAGAGACAGCU 305 54790_4_668 TET2 EXON + chr4:105237708-105237728 AAUGAGAAAAUAAACGACCAU 306 54790_4_670 TET2 EXON + chr4:105237748-105237768 UUUAAAUAUUUUUUAAUUCA 307 54790_4_679 TET2 EXON + chr4:105237778-105237798 UAUUAGUUUCACAAGAUUUC 308 54790_4_682 TET2 EXON + chr4:105237786-105237806 UCACAAGAUUUCUGGCUAAU 309 54790_4_686 TET2 EXON + chr4:105237787-105237807 CACAAGAUUUCUGGCUAAUA 310 54790_4_693 TET2 EXON + chr4:105237817-105237837 UAUCUUCAGUCUUCAUGAGU 311 54790_4_695 TET2 EXON + chr4:105237818-105237838 AUCUUCAGUCUUCAUGAGUU 312 54790_4_697 TET2 EXON + chr4:105237819-105237839 UCUUCAGUCUUCAUGAGUUG 313 54790_4_700 TET2 EXON + chr4:105237820-105237840 CUUCAGCUUCAUGAGUUGG 314 54790_4_709 TET2 EXON + chr4:105237882-105237902 CUUUUCUCCAUUUAUACAUU 315 54790_4_741 TET2 EXON + chr4:105240332-105240352 AAAGCUUUUUGUUAAAAUUC 316 54790_4_746 TET2 EXON + chr4:105240344-105240364 UAAAAUUCAGGAUAUGUAAU 317 54790_4_750 TET2 EXON + chr4:105240352-105240372 AGGAUAUGUAAUAGGUCUGU 318 54790_4_754 TET2 EXON + chr4:105240377-105240397 UAGUGAAAUAUUUUUGCUGA 319 54790_4_760 TET2 EXON + chr4:105240395-105240415 GAUGGAUGUAGAUAUAUACG 320 54790_4_770 TET2 EXON + chr4:105240478-105240498 AGACAAAUGUUAAAUUAGUG 321 54790_4_780 TET2 EXON + chr4:105240541-105240561 GAUACCCCACACUGUGUAG 322 54790_4_783 TET2 EXON + chr4:105240545-105240565 CCCCACACUGUGUAGGAAGGA 323 54790_4_785 TET2 EXON + chr4:105240548-105240568 CACACUGUGUAGAAAGGAUGG 324 54790_4_787 TET2 EXON + chr4:105240549-105240569 ACACUGUGUAGAAAGGAUGGA 325 54790_4_790 TET2 EXON + chr4:105240552-105240572 CUGUGUAGAGGAUGGAGGG 326 54790_4_791 TET2 EXON + chr4:105240579-105240599 CUACUGUCCCUCUUUGCGUG 327 54790_4_795 TET2 EXON + chr4:105240599-105240619 UGGUUAUUAAGUUGCCUCAC 328 54790_4_796 TET2 EXON + chr4:105240600-105240620 GGUUAUUAAGUUGCCUCACU 329 54790_4_800 TET2 EXON + chr4:105240634-105240654 CACAUCUCAUAGAUAAUAUU 330 54790_4_807 TET2 EXON + chr4:105240703-105240723 UCCCACUUUUCCAUCUUGU 331 54790_4_818 TET2 EXON + chr4:105240740-105240760 UUCUUUUUGCCUGACUCUCC 332 54790_4_829 TET2 EXON + chr4:105240784-105240804 UUCUAAAGUACAUACUAAUA 333 54790_4_830 TET2 EXON + chr4:105240785-105240805 UCUAAAGUACAUACUAAUAU 334 54790_4_833 TET2 EXON + chr4:105240790-105240810 AGUACAUACUAAUAUGGGUC 335 54790_4_841 TET2 EXON + chr4:105240833-105240853 AAACAGCAAUUAAAUGUUAU 336 54790_4_842 TET2 EXON + chr4:105240834-105240854 AACAGCAAUUAAAUGUUAUA 337 54790_4_845 TET2 EXON + chr4:105240841-105240861 AUUAAAUGUUAUAGGGAAGU 338 54790_4_851 TET2 EXON + chr4:105240851-105240871 AUAGGGAAGUAAGGAAGAAAA 339 54790_4_853 TET2 EXON + chr4:105240852-105240872 UAGGGAAGUAAGGAAGAAAAA 340 54790_4_855 TET2 EXON + chr4:105240853-105240873 AGGGAAGUAAGGAAGAAAAAG 341 54790_4_858 TET2 EXON + chr4:105240885-105240905 CAAUAAACCAAGCAAUAUUC 342 54790_4_861 TET2 EXON + chr4:105240886-105240906 AAUAAACCAAGCAAUAUUCU 343 54790_4_862 TET2 EXON + chr4:105240887-105240907 AUAAACCAAGCAAUAUUCUG 344 54790_4_863 TET2 EXON + chr4:105240888-105240908 UAAACCAAGCAAUAUUCUGG 345 54790_4_865 TET2 EXON + chr4:105240891-105240911 ACCAAGCAAUAUUCUGGGGG 346 54790_4_867 TET2 EXON + chr4:105240892-105240912 CCAAGCAAUAUUCUGGGGGU 347 54790_4_870 TET2 EXON + chr4:105240902-105240922 UUCUGGGGGUGGGAUAGAGC 348 54790_4_880 TET2 EXON + chr4:105240940-105240960 UCUUUUAAAAUCCAAGUAAU 349 54790_4_881 TET2 EXON + chr4:105240944-105240964 UUAAAAUUCCAAGUAAUAGGU 350 54790_4_891 TET2 EXON + chr4:105240991-105241011 UUUUUUCCAGCUCAAAAAAU 351 54790_4_905 TET2 EXON + chr4:105241063-105241083 UUUGUUUAGUUUCAUUUAUU 352 54790_4_929 TET2 EXON + chr4:105241146-105241166 UGUACAUAUACUUAAUUAUG 353 54790_4_945 TET2 EXON + chr4:105241237-105241257 UAGAGCCCUUAAUGUGUAGGU 354 54790_4_949 TET2 EXON + chr4:105241238-105241258 AGAGCCCUUAAUGUGUAGUU 355 54790_4_951 TET2 EXON + chr4:105241239-105241259 GAGCCCUUAAUGUGUAGUUG 356 54790_4_953 TET2 EXON + chr4:105241240-105241260 AGCCCUUAAUGUGUAGUUGG 357 54790_4_956 TET2 EXON + chr4:105241253-105241273 UAGUUGGGGGUUAAGCUUUG 358 54790_4_962 TET2 EXON + chr4:105241283-105241303 CUUUAUAUUUAGUAUAAUUG 359 54790_4_973 TET2 EXON + chr4:105241340-105241360 CAAAUUAUUGAAAAAAGAUGA 360 54790_4_977 TET2 EXON + chr4:105241361-105241381 GGUCCUUUUUAUACCCAUCU 361 54790_4_979 TET2 EXON + chr4:105241367-105241387 UUUUAAUACCCAUCUAGGAGC 362 54790_4_984 TET2 EXON + chr4:105241378-105241398 UCUAGGAGCAGGUCCUAAUG 363 54790_4_990 TET2 EXON + chr4:105241399-105241419 GGCAGCUAUUAGAGAAAUCA 364 54790_4_993 TET2 EXON + chr4:105241407-105241427 UUAGAGAAAUCAUGGAAGAA 365 54790_4_995 TET2 EXON + chr4:105241422-105241442 AAGAAAGGUAAUUAACGCAA 366 54790_4_997 TET2 EXON + chr4:105241428-105241448 GGUAAUUAACGCAAAGGCAC 367 54790_4_998 TET2 EXON + chr4:105241429-105241449 GUAAUUAACGCAAAGGCACA 368 54790_4_1014 TET2 EXON + chr4:105241523-105241543 UAAAUUGAGUAAUUAUUAGU 369 54790_4_1019 TET2 EXON + chr4:105241538-105241558 UUAGUAGGCUUAGCUAUUCU 370 54790_4_1020 TET2 EXON + chr4:105241539-105241559 UAGUAGGCUUAGCUAUUCUA 371 54790_4_1029 TET2 EXON + chr4:105241592-105241612 AGAGAGUCACAAUAUUUGAC 372 54790_4_1032 TET2 EXON + chr4:105241612-105241632 AGGACUAAUAGUCUGCUAGC 373 54790_4_1033 TET2 EXON + chr4:105241618-105241638 AAUAGUCUGCUAGCUGGCAC 374 54790_4_1035 TET2 EXON + chr4:105241636-105241656 ACAGGCUGCCCACUUUGCGA 375 54790_4_1040 TET2 EXON + chr4:105241653-105241673 CGAUGGAUGCCAGAAAACCC 376 54790_4_1043 TET2 EXON + chr4:105241663-105241683 CAGAAAACCCAGGCAUGAAC 377 54790_4_1045 TET2 EXON + chr4:105241669-105241689 ACCCAGGCAUGAACAGGAAU 378 54790_4_1046 TET2 EXON + chr4:105241678-105241698 UGAACAGGAAUCGGCCAGCC 379 54790_4_1047 TET2 EXON + chr4:105241693-105241713 CAGCCAGGCUGCCAGCCACA 380 54790_4_1048 TET2 EXON + chr4:105241699-105241719 GGCUGCCAGCCACAAGGUAC 381 54790_4_1049 TET2 EXON + chr4:105241705-105241725 CAGCCACAAGGUACUGGCAC 382 54790_4_1052 TET2 EXON + chr4:105241718-105241738 CUGGCACAGGCUCCAACGAG 383 54790_4_1053 TET2 EXON + chr4:105241729-105241749 UCCAACGAAGGUCCCACUC 384 54790_4_1058 TET2 EXON + chr4:105241770-105241790 AAGUGUCAAAGCAGAAAGAC 385 54790_4_1059 TET2 EXON + chr4:105241780-105241800 GCAGAAAGACUGGUAAAGUG 386 54790_4_1092 TET2 EXON + chr4:105241946-105241966 UUUUUUUCGCUAUCAAUCAC 387 54790_4_1109 TET2 EXON + chr4:105242012-105242032 UGAGCGAGAUAAUGCAGAGA 388 54790_4_1117 TET2 EXON + chr4:105242057-105242077 CUCUGAGCUGUUCUUCUUCU 389 54790_4_1118 TET2 EXON + chr4:105242058-105242078 UCUGAGCUGUUCUUCUUCUA 390 54790_4_1123 TET2 EXON + chr4:105242076-105242096 UAGGGUGCCUUUUCAUUAAG 391 54790_4_1124 TET2 EXON + chr4:105242080-105242100 GUGCCUUUUCAUUAAGAGGU 392 54790_4_1130 TET2 EXON + chr4:105242105-105242125 GUAUUAUUAUUAAAGUACUU 393 54790_4_1135 TET2 EXON + chr4:105242114-105242134 UUAAAGUACUUAGGAUACAU 394 54790_4_1136 TET2 EXON + chr4:105242115-105242135 UAAAGUACUUAGGAUACAUU 395 54790_4_1137 TET2 EXON + chr4:105242116-105242136 AAAGUACUUAGGAUACAUUG 396 54790_4_1140 TET2 EXON + chr4:105242124-105242144 UAGGAUACAUUGGGGCAGCU 397 54790_4_1154 TET2 EXON + chr4:105242210-105242230 UUCACUAAAAUAAUCAUCUAG 398 54790_4_1156 TET2 EXON + chr4:105242215-105242235 UAAAUAAUCAUCUAGUGGCC 399 54790_4_1162 TET2 EXON + chr4:105242287-105242307 UUGUUUUUUAAACAAGCAGU 400 54790_4_1163 TET2 EXON + chr4:105242290-105242310 UUUUUUAAACAAGCAGUAGG 401 54790_4_1164 TET2 EXON + chr4:105242298-105242318 ACAAGCAGUAGGUGGUGCUU 402 54790_4_1167 TET2 EXON + chr4:105242306-105242326 UAGGUGGUGCUUUGGUCAUA 403 54790_4_1169 TET2 EXON + chr4:105242307-105242327 AGGUGGUGCUUUGGUCAUAAA 404 54790_4_1173 TET2 EXON + chr4:105242328-105242348 GGAAGAUAUAGCUAUUUCU 405 54790_4_1176 TET2 EXON + chr4:105242351-105242371 ACUAUUCCAUAUUUUCCAUG 406 54790_4_1178 TET2 EXON + chr4:105242355-105242375 UUCCAAUAUUUUCCAUGUGGC 407 54790_4_1187 TET2 EXON + chr4:105242404-105242424 UCUAAAUUGUGAGACAUUCU 408 54790_4_1193 TET2 EXON + chr4:105242407-105242427 AAAUUGUGAGACAUUCUUGG 409 54790_4_1201 TET2 EXON + chr4:105242469-105242489 UAAAAUAGCUAAAUUUAGUA 410 54790_4_1205 TET2 EXON + chr4:105242470-105242490 AAAAUAGCUAAAUUUAGUAA 411 54790_4_1241 TET2 EXON + chr4:105242625-105242645 AUCUGUACAUUUUGAUAUUG 412 54790_4_1244 TET2 EXON + chr4:105242635-105242655 UUUGAUAUUGAGGAAAAACA 413 54790_4_1250 TET2 EXON + chr4:105242663-105242683 AAACCAUUAUCCAGUUUGCU 414 54790_4_1258 TET2 EXON + chr4:105242705-105242725 UAAUAAACCGUUCAUUUCUC 415 54790_4_1259 TET2 EXON + chr4:105242711-105242731 ACCGUUCAUUUCUCAGGAUG 416 54790_4_1269 TET2 EXON - chr4:105233886-105233906 UAGUUGAAUUCUAAAAGAGCA 417 54790_4_1276 TET2 EXON - chr4:105233917-105233937 UUGCUUCGGGGCCAUCCACA 418 54790_4_1278 TET2 EXON - chr4:105233929-105233949 GUUCCAUCAGGCUUGCUUCG 419 54790_4_1279 TET2 EXON - chr4:105233930-105233950 UGUUCCAUCAGGCUUGCUUC 420 54790_4_1281 TET2 EXON - chr4:105233931-105233951 CUGUUCCAUCAGGCUUGCUU 421 54790_4_1285 TET2 EXON - chr4:105233941-105233961 UGGUUCUAUCCUGUUCCAUC 422 54790_4_1288 TET2 EXON - chr4:105233961-105233981 UCUGUUGCCCUCAACAUGGU 423 54790_4_1289 TET2 EXON - chr4:105233965-105233985 UUAGUCUGUUGCCCUCAACA 424 54790_4_1290 TET2 EXON - chr4:105233990-105234010 GGAGGUGAUGGUAUUCAGGAA 425 54790_4_1293 TET2 EXON - chr4:105233995-105234015 AAAUGGGAGGUGAUGGUAUUC 426 54790_4_1296 TET2 EXON - chr4:105234002-105234022 GUCUGGCAAAUGGGAGGUGA 427 54790_4_1297 TET2 EXON - chr4:105234008-105234028 GGUUCUGUCUGGCAAAUGGG 428 54790_4_1298 TET2 EXON - chr4:105234011-105234031 AGAGGUUCUGUCUGGCAAAU 429 54790_4_1300 TET2 EXON - chr4:105234012-105234032 CAGAGGUUCUGUCUGGCAAA 430 54790_4_1305 TET2 EXON - chr4:105234019-105234039 UUGUAGCCAGAGGUUCUGUC 431 54790_4_1308 TET2 EXON - chr4:105234029-105234049 UUCUGGAGCUUUGUAGCCAG 432 54790_4_1310 TET2 EXON - chr4:105234046-105234066 CAGGCAGUGGGCUUCCAUUC 433 54790_4_1314 TET2 EXON - chr4:105234058-105234078 GAUGAGCUCUCUCAGGCAGU 434 54790_4_1315 TET2 EXON - chr4:105234059-105234079 GGAUGAGCUCUCUCAGGCAG 435 54790_4_1319 TET2 EXON - chr4:105234065-105234085 ACUUCUGGAUGAGCUCUCUC 436 54790_4_1322 TET2 EXON - chr4:105234080-105234100 UUGGUGUCUCCAUUUACUUC 437 54790_4_1327 TET2 EXON - chr4:105234099-105234119 ACUUUUGAAAGAGUGCCACU 438 54790_4_1334 TET2 EXON - chr4:105234134-105234154 UUCUGGCUUCCCUUCAUACA 439 54790_4_1335 TET2 EXON - chr4:105234135-105234155 AUUCUGGCUUCCCUUCAUAC 440 54790_4_1337 TET2 EXON - chr4:105234151-105234171 CAGGACUCAACACGACUAUUC 441 54790_4_1341 TET2 EXON - chr4:105234170-105234190 CUACUUUCUUGUGUAAAGUC 442 54790_4_1351 TET2 EXON - chr4:105234201-105234221 UCCUCCAUUUUGCAAACACU 443 54790_4_1355 TET2 EXON - chr4:105234245-105234265 UGAAGGAGCCCAGAGAGAGA 444 54790_4_1367 TET2 EXON - chr4:105234262-105234282 GUUUCAAUUUCUUGAUCUGA 445 54790_4_1378 TET2 EXON - chr4:105234289-105234309 GUCUUUCUCCAUUAGCCUUU 446 54790_4_1388 TET2 EXON - chr4:105234328-105234348 UUUCACCUGGAUUUCUUUCU 447 54790_4_1392 TET2 EXON - chr4:105234341-105234361 UUUGGUUGACUGCUUUCACCC 448 54790_4_1396 TET2 EXON - chr4:105234359-105234379 UCACUCAAAUCGGAGACAUU 449 54790_4_1399 TET2 EXON - chr4:105234369-105234389 UUCUUUCUUAUCACUCAAAU 450 54790_4_1410 TET2 EXON - chr4:105234408-105234428 AUCUUUAACUGCAUUUUCUU 451 54790_4_1411 TET2 EXON - chr4:105234409-105234429 AAUCUUUAACUGCAUUUUCU 452 54790_4_1416 TET2 EXON - chr4:105234435-105234455 GCAGUUAUGUGUUGAAAAAAC 453 54790_4_1422 TET2 EXON - chr4:105234464-105234484 AUCUGAAGCUCUGGAUUUUC 454 54790_4_1423 TET2 EXON - chr4:105234473-105234493 UCAUUCAGAAUCUGAAGCUC 455 54790_4_1435 TET2 EXON - chr4:105234520-105234540 GUAAUACAAUGUUCUUGUCA 456 54790_4_1441 TET2 EXON - chr4:105234566-105234586 GCAGAAACUGUAGCACCAUU 457 54790_4_1444 TET2 EXON - chr4:105234588-105234608 AUGUGUGUGUUCCACGGAAG 458 54790_4_1448 TET2 EXON - chr4:105234594-105234614 UUCACCAUGUGUGUGUUCCA 459 54790_4_1457 TET2 EXON - chr4:105234619-105234639 AUUGAGACAGUGUUUUUUCC 460 54790_4_1461 TET2 EXON - chr4:105234647-105234667 ACCGCAAUGGAAACACAAUUC 461 54790_4_1466 TET2 EXON - chr4:105234660-105234680 UGUGGUUUUCUGCACCGCAA 462 54790_4_1471 TET2 EXON - chr4:105234678-105234698 AAUGGCAUUUAUGUGAGAUG 463 54790_4_1474 TET2 EXON - chr4:105234696-105234716 AUUAGUAGCCUGACUGUUAA 464 54790_4_1475 TET2 EXON - chr4:105234726-105234746 CGAUGGGUGAGUGAUCUCAC 465 54790_4_1479 TET2 EXON - chr4:105234742-105234762 UCUGCCCUGAGGUAUGCGAU 466 54790_4_1480 TET2 EXON - chr4:105234743-105234763 AUCUGCCCUGAGGUAUGCGA 467 54790_4_1482 TET2 EXON - chr4:105234753-105234773 UGCGGAAUUGAUCUGCCCUG 468 54790_4_1485 TET2 EXON - chr4:105234771-105234791 CUCAGAGUUAGAGGUCUGUG 469 54790_4_1490 TET2 EXON - chr4:105234780-105234800 UGGAGGCAGCUCAGAGUUAG 470 54790_4_1493 TET2 EXON - chr4:105234797-105234817 ACCACUGCAGCUGGCUUUGG 471 54790_4_1495 TET2 EXON - chr4:105234800-105234820 CUCACCACUGCAGCUGGCUU 472 54790_4_1497 TET2 EXON - chr4:105234806-105234826 GCCUCACUCACCACUGCAGC 473 54790_4_1499 TET2 EXON - chr4:105234828-105234848 AUCAGCAUCAUCAGCAUCAC 474 54790_4_1505 TET2 EXON - chr4:105234855-105234875 UAGCAUUGCAGCUAGUUUAC 475 54790_4_1510 TET2 EXON - chr4:105234882-105234902 UUCUGGUUUCUGAAAGGAAC 476 54790_4_1514 TET2 EXON - chr4:105234888-105234908 UAGUUGUUCUGGUUUCUGAA 477 54790_4_1521 TET2 EXON - chr4:105234899-105234919 UUUUGUUGUUGUAGUUGUUC 478 54790_4_1526 TET2 EXON - chr4:105234940-105234960 UGUUAUUUUCUGCAGGAGAU 479 54790_4_1527 TET2 EXON - chr4:105234941-105234961 AUGUUAUUUUCUGCAGGAGA 480 54790_4_1531 TET2 EXON - chr4:105234947-105234967 CCCUGGAUGUUAUUUUCUGC 481 54790_4_1535 TET2 EXON - chr4:105234964-105234984 ACGCUAGCUUUGUGGUUCCC 482 54790_4_1539 TET2 EXON - chr4:105234972-105234992 UUCACCAGACGCUAGCUUUG 483 54790_4_1545 TET2 EXON - chr4:105235011-105235031 AGGAGCUUGCAAAUUGCUGC 484 54790_4_1551 TET2 EXON - chr4:105235031-105235051 UACCGUUCAGAGCUGCCACC 485 54790_4_1569 TET2 EXON - chr4:105235116-105235136 ACCACACCAUCACCCAGAAA 486 54790_4_1577 TET2 EXON - chr4:105235166-105235186 ACCUGUGGAAGAGGAGGAGG 487 54790_4_1579 TET2 EXON - chr4:105235167-105235187 AACCUGUGGAAGAGGAGGAG 488 54790_4_1581 TET2 EXON - chr4:105235168-105235188 GAACCUGUGGAAGAGGAGGA 489 54790_4_1582 TET2 EXON - chr4:105235169-105235189 GGAACCUGUGGAAGAGGAGG 490 54790_4_1586 TET2 EXON - chr4:105235172-105235192 UGAGGAACCUGUGGAAGAGG 491 54790_4_1588 TET2 EXON - chr4:105235175-105235195 AGCUGAGGAACCUGUGGAAG 492 54790_4_1593 TET2 EXON - chr4:105235181-105235201 GAAGGAAGCUGAGGAACCUG 493 54790_4_1600 TET2 EXON - chr4:105235190-105235210 UUUCCUUCUGAAGGAAGCUG 494 54790_4_1606 TET2 EXON - chr4:105235199-105235219 AGAGUGCUUUUUCCUUCUGA 495 54790_4_1617 TET2 EXON - chr4:105235246-105235266 UACUUUGGUUGGGGUAGUGG 496 54790_4_1618 TET2 EXON - chr4:105235249-105235269 UGUUACUUUGGUUGGGGUAG 497 54790_4_1620 TET2 EXON - chr4:105235255-105235275 GUGUUGUGUUACUUUGGUUG 498 54790_4_1621 TET2 EXON - chr4:105235256-105235276 AGUGUUGUGUUACUUUGGUU 499 54790_4_1623 TET2 EXON - chr4:105235257-105235277 AAGUGUUGUGUUACUUUGGU 500 54790_4_1626 TET2 EXON - chr4:105235261-105235281 UUAAAAGGUGUUGUGUUACUU 501 54790_4_1633 TET2 EXON - chr4:105235307-105235327 CUCUGGGAAGGUGGUGCCUC 502 54790_4_1634 TET2 EXON - chr4:105235316-105235336 GGAUUAGGACUCUGGGAAGG 503 54790_4_1635 TET2 EXON - chr4:105235319-105235339 GAUGGAUUAGGACUCUGGGA 504 54790_4_1636 TET2 EXON - chr4:105235323-105235343 UGUAGAUGGAUUAGGACUCU 505 54790_4_1638 TET2 EXON - chr4:105235324-105235344 GUGUAGAUGGAUUAGGACUC 506 54790_4_1641 TET2 EXON - chr4:105235331-105235351 CAUACAUGUGUAGAUGGAUU 507 54790_4_1643 TET2 EXON - chr4:105235337-105235357 GGGCUGCAUACAUGUGUAG 508 54790_4_1647 TET2 EXON - chr4:105235357-105235377 UUUCAGAAAGCAUCGGAGAA 509 54790_4_1648 TET2 EXON - chr4:105235358-105235378 CUUUCAGAAAGCAUCGGAGA 510 54790_4_1653 TET2 EXON - chr4:105235364-105235384 UGAGGCCUUUCAGAAAGCAU 511 54790_4_1660 TET2 EXON - chr4:105235382-105235402 CUGUUCACACAAUUAUUCUG 512 54790_4_1668 TET2 EXON - chr4:105235439-105235459 CUUGUUUUCUCAGAACACAA 513 54790_4_1676 TET2 EXON - chr4:105235463-105235483 UGCUUGAGGUGUUCUGACAU 514 54790_4_1678 TET2 EXON - chr4:105235477-105235497 AAAUUGGUGGGUUAUGCUUG 515 54790_4_1680 TET2 EXON - chr4:105235489-105235509 CACUGCUACCAAAAAUUGGU 516 54790_4_1681 TET2 EXON - chr4:105235490-105235510 CCACUGCUACCAAAAAUUGG 517 54790_4_1683 TET2 EXON - chr4:105235493-105235513 UCUCCACUGCUACCAAAAAU 518 54790_4_1690 TET2 EXON - chr4:105235531-105235551 CUUUGUUUCUCAUCAACUGC 519 54790_4_1699 TET2 EXON - chr4:105235604-105235624 UUCAGAUAGUGCUGUGUUGG 520 54790_4_1700 TET2 EXON - chr4:105235605-105235625 UUUCAGAUAGUGCUGUGUUG 521 54790_4_1702 TET2 EXON - chr4:105235606-105235626 GUUUCAGAUAGUGCUGUGUU 522 54790_4_1703 TET2 EXON - chr4:105235607-105235627 GGUUUCAGAUAGUGCUGUGU 523 54790_4_1708 TET2 EXON - chr4:105235628-105235648 GCCUUCAAUUCAAUCCAUCC 524 54790_4_1711 TET2 EXON - chr4:105235650-105235670 UUCCGCUUGGUGAAAACGAG 525 54790_4_1712 TET2 EXON - chr4:105235651-105235671 AUUCCGCUUGGUGAAAACGA 526 54790_4_1713 TET2 EXON - chr4:105235652-105235672 GAUUCCGCUUGGUGAAAACG 527 54790_4_1722 TET2 EXON - chr4:105235663-105235683 GUUUUAGAUGGGAUUCCGCU 528 54790_4_1723 TET2 EXON - chr4:105235674-105235694 UGCCUCAUUACGUUUUAGAU 529 54790_4_1724 TET2 EXON - chr4:105235675-105235695 AUGCCUCAUUACGUUUUAGA 530 54790_4_1730 TET2 EXON - chr4:105235703-105235723 GGUUGAUACUGAAGAAUUGA 531 54790_4_1737 TET2 EXON - chr4:105235724-105235744 GUCAUUUGAUUGGAGAGAUU 532 54790_4_1738 TET2 EXON - chr4:105235725-105235745 GGUCAUUUGAUUGGAGAGAU 533 54790_4_1743 TET2 EXON - chr4:105235734-105235754 UUGUUUGGAGGUCAUUUGAU 534 54790_4_1749 TET2 EXON - chr4:105235746-105235766 AUUUCCAGUGUAUUGUUUGG 535 54790_4_1751 TET2 EXON - chr4:105235749-105235769 GGAAUUUCCAGUGUAUUGUU 536 54790_4_1756 TET2 EXON - chr4:105235770-105235790 UGGGAGCCCCCCAGGCAUGU 537 54790_4_1758 TET2 EXON - chr4:105235778-105235798 GCUUGCCUUGGGAGCCCCCC 538 54790_4_1763 TET2 EXON - chr4:105235789-105235809 UCUGGGUGUAAGCUUGCCUU 539 54790_4_1766 TET2 EXON - chr4:105235790-105235810 UUCUGGGUGUAAGCUUGCCU 540 54790_4_1769 TET2 EXON - chr4:105235806-105235826 CUCCAGCUGUGUUGUUUUCU 541 54790_4_1770 TET2 EXON - chr4:105235807-105235827 GCUCCAGCUGUGUUGUUUUC 542 54790_4_1779 TET2 EXON - chr4:105235846-105235866 GCCCUUGAUUCAUUUCAACU 543 54790_4_1782 TET2 EXON - chr4:105235872-105235892 AUGUUGGUCCCACUGUACCUU 544 54790_4_1783 TET2 EXON - chr4:105235873-105235893 GAUGUUGGUCCCACUGUACCU 545 54790_4_1790 TET2 EXON - chr4:105235888-105235908 GUUUUUGGAACUGGAGAUGU 546 54790_4_1791 TET2 EXON - chr4:105235897-105235917 GGUGUGAGGGUUUUUGGAAC 547 54790_4_1795 TET2 EXON - chr4:105235903-105235923 GCACCUGGUGUGAGGGUUUU 548 54790_4_1800 TET2 EXON - chr4:105235910-105235930 GAGAAGUGCACCUGGUGUGA 549 54790_4_1801 TET2 EXON - chr4:105235911-105235931 GGAGAAGUGCACCUGGUGUG 550 54790_4_1804 TET2 EXON - chr4:105235918-105235938 CUGUUUUGGAGAAGUGCACC 551 54790_4_1811 TET2 EXON - chr4:105235932-105235952 UUUUGGUAAAUGGUCUGUUU 552 54790_4_1813 TET2 EXON - chr4:105235942-105235962 GCACAUGAGCUUUUGGUAAAA 553 54790_4_1814 TET2 EXON - chr4:105235949-105235969 AGUGACUGCACAUGAGCUUU 554 54790_4_1828 TET2 EXON - chr4:105236010-105236030 GGACAUAAAGUUUUUCAGUUU 555 54790_4_1829 TET2 EXON - chr4:105236011-105236031 GGGACAAUAAGUUUUUCAGUU 556 54790_4_1836 TET2 EXON - chr4:105236031-105236051 CAAGUGCUGUUUCAACACUG 557 54790_4_1838 TET2 EXON - chr4:105236032-105236052 UCAAGUGCUGUUUCAACACU 558 54790_4_1839 TET2 EXON - chr4:105236033-105236053 UUCAAGUGCUGUUUCAACAC 559 54790_4_1846 TET2 EXON - chr4:105236078-105236098 AAAAGGUGUGAGUUUGAAAAA 560 54790_4_1852 TET2 EXON - chr4:105236095-105236115 UAUGAGGCUUAUGUUGCAAA 561 54790_4_1856 TET2 EXON - chr4:105236111-105236131 GUUUGUGCUGCCUGUUUAUG 562 54790_4_1861 TET2 EXON - chr4:105236138-105236158 GGGAGAUGUGAACUCUGGGA 563 54790_4_1862 TET2 EXON - chr4:105236142-105236162 UUGAGGGAGAUGUGAACCUCU 564 54790_4_1864 TET2 EXON - chr4:105236143-105236163 UUUGAGGGAGAUGUGAACUC 565 54790_4_1873 TET2 EXON - chr4:105236158-105236178 GCUGCUGUUGCUGGUUUUGA 566 54790_4_1875 TET2 EXON - chr4:105236159-105236179 UGCUGCUGUUGCUGGUUUUG 567 54790_4_1880 TET2 EXON - chr4:105236167-105236187 GUAAUUUUUGCUGCUGUUGC 568 54790_4_1892 TET2 EXON - chr4:105236215-105236235 UUUGGGGGUGAGGAAAAGUC 569 54790_4_1896 TET2 EXON - chr4:105236225-105236245 UCAUUGUUGCUUUGGGGGUG 570 54790_4_1901 TET2 EXON - chr4:105236230-105236250 GCUGAUCAUUGUUGCUUUGG 571 54790_4_1902 TET2 EXON - chr4:105236231-105236251 UGCUGAUCAUUGUUGCUUUG 572 54790_4_1904 TET2 EXON - chr4:105236232-105236252 UUGCUGAUCAUUGUUGCUUU 573 54790_4_1906 TET2 EXON - chr4:105236233-105236253 UUUGCUGAUCAUUGUUGCUU 574 54790_4_1914 TET2 EXON - chr4:105236275-105236295 AACAUUCUUCCACUUUAGUC 575 54790_4_1931 TET2 EXON - chr4:105236350-105236370 GUACUUCCUCCAGUCCCAUU 576 54790_4_1941 TET2 EXON - chr4:105236394-105236414 UUUCAUGGUCUGACUAUAAG 577 54790_4_1943 TET2 EXON - chr4:105236395-105236415 AUUUCAUGGUCUGACUAUAA 578 54790_4_1944 TET2 EXON - chr4:105236396-105236416 GAUUUCAUGGUCUGACUUA 579 54790_4_1950 TET2 EXON - chr4:105236409-105236429 UUUGCAUGCACUUGAUUUCA 580 54790_4_1960 TET2 EXON - chr4:105236461-105236481 GUUCUUUAUUCUCUGAAACU 581 54790_4_1966 TET2 EXON - chr4:105236495-105236515 UUGUUUCCUGCAAAAAGUUC 582 54790_4_1972 TET2 EXON - chr4:105236520-105236540 UUGCAUGUGAUGCAAGUUUU 583 54790_4_1973 TET2 EXON - chr4:105236521-105236541 AUUGCAUGUGAUGCAAGUUU 584 54790_4_1982 TET2 EXON - chr4:105236549-105236569 UGCUUUGGGAUCACAUUAUU 585 54790_4_1984 TET2 EXON - chr4:105236563-105236583 UGUGAAGAGAUCUUGCUUU 586 54790_4_1985 TET2 EXON - chr4:105236564-105236584 CUGUGAAGAAAGAUCUUGCUU 587 54790_4_2009 TET2 EXON - chr4:105236653-105236673 CUUGUUGACCAGACAUAUCU 588 54790_4_2017 TET2 EXON - chr4:105236713-105236733 GCACAGGAAAAAACAUUUGCA 589 54790_4_2019 TET2 EXON - chr4:105236729-105236749 CUUCCUCCCUGGUCAGGCAC 590 54790_4_2022 TET2 EXON - chr4:105236735-105236755 GUGUGACUUCCUCCCUGGUC 591 54790_4_2023 TET2 EXON - chr4:105236740-105236760 UCUGAGUGUGACUUCCUCCC 592 54790_4_2029 TET2 EXON - chr4:105236763-105236783 UUGAGUGUCCUUCUGGGGAG 593 54790_4_2030 TET2 EXON - chr4:105236764-105236784 UUUGAGUGUCCUUCUGGGGA 594 54790_4_2031 TET2 EXON - chr4:105236765-105236785 UUUUGAGUGUCCUUCUGGGG 595 54790_4_2034 TET2 EXON - chr4:105236768-105236788 UGCUUUUGAGUGUCCUUCUG 596 54790_4_2037 TET2 EXON - chr4:105236769-105236789 AUGCUUUUGAGUGUCCUUCU 597 54790_4_2039 TET2 EXON - chr4:105236770-105236790 CAUGCUUUUGAGUGUCCUUC 598 54790_4_2053 TET2 EXON - chr4:105236846-105236866 CUAUGGCAAGACUCAGUUUG 599 54790_4_2054 TET2 EXON - chr4:105236847-105236867 ACUAUGGCAAGACUCAGUUU 600 54790_4_2055 TET2 EXON - chr4:105236848-105236868 GACUAUGGCAAGACUCAGUU 601 54790_4_2060 TET2 EXON - chr4:105236863-105236883 UUGGCCUGUGCAUCUGACUA 602 54790_4_2063 TET2 EXON - chr4:105236882-105236902 CAUCCAGGUUCCACCUUAAU 603 54790_4_2064 TET2 EXON - chr4:105236897-105236917 CAGGCAUGUGGCUUGCAUCC 604 54790_4_2065 TET2 EXON - chr4:105236909-105236929 GCUGUGUGCAUACAGGCAUG 605 54790_4_2069 TET2 EXON - chr4:105236916-105236936 UGGUGGUGCUGUGUGCAUAC 606 54790_4_2077 TET2 EXON - chr4:105236933-105236953 UUCCAGUUUUGUUUUCUGG 607 54790_4_2079 TET2 EXON - chr4:105236936-105236956 UUUUUCCAUGUUUUGUUUUC 608 54790_4_2085 TET2 EXON - chr4:105236978-105236998 ACAUUAUCACAGCUUGCAGG 609 54790_4_2089 TET2 EXON - chr4:105236981-105237001 UGCACAUUAUCACAGCUUGC 610 54790_4_2092 TET2 EXON - chr4:105237024-105237044 CUGCUUCAGAUGCUGCUCCA 611 54790_4_2096 TET2 EXON - chr4:105237054-105237074 CUUAUGGUCAAAUAACGACU 612 54790_4_2099 TET2 EXON - chr4:105237070-105237090 AUUUGAGAGUAAGAGCCUUA 613 54790_4_2112 TET2 EXON - chr4:105237125-105237145 UGUCUAGUCAAAACUGUGAC 614 54790_4_2114 TET2 EXON - chr4:105237150-105237170 GCUAUCAAGUUCUGCAGCAG 615 54790_4_2118 TET2 EXON - chr4:105237172-105237192 GCUGCUCUAAAGCUGGGGUG 616 54790_4_2119 TET2 EXON - chr4:105237177-105237197 UGUUUGCUGCUCUAAAGCUG 617 54790_4_2120 TET2 EXON - chr4:105237178-105237198 UUGUUUGCUGCUCUAAAGCU 618 54790_4_2122 TET2 EXON - chr4:105237179-105237199 GUUGUUUGCUGCUCUAAAGC 619 54790_4_2135 TET2 EXON - chr4:105237218-105237238 GAAGCAGCUGUUCUUUUGGU 620 54790_4_2137 TET2 EXON - chr4:105237222-105237242 AACAGAAGCAGCUGUUCUUU 621 54790_4_2148 TET2 EXON - chr4:105237266-105237286 GGAGUAUCUAGUAAUUUGGA 622 54790_4_2153 TET2 EXON - chr4:105237270-105237290 UAUAGGAGUAUCUAGUAAUU 623 54790_4_2156 TET2 EXON - chr4:105237287-105237307 GUAUCCAAUAAAUUUUUUAU 624 54790_4_2160 TET2 EXON - chr4:105237311-105237331 AAAUCAUAUUGAGUCUUGAC 625 54790_4_2163 TET2 EXON - chr4:105237334-105237354 UACCUACACAUCUGCAAGAU 626 54790_4_2165 TET2 EXON - chr4:105237335-105237355 UUACCUACACAAUCUGCAAGA 627 54790_4_2170 TET2 EXON - chr4:105237361-105237381 CAUGUGUCUCAGUACAUUUC 628 54790_4_2174 TET2 EXON - chr4:105237392-105237412 GAAGAUAAAUUUGCUAAUUC 629 54790_4_2180 TET2 EXON - chr4:105237429-105237449 ACUCAAGAUUUAAAAAAAGA 630 54790_4_2197 TET2 EXON - chr4:105237510-105237530 CUUUCACAAGACACAAGCAU 631 54790_4_2206 TET2 EXON - chr4:105237558-105237578 GCACGAUUAUUUAAUUCUUU 632 54790_4_2213 TET2 EXON - chr4:105237593-105237613 UUUUCAGGAUCUGAGAGA 633 54790_4_2215 TET2 EXON - chr4:105237594-105237614 AUUUUACAGGAUCUGAAGAG 634 54790_4_2221 TET2 EXON - chr4:105237607-105237627 CAGAUACAUUCAAAUUUUAC 635 54790_4_2225 TET2 EXON - chr4:105237645-105237665 UAAUAUACAAAGAGCUAAAU 636 54790_4_2233 TET2 EXON - chr4:105237671-105237691 UGCUGCCUAGCUGUCUCUCC 637 54790_4_2247 TET2 EXON - chr4:105237727-105237747 UUCGUACAUUAGACUGCCUA 638 54790_4_2270 TET2 EXON - chr4:105237874-105237894 AAUGGAGAAAAGGAAACUUU 639 54790_4_2274 TET2 EXON - chr4:105237884-105237904 CAAAUGUAUAAAUGGAGAAA 640 54790_4_2277 TET2 EXON - chr4:105237892-105237912 CAACAUUCCAAAUGUAUAAAA 641 54790_4_2284 TET2 EXON - chr4:105237936-105237956 AGAUGAAAUUUUGAGAAAAA 642 54790_4_2287 TET2 EXON - chr4:105237937-105237957 AAGAUGAAAUUUUAGAGAAA 643 54790_4_2323 TET2 EXON - chr4:105240511-105240531 AGGGAAAACAUGGCACGGGU 644 54790_4_2325 TET2 EXON - chr4:105240515-105240535 CAAGAGGGAAAACAUGGCAC 645 54790_4_2326 TET2 EXON - chr4:105240516-105240536 GCAAGAGGGAAAACAUGGCA 646 54790_4_2328 TET2 EXON - chr4:105240521-105240541 UCAUUGCAAGAGGGAAAAACA 647 54790_4_2330 TET2 EXON - chr4:105240530-105240550 UGGGGUAUCUCAUUGCAAGA 648 54790_4_2331 TET2 EXON - chr4:105240531-105240551 GUGGGGUAUCUCAUUGCAAG 649 54790_4_2336 TET2 EXON - chr4:105240548-105240568 CCAUCCUUCUACACAGUGUG 650 54790_4_2337 TET2 EXON - chr4:105240549-105240569 UCCAUCCUUCUACACAGUGU 651 54790_4_2338 TET2 EXON - chr4:105240550-105240570 CUCCAUCCUUCUACACAGUG 652 54790_4_2342 TET2 EXON - chr4:105240581-105240601 CACACGCAAAGAGGGACAGU 653 54790_4_2345 TET2 EXON - chr4:105240589-105240609 UUAAUAACCCACGCAAAGA 654 54790_4_2347 TET2 EXON - chr4:105240590-105240610 CUUAAUAACCACACGCAAAG 655 54790_4_2353 TET2 EXON - chr4:105240616-105240636 UGUGGUGUUUUAGCCCAGUG 656 54790_4_2357 TET2 EXON - chr4:105240634-105240654 AAUAUUAUCUAUGAGAUGUG 657 54790_4_2365 TET2 EXON - chr4:105240693-105240713 AAAAGUGGGAAGAUAGGGGU 658 54790_4_2366 TET2 EXON - chr4:105240694-105240714 GAAAAGUGGGAAGAUAGGGG 659 54790_4_2368 TET2 EXON - chr4:105240697-105240717 AUGGAAAAGGUGGGAAGAUAG 660 54790_4_2369 TET2 EXON - chr4:105240698-105240718 GAUGGAAAAAGUGGGAAGAUA 661 54790_4_2370 TET2 EXON - chr4:105240699-105240719 AGAUGGAAAAAGUGGGAAGAU 662 54790_4_2373 TET2 EXON - chr4:105240707-105240727 ACCAACAAAGAUGGAAAAGGU 663 54790_4_2377 TET2 EXON - chr4:105240708-105240728 AACCAACAAAGAUGGAAAAG 664 54790_4_2380 TET2 EXON - chr4:105240716-105240736 CUGUUGCAAACCAACAAAGA 665 54790_4_2382 TET2 EXON - chr4:105240739-105240759 GAGAGUCAGGCAAAAAGAAG 666 54790_4_2383 TET2 EXON - chr4:105240740-105240760 GGAGAGUCAGGCAAAAAGAA 667 54790_4_2384 TET2 EXON - chr4:105240741-105240761 UGGAGAGUCAGGCAAAAAAGA 668 54790_4_2389 TET2 EXON - chr4:105240752-105240772 AGAGAAAAAUCCUGGAGAGUC 669 54790_4_2393 TET2 EXON - chr4:105240761-105240781 UUUAUGAUGAGAGAAAAUCC 670 54790_4_2422 TET2 EXON - chr4:105240882-105240902 UAUUGCUUGGUUUAUUGUCA 671 54790_4_2424 TET2 EXON - chr4:105240895-105240915 CCCACCCCCAGAAUAUUGCU 672 54790_4_2434 TET2 EXON - chr4:105240954-105240974 CUGGAAGCCUACCUAUUACU 673 54790_4_2439 TET2 EXON - chr4:105240973-105240993 AAAAAACAUUUAAAGCUAAC 674 54790_4_2446 TET2 EXON - chr4:105241000-105241020 UACAAUCCAAUUUUUUGAGC 675 54790_4_2454 TET2 EXON - chr4:105241052-105241072 CUAAACAAAGAAUACAGUGA 676 54790_4_2456 TET2 EXON - chr4:105241053-105241073 ACUAAACAAAGAAUACAGUG 677 54790_4_2468 TET2 EXON - chr4:105241107-105241127 AUAUAUUACAUUUCAGAUAU 678 54790_4_2469 TET2 EXON - chr4:105241108-105241128 AAUAUAUUACAUUUCAGAUA 679 54790_4_2475 TET2 EXON - chr4:105241136-105241156 UAUAUGUACAUGCUGGUUGU 680 54790_4_2477 TET2 EXON - chr4:105241143-105241163 AAUUAAGUAUAUGUACAUGC 681 54790_4_2488 TET2 EXON - chr4:105241193-105241213 CUUUAAAAUGAGUAGAUUGA 682 54790_4_2498 TET2 EXON - chr4:105241245-105241265 AACCCCCAACUACACAUUAA 683 54790_4_2499 TET2 EXON - chr4:105241246-105241266 UAACCCCCAACUACACAUUA 684 54790_4_2503 TET2 EXON - chr4:105241285-105241305 CUCAAUUAUACUAAAUAUAA 685 54790_4_2519 TET2 EXON - chr4:105241367-105241387 GCUCCUAGAUGGGUAUAAAA 686 54790_4_2522 TET2 EXON - chr4:105241377-105241397 AUUAGGACCUGCUCCUAGAU 687 54790_4_2523 TET2 EXON - chr4:105241378-105241398 CAUUAGGACCUGCUCCUAGA 688 54790_4_2527 TET2 EXON - chr4:105241394-105241414 UCUCUAAUAGCUGCCACAUU 689 54790_4_2538 TET2 EXON - chr4:105241470-105241490 AAAAUUCUGACAUAUACAAA 690 54790_4_2546 TET2 EXON - chr4:105241494-105241514 ACUGCUUUGUGUGUGAAGGC 691 54790_4_2548 TET2 EXON - chr4:105241498-105241518 GUUUACUGCUUUGUGUGUGA 692 54790_4_2555 TET2 EXON - chr4:105241568-105241588 AAUAGCACAGUGUGUAGGUGU 693 54790_4_2558 TET2 EXON - chr4:105241593-105241613 UGUCAAAUAUUGUGACUCUC 694 54790_4_2563 TET2 EXON - chr4:105241647-105241667 UCUGGCAUCCAUCGCAAAGU 695 54790_4_2564 TET2 EXON - chr4:105241648-105241668 UUCUGGCAUCCAUCGCAAAG 696 54790_4_2568 TET2 EXON - chr4:105241665-105241685 CUGUUCAUGCCUGGGUUUUC 697 54790_4_2569 TET2 EXON - chr4:105241673-105241693 GCCGAUUCCUGUUCAUGCCU 698 54790_4_2570 TET2 EXON - chr4:105241674-105241694 GGCCGAUUCCUGUUCAUGCC 699 54790_4_2573 TET2 EXON - chr4:105241695-105241715 CUUGUGGCUGGCAGCCUGGC 700 54790_4_2574 TET2 EXON - chr4:105241699-105241719 GUACCUUGUGGCUGGCAGCC 701 54790_4_2575 TET2 EXON - chr4:105241707-105241727 CUGUGCCAGUACCUUGUGGC 702 54790_4_2577 TET2 EXON - chr4:105241711-105241731 GAGCCUGUGCCAGUACCUUG 703 54790_4_2578 TET2 EXON - chr4:105241733-105241753 GCCAGAGUGGGACCUCUCGU 704 54790_4_2582 TET2 EXON - chr4:105241745-105241765 UCAGGUGGGAAAGCCAGAGU 705 54790_4_2585 TET2 EXON - chr4:105241746-105241766 AUCAGGUGGGAAAGCCAGAG 706 54790_4_2591 TET2 EXON - chr4:105241759-105241779 UUGACACUUUAUUAUCAGGU 707 54790_4_2595 TET2 EXON - chr4:105241760-105241780 UUUGACACUUUAUUAUCAGG 708 54790_4_2598 TET2 EXON - chr4:105241763-105241783 UGCUUUGACACUUUAUUAUC 709 54790_4_2609 TET2 EXON - chr4:105241819-105241839 ACUAGGUGAAUUUAAUUCAG 710 54790_4_2613 TET2 EXON - chr4:105241836-105241856 AAGUACUCAUUUGCAACACU 711 54790_4_2622 TET2 EXON - chr4:105241878-105241898 UCACACUUGCUCUCUUUUUA 712 54790_4_2629 TET2 EXON - chr4:105241939-105241959 AUAGCGAAAAAAAAAAAAAA 713 54790_4_2633 TET2 EXON - chr4:105241986-105242006 UCUUCUACAUGCAGGAGUAA 714 54790_4_2635 TET2 EXON - chr4:105241994-105242014 CAUAAGAGUCUUCUACAUGC 715 54790_4_2642 TET2 EXON - chr4:105242038-105242058 GCUGUAUAAAUUUAUAUGAA 716 54790_4_2652 TET2 EXON - chr4:105242086-105242106 CUGCCUACCUCUUAAUGAAA 717 54790_4_2663 TET2 EXON - chr4:105242173-105242193 AGAAAUGAAUAAUUUGGAAA 718 54790_4_2665 TET2 EXON - chr4:105242179-105242199 UAAUUUAGAAAUGAAUAAUU 719 54790_4_2679 TET2 EXON - chr4:105242236-105242256 GGAAAUUCACUAUUUCUGCC 720 54790_4_2681 TET2 EXON - chr4:105242257-105242277 GUUGUUUUUUUUGGCACUUA 721 54790_4_2683 TET2 EXON - chr4:105242258-105242278 UGUUGUUUUUUUUGGCACUU 722 54790_4_2685 TET2 EXON - chr4:105242266-105242286 UGUUUUUUUGUUGUUUUUU 723 54790_4_2694 TET2 EXON - chr4:105242360-105242380 AUCCAGCCACAUGGAAAAUA 724 54790_4_2697 TET2 EXON - chr4:105242369-105242389 AUAGUUAGUAUCCAGCCCACA 725 54790_4_2701 TET2 EXON - chr4:105242395-105242415 CACAAUUUAGAAAAGGAGGC 726 54790_4_2702 TET2 EXON - chr4:105242399-105242419 GUCUCACAAUUUAGAAAAGG 727 54790_4_2703 TET2 EXON - chr4:105242402-105242422 AAUGUCUCACAAUUUAGAAA 728 54790_4_2721 TET2 EXON - chr4:105242462-105242482 UUUAGCUAUUUUAAAAACUUG 729 54790_4_2723 TET2 EXON - chr4:105242463-105242483 AUUUAGCUAUUUUAAAAACUU 730 54790_4_2726 TET2 EXON - chr4:105242464-105242484 AAUUUAGCUAUUUUAAAAACU 731 54790_4_2742 TET2 EXON - chr4:105242539-105242559 UUUCACAAAGCACAAAAUUC 732 54790_4_2749 TET2 EXON - chr4:105242583-105242603 AAUUACAUGUGGGUGAAAAU 733 54790_4_2752 TET2 EXON - chr4:105242584-105242604 AAAUUACAUGUGGGUGAAAAA 734 54790_4_2755 TET2 EXON - chr4:105242593-105242613 CUAUUUUGUAAAUUACAUGU 735 54790_4_2756 TET2 EXON - chr4:105242594-105242614 ACUAUUUUGUAAAUUACAUG 736 54790_4_2769 TET2 EXON - chr4:105242669-105242689 ACGCCAAGCAAACUGGAUAAA 737 54790_4_2772 TET2 EXON - chr4:105242676-105242696 CAGGUCUACGCCAAGCAAAC 738 54790_4_2780 TET2 EXON - chr4:105242695-105242715 CGGUUUAUUAUUUUUAAAC 739 54790_4_2781 TET2 EXON - chr4:105242715-105242735 ACCACAUCCUGAGAAAUGAA 740 54790_5_3 TET2 EXON + chr4:105242816-105242836 CUGUGGGUUUCUUUAAGGUU 741 54790_5_7 TET2 EXON + chr4:105242824-105242844 UUCUUUAAGGUUUGGACAGA 742 54790_5_8 TET2 EXON + chr4:105242825-105242845 UCUUUAAGGUUUGGACAGAA 743 54790_5_15 TET2 EXON + chr4:105242838-105242858 GACAGAAGGGUAAAGCUAUU 744 54790_5_20 TET2 EXON + chr4:105242861-105242881 AUUGAAAGAGUCAUCUAUAC 745 54790_5_23 TET2 EXON + chr4:105242870-105242890 GUCAUCUAUACUGGUAAAAGA 746 54790_5_26 TET2 EXON + chr4:105242884-105242904 UAAAGAAGGCAAAAGUUCUC 747 54790_5_27 TET2 EXON + chr4:105242885-105242905 AAAGAAGGCAAAAGUUCUCA 748 54790_5_30 TET2 EXON + chr4:105242904-105242924 AGGGAUGUCCUAUUGCUAAG 749 54790_5_31 TET2 EXON + chr4:105242905-105242925 GGGAUGUCCCUAUUGCUAAGU 750 54790_5_51 TET2 EXON - chr4:105242915-105242935 ACACUUACCCACUUAGCAAU 751 54790_6_1 TET2 EXON + chr4:105243550-105243570 GGAAUGGUGAUCCAGCAGG 752 54790_6_7 TET2 EXON + chr4:105243589-105243609 UGAAGAGAAGCUACUGUGUU 753 54790_6_9 TET2 EXON + chr4:105243594-105243614 AGAAGCUACUGUGUUUGGUG 754 54790_6_12 TET2 EXON + chr4:105243595-105243615 GAAGCUACUGUGUUUGGUGC 755 54790_6_14 TET2 EXON + chr4:105243605-105243625 UGUUUGGUGCGGGAGCGAGC 756 54790_6_18 TET2 EXON + chr4:105243619-105243639 GCGAGCUGGCCACACCUGUG 757 54790_6_19 TET2 EXON + chr4:105243646-105243666 AGUGAUUGUGAUUCUCAUCC 758 54790_6_21 TET2 EXON + chr4:105243651-105243671 UUGUGAUUUCUCAUCCUGGUG 759 54790_6_24 TET2 EXON + chr4:105243652-105243672 UGUGAUUUCUCAUCCUGGUGU 760 54790_6_27 TET2 EXON + chr4:105243656-105243676 AUUCUCAUCCUGGUGUGGGA 761 54790_6_30 TET2 EXON + chr4:105243673-105243693 GGAAGGAAUCCCGCUGUCUC 762 54790_6_32 TET2 EXON + chr4:105243691-105243711 UCUGGCUGACAAACUCUACU 763 54790_6_37 TET2 EXON + chr4:105243711-105243731 CGGAGCUUACCGAGACGCUG 764 54790_6_39 TET2 EXON + chr4:105243719-105243739 ACCGAGACGCUGAGGAAAUA 765 54790_6_41 TET2 EXON + chr4:105243738-105243758 ACGGCACCGCUCACCAAUCGC 766 54790_6_48 TET2 EXON + chr4:105243771-105243791 AUGAAGUAAGUGAAGCCCC 767 54790_6_49 TET2 EXON + chr4:105243772-105243792 UGAAGUAGUGAAGCCCA 768 54790_6_51 TET2 EXON - chr4:105243564-105243584 GCUUCUGCGAACCACCUGCG 769 54790_6_56 TET2 EXON - chr4:105243631-105243651 UCACUGCAGCCUCACAGGUG 770 54790_6_57 TET2 EXON - chr4:105243636-105243656 CACAAUCACUGCAGCCUCAC 771 54790_6_62 TET2 EXON - chr4:105243667-105243687 GCGGGAUUCCUUCCCACACCC 772 54790_6_66 TET2 EXON - chr4:105243685-105243705 GUUUGUCAGCCAGAGACAGC 773 54790_6_67 TET2 EXON - chr4:105243686-105243706 AGUUUGUCAGCCAGAGACAG 774 54790_6_75 TET2 EXON - chr4:105243723-105243743 GCCGUAUUUCCUCAGCGUCU 775 54790_6_80 TET2 EXON - chr4:105243753-105243773 AUUCAAGGCACACCGGCGAU 776 54790_6_82 TET2 EXON - chr4:105243760-105243780 ACUCUUCAUUCAAGGCACAC 777 54790_6_84 TET2 EXON - chr4:105243768-105243788 CUUCACCUUACUCUUCAUUCA 778 54790_7_10 TET2 EXON + chr4:105259615-105259635 CAGGAGAACUUGCGCCUGUC 779 54790_7_12 TET2 EXON + chr4:105259616-105259636 AGGAGAACUUGCGCCUGUCA 780 54790_7_14 TET2 EXON + chr4:105259617-105259637 GGAGAACUUGCGCCUGUCAG 781 54790_7_16 TET2 EXON + chr4:105259621-105259641 AACUUGCGCCUGUCAGGGGC 782 54790_7_20 TET2 EXON + chr4:105259637-105259657 GGGCUGGAUCCAGAAACCUG 783 54790_7_21 TET2 EXON + chr4:105259655-105259675 UGUGGUGCCUCCUUCUCUUU 784 54790_7_23 TET2 EXON + chr4:105259665-105259685 CCUUCUCUUUUGGUUGUUCA 785 54790_7_31 TET2 EXON + chr4:105259682-105259702 UCAUGGAGCAUGUACUACAAA 786 54790_7_35 TET2 EXON + chr4:105259713-105259733 UUGCCAGAAGCAAGAUCCCA 787 54790_7_41 TET2 EXON + chr4:105259730-105259750 CCAAGGAAGUUUAAGCUGCU 788 54790_7_42 TET2 EXON + chr4:105259731-105259751 CAAGGAAGUUUAAGCUGCUU 789 54790_7_44 TET2 EXON + chr4:105259732-105259752 AAGGAAGUUUAAGCUGCUUG 790 54790_7_48 TET2 EXON + chr4:105259747-105259767 GCUUGGGGAUGACCCAAAAG 791 54790_7_53 TET2 EXON - chr4:105259632-105259652 UUCUGGAUCCAGCCCCUGAC 792 54790_7_54 TET2 EXON - chr4:105259649-105259669 AAGGAGGCACCAGAGUUUC 793 54790_7_56 TET2 EXON - chr4:105259656-105259676 AAAAGAGAAGGAGGCACAC 794 54790_7_57 TET2 EXON - chr4:105259665-105259685 UGAACAACCAAAAGAGAAGG 795 54790_7_58 TET2 EXON - chr4:105259668-105259688 CCAUGAACAACCAAAAGAGA 796 54790_7_72 TET2 EXON - chr4:105259719-105259739 CUUCCUUGGGAUCUUGCUUC 797 54790_7_73 TET2 EXON - chr4:105259732-105259752 CAAGCAGCUUAAACUUCCUU 798 54790_7_74 TET2 EXON - chr4:105259733-105259753 CCAAGCAGCUUAAACUUCCU 799 54790_7_80 TET2 EXON - chr4:105259762-105259782 GAAGUAAACAAACCUCUUUU 800 54790_7_81 TET2 EXON - chr4:105259763-105259783 GGAAGUAAAACAAACCUCUUU 801 54790_8_8 TET2 EXON + chr4:105261748-105261768 CUUUAUACAGGAAGAGAAAC 802 54790_8_12 TET2 EXON + chr4:105261781-105261801 GCAAAACCUGUCCACUCUUA 803 54790_8_18 TET2 EXON + chr4:105261826-105261846 ACCUGAUGCAUAUAAAUAAUC 804 54790_8_27 TET2 EXON - chr4:105261790-105261810 UUGGUGCCAUAAGAGUGGAC 805 54790_8_30 TET2 EXON - chr4:105261795-105261815 AUAUGUUGGUGCCAUAAGAG 806 54790_8_34 TET2 EXON - chr4:105261809-105261829 GGUGCAAGUUUCUUAUAUGU 807 54790_8_38 TET2 EXON - chr4:105261830-105261850 ACCUGAUUAUUAUAUGCAUC 808 54790_9_14 TET2 EXON + chr4:105269623-105269643 CAGAGCACCAGAGUGCCGUC 809 54790_9_15 TET2 EXON + chr4:105269624-105269644 AGAGCACCAGAGUGCCGUCU 810 54790_9_19 TET2 EXON + chr4:105269632-105269652 AGAGUGCCGUCUGGGUCUGA 811 54790_9_20 TET2 EXON + chr4:105269636-105269656 UGCCGUCUGGGUCUGAAGGA 812 54790_9_22 TET2 EXON + chr4:105269651-105269671 AAGGAAGGCCGUCCAUUCUC 813 54790_9_24 TET2 EXON + chr4:105269652-105269672 AGGAAGGCCCGUCCAUUCUCA 814 54790_9_25 TET2 EXON + chr4:105269653-105269673 GGAAGGCCGUCCAUUCUCAG 815 54790_9_27 TET2 EXON + chr4:105269668-105269688 CUCAGGGGUCACUGCAUGUU 816 54790_9_35 TET2 EXON + chr4:105269714-105269734 GACUUGCACAACAUGCAGAA 817 54790_9_37 TET2 EXON + chr4:105269725-105269745 CAUGCAGAAUGGCAGCACA 818 54790_9_39 TET2 EXON + chr4:105269733-105269753 AUGGCAGCACAUUGGUAAGU 819 54790_9_40 TET2 EXON + chr4:105269734-105269754 UGGCAGCACAUUGGUAAGUU 820 54790_9_43 TET2 EXON + chr4:105269740-105269760 CACAUUGGUAAGUUGGGCUG 821 54790_9_49 TET2 EXON - chr4:105269633-105269653 UUCAGACCCAGACGGCACUC 822 54790_9_50 TET2 EXON - chr4:105269641-105269661 GGCCUUCCUUCAGACCCAGA 823 54790_9_51 TET2 EXON - chr4:105269662-105269682 CAGUGACCCCCUGAGAAUGGA 824 54790_9_52 TET2 EXON - chr4:105269666-105269686 CAUGCAGUGACCCCCUGAGAA 825 54790_9_61 TET2 EXON - chr4:105269709-105269729 CAUGUUGUGCAAGUCUCUGU 826 54790_9_62 TET2 EXON - chr4:105269710-105269730 GCAUGUUGUGCAAGUCUCUG 827 54790_10_10 TET2 EXON + chr4:105272578-105272598 AGAGAAGACAAUCGAGAAUU 828 54790_10_13 TET2 EXON + chr4:105272581-105272601 GAAGACAAUCGAGAAUUUGG 829 54790_10_16 TET2 EXON + chr4:105272592-105272612 AGAAUUUGGAGGAAAACCUG 830 54790_10_23 TET2 EXON + chr4:105272637-105272657 UUUAUACAAAGUCUCUGACG 831 54790_10_29 TET2 EXON + chr4:105272647-105272667 GUCUCUGACGUGGAUGAGUU 832 54790_10_30 TET2 EXON + chr4:105272648-105272668 UCUCUGACGUGGAUGAGUUU 833 54790_10_33 TET2 EXON + chr4:105272655-105272675 CGUGGAUGAGUUUGGGAGUG 834 54790_10_36 TET2 EXON + chr4:105272664-105272684 GUUUGGGAGUGUGGAAGCUC 835 54790_10_40 TET2 EXON + chr4:105272667-105272687 UGGGAGUGUGGAAGCUCAGG 836 54790_10_46 TET2 EXON + chr4:105272678-105272698 AAGCUCAGGAGGAGAAAAAAA 837 54790_10_48 TET2 EXON + chr4:105272683-105272703 CAGGAGGAGAAAAAACGGAG 838 54790_10_49 TET2 EXON + chr4:105272694-105272714 AAAACGGAGUGGUGCCAUUC 839 54790_10_51 TET2 EXON + chr4:105272711-105272731 UUCAGGUACUGAGUUCUUUU 840 54790_10_55 TET2 EXON + chr4:105272723-105272743 GUUCUUUUCGGCGAAAAGUC 841 54790_10_64 TET2 EXON + chr4:105272759-105272779 CAGUCAAGACUUGCCGACAA 842 54790_10_71 TET2 EXON + chr4:105272805-105272825 AGCUGAAAAGCUUUCCUCCCC 843 54790_10_78 TET2 EXON + chr4:105272832-105272852 CAGCUCAAAUAAAAAUGAAA 844 54790_10_81 TET2 EXON + chr4:105272880-105272900 ACAAACUGAAAACGCAAGCC 845 54790_10_82 TET2 EXON + chr4:105272892-105272912 CGCAAGCCAGGCUAAACAGU 846 54790_10_83 TET2 EXON + chr4:105272896-105272916 AGCCAGGCUAAACAGUUGGC 847 54790_10_85 TET2 EXON - chr4:105272557-105272577 GUGAGAGUGCAUACCUGGUA 848 54790_10_87 TET2 EXON - chr4:105272558-105272578 AGUGAGAGUGCAUACCUGGU 849 54790_10_91 TET2 EXON - chr4:105272562-105272582 CUCUAGUGAGAGUGCAUACC 850 54790_10_99 TET2 EXON - chr4:105272611-105272631 ACGUGAAGCUGCUCAUCCUC 851 54790_10_105 TET2 EXON - chr4:105272638-105272658 ACGUCAGAGACUUUGUAUAA 852 54790_10_114 TET2 EXON - chr4:105272711-105272731 AAAAGAACUCAGUACCUGAA 853 54790_10_127 TET2 EXON - chr4:105272761-105272781 CUUUGUCGGCAAGUCUUGAC 854 54790_10_132 TET2 EXON - chr4:105272775-105272795 UGGCUUCUAGUUUCCUUUGU 855 54790_10_136 TET2 EXON - chr4:105272795-105272815 CUUUUCAGCUGCAGCUUUCU 856 54790_10_145 TET2 EXON - chr4:105272822-105272842 AUUUGAGCUGUUCUCCAGGG 857 54790_10_147 TET2 EXON - chr4:105272825-105272845 UUUAUUUGAGCUGUUCUCCA 858 54790_10_150 TET2 EXON - chr4:105272826-105272846 UUUUAUUUGAGCUGUUCUCC 859 54790_10_167 TET2 EXON - chr4:105272867-105272887 AGUUUGUUUUGUACGUGAUG 860 54790_10_168 TET2 EXON - chr4:105272868-105272888 CAGUUUGUUUUGUACGUGAU 861 54790_10_169 TET2 EXON - chr4:105272869-105272889 UCAGUUUGUUUUGUACGUGA 862 54790_10_177 TET2 EXON - chr4:105272901-105272921 UACCUGCCAACUGUUUAGCC 863 54790_11_9 TET2 EXON + chr4:105275178-105275198 GUCAACUCUUAUUCUGCUUC 864 54790_11_14 TET2 EXON + chr4:105275203-105275223 CCACCAAUCCAUACAUGAGA 865 54790_11_19 TET2 EXON + chr4:105275256-105275276 UCACACACUUCAGAUAUCUA 866 54790_11_24 TET2 EXON + chr4:105275304-105275324 UCCACCUCAUCUCAAGCUGC 867 54790_11_34 TET2 EXON + chr4:105275346-105275366 AAUCCCAUGAACCCCUUACCC 868 54790_11_35 TET2 EXON + chr4:105275347-105275367 AUCCCAUGAACCCUUACCCU 869 54790_11_44 TET2 EXON + chr4:105275391-105275411 UAUCCAUCAUAUCAAUGCAA 870 54790_11_47 TET2 EXON + chr4:105275405-105275425 AUGCAAUGGAAACCUAUCAG 871 54790_11_49 TET2 EXON + chr4:105275426-105275446 GGACAACCUGCUCCCCAUAUC 872 54790_11_50 TET2 EXON + chr4:105275427-105275447 GACAACUGCUCCCCAUAUCU 873 54790_11_53 TET2 EXON + chr4:105275456-105275476 UUCUCCCCAGUCUCAGCGA 874 54790_11_55 TET2 EXON + chr4:105275467-105275487 CUCAGCCGAUGGAUCUGUAU 875 54790_11_56 TET2 EXON + chr4:105275533-105275553 UCCAUACACUUUACCAGCCCA 876 54790_11_59 TET2 EXON + chr4:105275538-105275558 ACACUUUACCAGCCAAGGUU 877 54790_11_65 TET2 EXON + chr4:105275571-105275591 AGUUUUACAUCUAAAUACUU 878 54790_11_68 TET2 EXON + chr4:105275577-105275597 ACAUCUAAAUACUUAGGUUA 879 54790_11_74 TET2 EXON + chr4:105275594-105275614 UUAUGGAAACCAAAAAUAUGC 880 54790_11_77 TET2 EXON + chr4:105275595-105275615 UAUGGAAAACCAAAAUAUGCA 881 54790_11_79 TET2 EXON + chr4:105275601-105275621 AACCAAAAUAUGCAGGGAGA 882 54790_11_85 TET2 EXON + chr4:105275643-105275663 AGACCAAAUGUACAUCAGUGU 883 54790_11_86 TET2 EXON + chr4:105275644-105275664 GACCAAAUGUACAUCAUGUA 884 54790_11_92 TET2 EXON + chr4:105275675-105275695 UCCUUAUCCCACUCAUGAGA 885 54790_11_93 TET2 EXON + chr4:105275679-105275699 UAUCCCACUCAUGAGAUGGA 886 54790_11_96 TET2 EXON + chr4:105275690-105275710 UGAGAUGGAUGGCCACUUCA 887 54790_11_99 TET2 EXON + chr4:105275691-105275711 GAGAUGGAUGGCCACUUCAU 888 54790_11_104 TET2 EXON + chr4:105275735-105275755 CAAUCUGAGCAAUCCAAACA 889 54790_11_105 TET2 EXON + chr4:105275748-105275768 CCAAACAUGGACUAUAAAAAA 890 54790_11_110 TET2 EXON + chr4:105275798-105275818 CCAUAACUACAGUGCAGCUC 891 54790_11_111 TET2 EXON + chr4:105275799-105275819 CAUAACUACAGUGCAGCUCC 892 54790_11_116 TET2 EXON + chr4:105275843-105275863 UGCCCUGCAUCUCCAAAACA 893 54790_11_120 TET2 EXON + chr4:105275874-105275894 AUGCUUUCCCACACAGCUAA 894 54790_11_121 TET2 EXON + chr4:105275875-105275895 UGCUUUCCCACACAGCUAAU 895 54790_11_129 TET2 EXON + chr4:105275928-105275948 GAUAGAACUGCUUGUGUGUCCA 896 54790_11_131 TET2 EXON + chr4:105275931-105275951 AGAACUGCUUGUGUGUCCAAGG 897 54790_11_133 TET2 EXON + chr4:105275958-105275978 CACAAAUUAAGUGAUGCUAA 898 54790_11_137 TET2 EXON + chr4:105275963-105275983 AUUAAGUGAUGCUAAUGGUC 899 54790_11_139 TET2 EXON + chr4:105275978-105275998 UGGUCAGGAAAAGCAGCCAU 900 54790_11_141 TET2 EXON + chr4:105275990-105276010 GCAGCCAUUGGCACUAGUCC 901 54790_11_142 TET2 EXON + chr4:105275991-105276011 CAGCCAUUGGCACUAGUCCA 902 54790_11_143 TET2 EXON + chr4:105275996-105276016 AUUGGCACUAGUCCAGGGUG 903 54790_11_145 TET2 EXON + chr4:105276003-105276023 CUAGUCCAGGGUGUGGCUUC 904 54790_11_148 TET2 EXON + chr4:105276011-105276031 GGGUGUGGCUUCUGGUGCAG 905 54790_11_150 TET2 EXON + chr4:105276023-105276043 UGGUGCAGAGGACAACGAUG 906 54790_11_152 TET2 EXON + chr4:105276028-105276048 CAGAGGACAACGAUGAGGUC 907 54790_11_156 TET2 EXON + chr4:105276053-105276073 AGACAGCGAGCAGAGCUUUC 908 54790_11_158 TET2 EXON + chr4:105276066-105276086 AGCUUUCUGGAUCCUGACAU 909 54790_11_160 TET2 EXON + chr4:105276067-105276087 GCUUUCUGGAUCCUGACAUU 910 54790_11_162 TET2 EXON + chr4:105276068-105276088 CUUUCUGGAUCCUGACAUUG 911 54790_11_165 TET2 EXON + chr4:105276069-105276089 UUUCUGGAUCCUGACAUUGG 912 54790_11_168 TET2 EXON + chr4:105276074-105276094 GGAUCCUGACAUUGGGGGAG 913 54790_11_169 TET2 EXON + chr4:105276080-105276100 UGACAUUGGGGGAGUGGCCG 914 54790_11_172 TET2 EXON + chr4:105276093-105276113 GUGGCCGUGGCUCCAACUCA 915 54790_11_173 TET2 EXON + chr4:105276094-105276114 UGGCCGUGGCUCCAACUCAU 916 54790_11_182 TET2 EXON + chr4:105276160-105276180 CCCCUUUAAAGAAUCCCAAU 917 54790_11_186 TET2 EXON + chr4:105276175-105276195 CCAAUAGGAAUCACCCCACCC 918 54790_11_193 TET2 EXON + chr4:105276225-105276245 AGCAUGAAUGAGCCAAAACA 919 54790_11_194 TET2 EXON + chr4:105276230-105276250 GAAUGAGCCAAAACAUGGCU 920 54790_11_196 TET2 EXON + chr4:105276238-105276258 CAAAACAUGGCUUGGCUCUU 921 54790_11_199 TET2 EXON + chr4:105276239-105276259 AAAACAUGGCUUGGCUCUUU 922 54790_11_200 TET2 EXON + chr4:105276251-105276271 GGCUCUUUGGGAAGCCAAAA 923 54790_11_210 TET2 EXON + chr4:105276275-105276295 UGAAAAAGCCCGUGAGAAAG 924 54790_11_214 TET2 EXON + chr4:105276294-105276314 GAGGAAGAGGUGUGAAAAGUA 925 54790_11_217 TET2 EXON + chr4:105276324-105276344 UAUGUGCCUCAGAAAUCCCA 926 54790_11_221 TET2 EXON + chr4:105276340-105276360 CCCAUGGCAAAAAAGUGAAA 927 54790_11_223 TET2 EXON + chr4:105276341-105276361 CCAUGGCAAAAAAGUGAAAC 928 54790_11_231 TET2 EXON + chr4:105276409-105276429 UCAUCAAGUCUCUUGCCGAA 929 54790_11_236 TET2 EXON + chr4:105276466-105276486 CAUCUCCAUAUGCCUUCACCU 930 54790_11_237 TET2 EXON + chr4:105276467-105276487 AUCUCCAUAUGCCUUCACUC 931 54790_11_239 TET2 EXON + chr4:105276474-105276494 UAUGCCUUCACUCGGGUUCAC 932 54790_11_240 TET2 EXON + chr4:105276475-105276495 AUGCCUUCACUCGGGUCACA 933 54790_11_243 TET2 EXON + chr4:105276515-105276535 AUGAUAUCACCCCCUUUUGU 934 54790_11_252 TET2 EXON + chr4:105276573-105276593 GUAGUAUAGUUCUCAUGACG 935 54790_11_253 TET2 EXON + chr4:105276574-105276594 UAGUUAGUUCUCAUGACGU 936 54790_11_256 TET2 EXON + chr4:105276580-105276600 AGUUCUCAUGACGUGGGCAG 937 54790_11_258 TET2 EXON + chr4:105276581-105276601 GUUCUCAUGACGUGGGCAGU 938 54790_11_259 TET2 EXON + chr4:105276582-105276602 UUCUCAUGACGUGGGCAGUG 939 54790_11_262 TET2 EXON + chr4:105276587-105276607 AUGACGUGGGCAGUGGGGAA 940 54790_11_263 TET2 EXON + chr4:105276611-105276631 CACAGUAUUCAUGACAAAUG 941 54790_11_265 TET2 EXON + chr4:105276614-105276634 AGUAUUCAUGACAAAAUGUGG 942 54790_11_267 TET2 EXON + chr4:105276615-105276635 GUAUUCAUGACAAAUGUGGU 943 54790_11_271 TET2 EXON + chr4:105276646-105276666 CAGCUCACCAGCAACAAAAG 944 54790_11_273 TET2 EXON + chr4:105276677-105276697 CCAUAGCACUUAAUUUUCAC 945 54790_11_275 TET2 EXON + chr4:105276688-105276708 AAUUUUCACUGGCUCCCAAG 946 54790_11_280 TET2 EXON + chr4:105276698-105276718 GGCUCCCAAGUGGUCACAGA 947 54790_11_283 TET2 EXON + chr4:105276706-105276726 AGUGGUCACAGAAUGGCAUCU 948 54790_11_285 TET2 EXON + chr4:105276738-105276758 AAGCAUUCUAUGCAAAAAGA 949 54790_11_288 TET2 EXON + chr4:105276741-105276761 CAUUCUAUGCAAAAAGAAGG 950 54790_11_289 TET2 EXON + chr4:105276742-105276762 AUUCUAUGCAAAAAGAAGGU 951 54790_11_291 TET2 EXON + chr4:105276743-105276763 UUCUAUGCAAAAAGAAGGUG 952 54790_11_297 TET2 EXON + chr4:105276780-105276800 CAAUUUACAUUUUUAACAC 953 54790_11_302 TET2 EXON + chr4:105276792-105276812 UUAAACACUGGUUCUAUUAU 954 54790_11_316 TET2 EXON + chr4:105276885-105276905 AUAUCAGUUUGCAUAGUCA 955 54790_11_321 TET2 EXON + chr4:105276925-105276945 UACUGUAGUAAUUACAGUGAC 956 54790_11_323 TET2 EXON + chr4:105276945-105276965 AGGAAUCUUAAAAUACCAUC 957 54790_11_329 TET2 EXON + chr4:105276975-105276995 UAUAUGAUGUACUGAAAUAC 958 54790_11_330 TET2 EXON + chr4:105276983-105277003 GUACUGAAAUACUGGAAUUA 959 54790_11_344 TET2 EXON + chr4:105277042-105277062 UUAUUUAUCAAAAUAGCUAC 960 54790_11_352 TET2 EXON + chr4:105277058-105277078 CUACAGGAAAACAUGAAUAGC 961 54790_11_356 TET2 EXON + chr4:105277078-105277098 AGGAAAAACACUGAAUUUGUU 962 54790_11_359 TET2 EXON + chr4:105277094-105277114 UGUUUGGAUGUUCUAAAAA 963 54790_11_367 TET2 EXON + chr4:105277108-105277128 AAGAAAUGGUGCUAAGAAAA 964 54790_11_377 TET2 EXON + chr4:105277187-105277207 CUCCAGUGCCCUUGAAUAAU 965 54790_11_378 TET2 EXON + chr4:105277188-105277208 UCCAGUGCCCUUGAAUAAUA 966 54790_11_379 TET2 EXON + chr4:105277189-105277209 CCAGUGCCCUUGAAUAAUAG 967 54790_11_393 TET2 EXON + chr4:105277255-105277275 CAAGCUUAGUUUUUAAAAUG 968 54790_11_395 TET2 EXON + chr4:105277267-105277287 UUAAAAUGUGGACAUUUUAA 969 54790_11_401 TET2 EXON + chr4:105277274-105277294 GUGGACAUUUUAAAGGCCUC 970 54790_11_410 TET2 EXON + chr4:105277304-105277324 UCAUCCAGUGAAGUCCUUGU 971 54790_11_419 TET2 EXON + chr4:105277438-105277458 UGACAACCUUGAACAAUGCUA 972 54790_11_437 TET2 EXON + chr4:105277501-105277521 AUGCAAAGUUGAUUUUUUUA 973 54790_11_465 TET2 EXON + chr4:105277599-105277619 ACAGCCAGUUAAAAUCCACCA 974 54790_11_466 TET2 EXON + chr4:105277600-105277620 CAGCCAGUUAAAUCCACCAU 975 54790_11_467 TET2 EXON + chr4:105277601-105277621 AGCCAGUUAAAUCCACCAUG 976 54790_11_469 TET2 EXON + chr4:105277609-105277629 AAAUCCACCAUGGGGCUUAC 977 54790_11_472 TET2 EXON + chr4:105277617-105277637 CAUGGGGCUUACUGGAUUCA 978 54790_11_474 TET2 EXON + chr4:105277618-105277638 AUGGGGCUUACUGGAUUCAA 979 54790_11_478 TET2 EXON + chr4:105277649-105277669 AGUCCACAAAACAUGUUUUC 980 54790_11_492 TET2 EXON + chr4:105277753-105277773 AAGAAUUUUCUAUUAACUGC 981 54790_11_503 TET2 EXON + chr4:105277818-105277838 CUGAAGCCUAUGCUAUUUUA 982 54790_11_504 TET2 EXON + chr4:105277826-105277846 UAUGCUAUUUUAUGGAUCAU 983 54790_11_511 TET2 EXON + chr4:105277846-105277866 AGGCUCUUCAGAGAACUGAA 984 54790_11_524 TET2 EXON + chr4:105277924-105277944 UAAGUGUCCUCUUUAACAAG 985 54790_11_532 TET2 EXON + chr4:105277963-105277983 CCUGCAUAAGAUGAAUAAAC 986 54790_11_533 TET2 EXON + chr4:105277964-105277984 CUGCAUAAGAUGAAUAAAACA 987 54790_11_539 TET2 EXON + chr4:105278008-105278028 AGUUAAAAAGAAAACAAAAAC 988 54790_11_541 TET2 EXON + chr4:105278015-105278035 AAGAAACAAAAACAGGCAGC 989 54790_11_542 TET2 EXON + chr4:105278025-105278045 AACAGGCAGCUGGUUUGCUG 990 54790_11_543 TET2 EXON + chr4:105278028-105278048 AGGCAGCUGGUUUGCUGUGG 991 54790_11_574 TET2 EXON + chr4:105278210-105278230 AAGCAGAAUUCACAUCAUGA 992 54790_11_587 TET2 EXON + chr4:105278310-105278330 CAUAUACCUCAACACUAGUU 993 54790_11_589 TET2 EXON + chr4:105278317-105278337 CUCAACACUAGUUUGGCAAU 994 54790_11_627 TET2 EXON + chr4:105278467-105278487 CCUUUUGUUCUAAAAAAUUC 995 54790_11_628 TET2 EXON + chr4:105278468-105278488 CUUUUUGUUCUAAAAAAUUCA 996 54790_11_637 TET2 EXON + chr4:105278532-105278552 UGUUUAUGUAAAAAUUGUUGU 997 54790_11_643 TET2 EXON + chr4:105278556-105278576 UAAUAAAUAUAUUCUUUGUC 998 54790_11_645 TET2 EXON + chr4:105278557-105278577 AAUAAAUAUAUUCUUUGUCA 999 54790_11_664 TET2 EXON + chr4:105278640-105278660 AACUAAUUUUGUAAAUCUGU 1000 54790_11_679 TET2 EXON + chr4:105278680-105278700 AAAAGCAUUUUAAAAAGUUUG 1001 54790_11_686 TET2 EXON + chr4:105278704-105278724 AUCUUUUGACUGUUUCAAGC 1002 54790_11_700 TET2 EXON + chr4:105278748-105278768 AGAAUGCACUGAGUUGAUAA 1003 54790_11_701 TET2 EXON + chr4:105278749-105278769 GAAUGCACUGAGUUGAUAAA 1004 54790_11_703 TET2 EXON + chr4:105278762-105278782 UGAUAAAGGGAAAAAUUGUA 1005 54790_11_707 TET2 EXON + chr4:105278766-105278786 AAAGGGAAAAAAUUGUAAGGC 1006 54790_11_708 TET2 EXON + chr4:105278773-105278793 AAAAUUGUAAGGCAGGAGUU 1007 54790_11_710 TET2 EXON + chr4:105278780-105278800 UAAGGCAGGAGUUUGGCAAG 1008 54790_11_711 TET2 EXON + chr4:105278787-105278807 GGAGUUUGGCAAGUGGCUGU 1009 54790_11_721 TET2 EXON + chr4:105278846-105278866 UUUGAUCCUGUAAUCACUGA 1010 54790_11_728 TET2 EXON + chr4:105278862-105278882 CUGAAGGUACAUACUCCCAUG 1011 54790_11_729 TET2 EXON + chr4:105278878-105278898 CAUGUGGACUUCCCUUAAAC 1012 54790_11_731 TET2 EXON + chr4:105278892-105278912 UUAAAACAGGCAAACACCUAC 1013 54790_11_733 TET2 EXON + chr4:105278897-105278917 CAGGCAAACACCUACAGGUA 1014 54790_11_734 TET2 EXON + chr4:105278927-105278947 CAGAUUGUACAAUUACAUUU 1015 54790_11_748 TET2 EXON + chr4:105278978-105278998 UAAAAUAAAUUCUUAAUCAG 1016 54790_11_751 TET2 EXON + chr4:105278981-105279001 AAUAAAUUCUUAAUCAGAGG 1017 54790_11_753 TET2 EXON + chr4:105278988-105279008 UCUUAAUCAGAGGAGGCCUU 1018 54790_11_754 TET2 EXON + chr4:105278989-105279009 CUUAAUCAGAGGAGGCCUUU 1019 54790_11_757 TET2 EXON + chr4:105278998-105279018 AGGAGGCCUUUGGGUUUUAU 1020 54790_11_762 TET2 EXON + chr4:105279017-105279037 UUGGUCAAAAUCUUUGUAAGC 1021 54790_11_772 TET2 EXON + chr4:105279052-105279072 UAAAAAAUUUCUUGAAUUUG 1022 54790_11_799 TET2 EXON + chr4:105279173-105279193 UUUGAUUACUACAUGUGCAU 1023 54790_11_813 TET2 EXON + chr4:105279240-105279260 ACUGUCAUUUGUUAAACUGC 1024 54790_11_818 TET2 EXON + chr4:105279254-105279274 AACUGCUGGCCAACAAGAAC 1025 54790_11_822 TET2 EXON + chr4:105279267-105279287 CAAGAACAGGAAGUAUAGUU 1026 54790_11_825 TET2 EXON + chr4:105279268-105279288 AAGAACAGGAAGUAUAGUUU 1027 54790_11_827 TET2 EXON + chr4:105279269-105279289 AGAACAGGAAGUAUAGUUUG 1028 54790_11_828 TET2 EXON + chr4:105279270-105279290 GAACAGGAAGUAUAGUUUGG 1029 54790_11_829 TET2 EXON + chr4:105279271-105279291 AACAGGAAGUAUAGUUUGGG 1030 54790_11_832 TET2 EXON + chr4:105279275-105279295 GGAAGUAUAGUUUUGGGGGU 1031 54790_11_833 TET2 EXON + chr4:105279276-105279296 GAAGUAUAGUUUGGGGGGUU 1032 54790_11_836 TET2 EXON + chr4:105279277-105279297 AAGUAUAGUUUGGGGGGUUG 1033 54790_11_841 TET2 EXON + chr4:105279292-105279312 GGUUGGGGAGAGUUUACAUA 1034 54790_11_851 TET2 EXON + chr4:105279311-105279331 AAGGAAGAGAGAGAAAUUGAG 1035 54790_11_859 TET2 EXON + chr4:105279373-105279393 CCUGCCUCAGUUAGAAUGAA 1036 54790_11_864 TET2 EXON + chr4:105279402-105279422 GAUCUACAAUUUGCUAAUAU 1037 54790_11_865 TET2 EXON + chr4:105279411-105279431 UUUGCUAAUAUAGGAAUAUUC 1038 54790_11_871 TET2 EXON + chr4:105279449-105279469 UACUUGAAAAUGCUUCUGAG 1039 54790_11_886 TET2 EXON + chr4:105279524-105279544 CAGUUCACUUCUGAAGCUAG 1040 54790_11_890 TET2 EXON + chr4:105279538-105279558 AGCUAGUGGUUAACUUGUGU 1041 54790_11_912 TET2 EXON + chr4:105279632-105279652 UUUCAUUUUCAUGAGAUGUU 1042 54790_11_920 TET2 EXON + chr4:105279648-105279668 UGUUUGGUUUAUAAGAUCUG 1043 54790_11_921 TET2 EXON + chr4:105279652-105279672 UGGUUUAUAAGAUCUGAGGA 1044 54790_11_928 TET2 EXON + chr4:105279691-105279711 UAUUGUAAUGUUAUGAAUGC 1045 54790_11_954 TET2 EXON - chr4:105275038-105275058 UCGCAAAAGUUCUGUGGACA 1046 54790_11_955 TET2 EXON - chr4:105275039-105275059 GUCGCAAAAGUUCUGUGGAC 1047 54790_11_957 TET2 EXON - chr4:105275044-105275064 ACAAAGUCGCAAAAGUUCUG 1048 54790_11_960 TET2 EXON - chr4:105275165-105275185 AGUUGACAGACUCUGUCUGA 1049 54790_11_961 TET2 EXON - chr4:105275166-105275186 GAGUUGACAGACUCUGUCUG 1050 54790_11_970 TET2 EXON - chr4:105275206-105275226 CCGUCUCAUGUAUGGAUUGG 1051 54790_11_972 TET2 EXON - chr4:105275209-105275229 GGGCCGUCUCAUGUAUGGAU 1052 54790_11_973 TET2 EXON - chr4:105275214-105275234 GGAUUGGGCCGUCUCAUGUA 1053 54790_11_977 TET2 EXON - chr4:105275229-105275249 GGAUAAGGACUAACUGGAUU 1054 54790_11_978 TET2 EXON - chr4:105275230-105275250 UGGAUAAGGACUAACUGGAU 1055 54790_11_980 TET2 EXON - chr4:105275235-105275255 GAGUUUGGAUAAGGACUAAC 1056 54790_11_982 TET2 EXON - chr4:105275244-105275264 GUGUGUGAAGAGUUUGGAUA 1057 54790_11_984 TET2 EXON - chr4:105275250-105275270 UCUGAAGGUGUGUGAAGAGUU 1058 54790_11_991 TET2 EXON - chr4:105275287-105275307 GGAAUAGAAGUUCAUAGGGC 1059 54790_11_992 TET2 EXON - chr4:105275291-105275311 AGGUGGAAUAGAAGUUCUA 1060 54790_11_993 TET2 EXON - chr4:105275292-105275312 GAGGUGGAAUAGAAGUUCAU 1061 54790_11_999 TET2 EXON - chr4:105275308-105275328 ACCUGCAGCUUGAGAUGAGG 1062 54790_11_1001 TET2 EXON - chr4:105275311-105275331 UGAACCUGCAGCUUGAGAUG 1063 54790_11_1012 TET2 EXON - chr4:105275352-105275372 AGCCCAGGGUAAGGGUUCAU 1064 54790_11_1013 TET2 EXON - chr4:105275353-105275373 AAGCCCAGGGUAAGGGUUCA 1065 54790_11_1017 TET2 EXON - chr4:105275360-105275380 GAUUCAAAAGCCCAGGGUAA 1066 54790_11_1018 TET2 EXON - chr4:105275361-105275381 UGAUUCAAAAGCCCAGGGUA 1067 54790_11_1021 TET2 EXON - chr4:105275366-105275386 UAUUCUGAUUCAAAAAGCCCA 1068 54790_11_1022 TET2 EXON - chr4:105275367-105275387 GUAUUCUGAUUCAAAAGCCC 1069 54790_11_1026 TET2 EXON - chr4:105275389-105275409 GCAUUGAUAUGAUGGAUAUU 1070 54790_11_1027 TET2 EXON - chr4:105275390-105275410 UGCAUUGAUAUGAUGGAUAU 1071 54790_11_1031 TET2 EXON - chr4:105275397-105275417 UUUCCAUUGCAUUGAUAUGA 1072 54790_11_1034 TET2 EXON - chr4:105275420-105275440 GGGAGCAGUUGUCCACUGAU 1073 54790_11_1035 TET2 EXON - chr4:105275440-105275460 AGAAUAGGAACCCAGAUAUG 1074 54790_11_1037 TET2 EXON - chr4:105275441-105275461 GAGAAUAGGAACCCAGAUAU 1075 54790_11_1040 TET2 EXON - chr4:105275442-105275462 GGAGAAUAGGAACCCAGAUA 1076 54790_11_1042 TET2 EXON - chr4:105275455-105275475 CGGCUGAGACUGGGGAGAAU 1077 54790_11_1046 TET2 EXON - chr4:105275463-105275483 AGAUCCAUCGGCUGAGACUG 1078 54790_11_1049 TET2 EXON - chr4:105275464-105275484 CAGAUCCAUCGGCUGAGACU 1079 54790_11_1050 TET2 EXON - chr4:105275465-105275485 ACAGAUCCAUCGGCUGAGAC 1080 54790_11_1055 TET2 EXON - chr4:105275475-105275495 GGAUACCUAUACAGAUCCAU 1081 54790_11_1058 TET2 EXON - chr4:105275496-105275516 UUAGACAGAGGGUCUUGGCU 1082 54790_11_1060 TET2 EXON - chr4:105275501-105275521 UGAGCUUAGACAGAGGGUCU 1083 54790_11_1061 TET2 EXON - chr4:105275507-105275527 GUAGACUGAGCUUAGACAGA 1084 54790_11_1062 TET2 EXON - chr4:105275508-105275528 GGUAGACUGAGCUUAGACAG 1085 54790_11_1067 TET2 EXON - chr4:105275529-105275549 UGGUAAAAGUGUAUGGAUGGG 1086 54790_11_1068 TET2 EXON - chr4:105275532-105275552 GGCUGGUAAAAGUGUAUGGAU 1087 54790_11_1069 TET2 EXON - chr4:105275533-105275553 UGGCUGGUAAAGGUGUAUGGA 1088 54790_11_1072 TET2 EXON - chr4:105275537-105275557 ACCUUGGCUGGUAAAGUGUA 1089 54790_11_1075 TET2 EXON - chr4:105275549-105275569 GGCUAUUUCCAAAACCUUGGC 1090 54790_11_1076 TET2 EXON - chr4:105275553-105275573 CUCUGGCUAUUUCCAAACCU 1091 54790_11_1079 TET2 EXON - chr4:105275570-105275590 AGUAUUUAGAUGUAAAAACUC 1092 54790_11_1085 TET2 EXON - chr4:105275606-105275626 AACCAUCUCCCUGCAUAUUU 1093 54790_11_1089 TET2 EXON - chr4:105275641-105275661 AUGAUGUACAUUUGGUCUAA 1094 54790_11_1092 TET2 EXON - chr4:105275649-105275669 UUCCCUACAUGAUGUACAUU 1095 54790_11_1093 TET2 EXON - chr4:105275676-105275696 AUCUCAUGAGUGGGAUAAGG 1096 54790_11_1095 TET2 EXON - chr4:105275679-105275699 UCCAUCUCAUGAGUGGGAUA 1097 54790_11_1097 TET2 EXON - chr4:105275685-105275705 UGGCCAUCCAUCUCAUGAGU 1098 54790_11_1098 TET2 EXON - chr4:105275686-105275706 GUGGCCAUCCAUCUCAUGAG 1099 54790_11_1102 TET2 EXON - chr4:105275705-105275725 UAGAGGUGGCUCCCAUGAAG 1100 54790_11_1105 TET2 EXON - chr4:105275719-105275739 AUUGGGUGGUAAUCUAGAGG 1101 54790_11_1107 TET2 EXON - chr4:105275722-105275742 CAGAUUGGGUGGUAAUCUAG 1102 54790_11_1111 TET2 EXON - chr4:105275733-105275753 UUUGGAUUGCUCAGAUUGGG 1103 54790_11_1112 TET2 EXON - chr4:105275736-105275756 AUGUUUGGAUUGCUCAGAUU 1104 54790_11_1113 TET2 EXON - chr4:105275737-105275757 CAUGUUUGGAUUGCUCAGAU 1105 54790_11_1120 TET2 EXON - chr4:105275751-105275771 CCAUUUUUAUAGUCCAUGUU 1106 54790_11_1125 TET2 EXON - chr4:105275787-105275807 UAGUUAUGGAUUAUGUGAGA 1107 54790_11_1129 TET2 EXON - chr4:105275801-105275821 CCGGAGCUGCACUGUAGUUA 1108 54790_11_1133 TET2 EXON - chr4:105275820-105275840 AGAGAGCUGUUGAACAUGCC 1109 54790_11_1144 TET2 EXON - chr4:105275848-105275868 CUCCUUGUUUUGGAGAUGCA 1110 54790_11_1145 TET2 EXON - chr4:105275849-105275869 UCUCCUUGUUUUGGAGAUGC 1111 54790_11_1148 TET2 EXON - chr4:105275858-105275878 GCAUGUCAUUCUCCUUGUUU 1112 54790_11_1154 TET2 EXON - chr4:105275884-105275904 UGAUAACCCAUUAGCUGUGU 1113 54790_11_1155 TET2 EXON - chr4:105275885-105275905 UUGAUAACCCAUUAGCUGUG 1114 54790_11_1161 TET2 EXON - chr4:105275916-105275936 GUUCUAUCAUGGUUAAGGC 1115 54790_11_1165 TET2 EXON - chr4:105275927-105275947 GGACACAAGCAGUUCUAUCA 1116 54790_11_1169 TET2 EXON - chr4:105275948-105275968 UUAAUUUGUGUAAGCCUCCU 1117 54790_11_1175 TET2 EXON - chr4:105275997-105276017 ACACCCUGGACUAGUGCCAA 1118 54790_11_1176 TET2 EXON - chr4:105276011-105276031 CUGCACCAGAAGCCACACCC 1119 54790_11_1182 TET2 EXON - chr4:105276081-105276101 ACGGCCACUCCCCCAAUGUC 1120 54790_11_1186 TET2 EXON - chr4:105276100-105276120 UGACCCAUGAGUUGGAGCCA 1121 54790_11_1188 TET2 EXON - chr4:105276108-105276128 AUGAGAAUUGACCCAUGAGU 1122 54790_11_1200 TET2 EXON - chr4:105276157-105276177 GGGAUUCUUUAAAGGGGUUG 1123 54790_11_1202 TET2 EXON - chr4:105276163-105276183 CCUAUUGGGAUUCUUUAAG 1124 54790_11_1203 TET2 EXON - chr4:105276164-105276184 UCCUAUUGGGAUUCUUUAAA 1125 54790_11_1205 TET2 EXON - chr4:105276165-105276185 UUCCUAUUGGGAUUCUUUAA 1126 54790_11_1207 TET2 EXON - chr4:105276177-105276197 CUGGUGGGGUGAUUCCUAUU 1127 54790_11_1209 TET2 EXON - chr4:105276178-105276198 CCUGGUGGGGUGAUUCCUAU 1128 54790_11_1211 TET2 EXON - chr4:105276191-105276211 AGACGAGGGAGAUCCUGGUG 1129 54790_11_1212 TET2 EXON - chr4:105276192-105276212 AAGACGAGGGAGAUCCUGGU 1130 54790_11_1214 TET2 EXON - chr4:105276193-105276213 AAAGACGAGGGAGAUCCUGG 1131 54790_11_1216 TET2 EXON - chr4:105276196-105276216 GUAAAAGACGAGGGAGAUCC 1132 54790_11_1219 TET2 EXON - chr4:105276205-105276225 CUUAUGCUGGUAAAAAGACGA 1133 54790_11_1221 TET2 EXON - chr4:105276206-105276226 UCUUAUGCUGGUAAAAGACG 1134 54790_11_1228 TET2 EXON - chr4:105276218-105276238 GCUCAUUCAUGCUCUUAUGC 1135 54790_11_1230 TET2 EXON - chr4:105276240-105276260 CAAAGAGCCAAGCCAUGUUU 1136 54790_11_1241 TET2 EXON - chr4:105276268-105276288 ACGGGCUUUUUCAGCCAUUU 1137 54790_11_1246 TET2 EXON - chr4:105276286-105276306 ACACUCUUCCUCUUUCUCAC 1138 54790_11_1247 TET2 EXON - chr4:105276287-105276307 CACACUCUUCCUCUUUCUCA 1139 54790_11_1251 TET2 EXON - chr4:105276320-105276340 AUUUCUGAGGCACAUAGUCU 1140 54790_11_1252 TET2 EXON - chr4:105276321-105276341 GAUUUCUGAGGCACAUAGUC 1141 54790_11_1260 TET2 EXON - chr4:105276333-105276353 UUUUUGCCAUGGGAUUUCUG 1142 54790_11_1263 TET2 EXON - chr4:105276343-105276363 CCGUUUCACUUUUUUGCCAU 1143 54790_11_1265 TET2 EXON - chr4:105276344-105276364 CCCGUUUCACUUUUUUGCCA 1144 54790_11_1269 TET2 EXON - chr4:105276369-105276389 GAAGUUUCAUGUGGCUCAGC 1145 54790_11_1270 TET2 EXON - chr4:105276378-105276398 GUGGGCUCUGAAGUUUCAUG 1146 54790_11_1273 TET2 EXON - chr4:105276396-105276416 UUGAUGAAAACGCAGGUAAGU 1147 54790_11_1274 TET2 EXON - chr4:105276397-105276417 CUUGAUGAAAACGCAGGUAAG 1148 54790_11_1277 TET2 EXON - chr4:105276404-105276424 CAAGAGACUUGAUGAAACGC 1149 54790_11_1281 TET2 EXON - chr4:105276427-105276447 GGUCACGGACAUGGUCCUUU 1150 54790_11_1282 TET2 EXON - chr4:105276436-105276456 GGAGUCUGUGGUCACGGACA 1151 54790_11_1284 TET2 EXON - chr4:105276442-105276462 UACUGUGGAGUCUGUGGUCA 1152 54790_11_1286 TET2 EXON - chr4:105276448-105276468 UGUAGUUACUGUGGAGUCUG 1153 54790_11_1288 TET2 EXON - chr4:105276457-105276477 AUAUGGAGAUGUAGUUACUG 1154 54790_11_1290 TET2 EXON - chr4:105276474-105276494 GUGACCCGAGUGAAGGCAUA 1155 54790_11_1294 TET2 EXON - chr4:105276481-105276501 AGGCCCUGUGACCCGAGUGA 1156 54790_11_1297 TET2 EXON - chr4:105276501-105276521 UAUCAUAUAUAUCUGUUGUA 1157 54790_11_1300 TET2 EXON - chr4:105276527-105276547 GUGAGGUAACCAACAAAAGG 1158 54790_11_1301 TET2 EXON - chr4:105276528-105276548 AGUGAGGUAACCAACAAAAG 1159 54790_11_1303 TET2 EXON - chr4:105276529-105276549 AAGUGAGGUAAACCAACAAAA 1160 54790_11_1305 TET2 EXON - chr4:105276530-105276550 CAAGUGAGGUAACCAACAAA 1161 54790_11_1310 TET2 EXON - chr4:105276544-105276564 GGUUGUGGUCUUUUCAAGUG 1162 54790_11_1312 TET2 EXON - chr4:105276559-105276579 UACUACUGACAGGUUGGUUG 1163 54790_11_1313 TET2 EXON - chr4:105276565-105276585 GAACUAUACUACUGACAGGU 1164 54790_11_1314 TET2 EXON - chr4:105276569-105276589 AUGAGAACUAAUACUACUGAC 1165 54790_11_1331 TET2 EXON - chr4:105276646-105276666 CUUUUGUUGCUGGUGAGCUG 1166 54790_11_1334 TET2 EXON - chr4:105276656-105276676 AAGAUAACCUCUUUUGUUGC 1167 54790_11_1336 TET2 EXON - chr4:105276680-105276700 CCAGUGAAAAUUAAGUGCUA 1168 54790_11_1339 TET2 EXON - chr4:105276705-105276725 GAUGCCAUCUGUGACCACCUU 1169 54790_11_1344 TET2 EXON - chr4:105276706-105276726 AGAUGCCAUCUGUGACCACCU 1170 54790_11_1354 TET2 EXON - chr4:105276738-105276758 UCUUUUUGCAUAGAAUGCUU 1171 54790_11_1363 TET2 EXON - chr4:105276780-105276800 GUGUUUAAAAAAUGUAAAUUG 1172 54790_11_1370 TET2 EXON - chr4:105276841-105276861 AGAGUUGUAAAGCGGGGGGGG 1173 54790_11_1371 TET2 EXON - chr4:105276842-105276862 UAGAGUUGUAAAGCGGGGGGG 1174 54790_11_1374 TET2 EXON - chr4:105276843-105276863 GUAGAGUUGUAAAGCGGGGGG 1175 54790_11_1376 TET2 EXON - chr4:105276844-105276864 UGUAGAGUUGUAAAGCGGGGG 1176 54790_11_1378 TET2 EXON - chr4:105276845-105276865 GUGUAGAGUUGUAAAGCGGGG 1177 54790_11_1379 TET2 EXON - chr4:105276846-105276866 UGUGUAGAGGUUGUAAAGCGGG 1178 54790_11_1382 TET2 EXON - chr4:105276847-105276867 AUGUGUAGAGGUUGUAAAGCGG 1179 54790_11_1383 TET2 EXON - chr4:105276848-105276868 GAUGUGUAGAGGUUGUAAAGCG 1180 54790_11_1386 TET2 EXON - chr4:105276849-105276869 AGAUGUGUAGAGGUUGUAAGC 1181 54790_11_1388 TET2 EXON - chr4:105276850-105276870 CAGAUGUGUAGAGUUGUAAG 1182 54790_11_1394 TET2 EXON - chr4:105276876-105276896 AAACUUGAUAUUAUUAAAAG 1183 54790_11_1406 TET2 EXON - chr4:105276963-105276983 AUCAUAUAUUCAGCACCAGA 1184 54790_11_1440 TET2 EXON - chr4:105277160-105277180 AUAGCAUCUUGAUGAAUAA 1185 54790_11_1444 TET2 EXON - chr4:105277192-105277212 CCCCUAUUAUUCAAGGGCAC 1186 54790_11_1446 TET2 EXON - chr4:105277198-105277218 AAGGUACCCCUAUUAUUCAA 1187 54790_11_1447 TET2 EXON - chr4:105277199-105277219 AAAGGUACCCCUAUUAUUCA 1188 54790_11_1451 TET2 EXON - chr4:105277217-105277237 UGAUAAAAACUUGAAUGAAA 1189 54790_11_1457 TET2 EXON - chr4:105277246-105277266 AACUAAGCUUGUGUAAGAAU 1190 54790_11_1463 TET2 EXON - chr4:105277293-105277313 ACUGGAUGAGCAAAAUCCAG 1191 54790_11_1469 TET2 EXON - chr4:105277311-105277331 UUGUCCUACAAGGACUUCAC 1192 54790_11_1471 TET2 EXON - chr4:105277321-105277341 AUAUCGUUUAUUGUCCUACA 1193 54790_11_1478 TET2 EXON - chr4:105277432-105277452 UGUUCAAGUUGUCAAAAGCUU 1194 54790_11_1501 TET2 EXON - chr4:105277563-105277583 AUUGCUCAUCAGCAGAUGCA 1195 54790_11_1505 TET2 EXON - chr4:105277591-105277611 UUAACUGGCUGUGUUAAAAA 1196 54790_11_1507 TET2 EXON - chr4:105277606-105277626 AGCCCCAUGGUGGAUUUAAC 1197 54790_11_1509 TET2 EXON - chr4:105277616-105277636 GAAUCCAGUAAGCCCCAUGG 1198 54790_11_1512 TET2 EXON - chr4:105277619-105277639 CUUGAAUCCAGUAAGCCCCA 1199 54790_11_1517 TET2 EXON - chr4:105277655-105277675 GCACCAGAAAACAUGUUUUG 1200 54790_11_1547 TET2 EXON - chr4:105277827-105277847 UAUGAUCCAUAAAAUAGCAU 1201 54790_11_1553 TET2 EXON - chr4:105277879-105277899 GUACAUAAUUAUCAACACAA 1202 54790_11_1558 TET2 EXON - chr4:105277934-105277954 GCUCAAUCCUCUUGUUAAG 1203 54790_11_1565 TET2 EXON - chr4:105277966-105277986 CCUGUUUAUUCAUCUUAUGC 1204 54790_11_1574 TET2 EXON - chr4:105277996-105278016 UUUUAACUGACAGAUUCACA 1205 54790_11_1613 TET2 EXON - chr4:105278246-105278266 CAUUAUGAUAUAUUUGUAGGC 1206 54790_11_1621 TET2 EXON - chr4:105278304-105278324 UGUUGAGGUAUAUGACAAGGU 1207 54790_11_1624 TET2 EXON - chr4:105278319-105278339 CUAUUGCCAAACUAGUGUUG 1208 54790_11_1630 TET2 EXON - chr4:105278373-105278393 AAGGACUUGGAAAAAAAAUGA 1209 54790_11_1636 TET2 EXON - chr4:105278386-105278406 UAACAAUAAAAAAAAGGACU 1210 54790_11_1643 TET2 EXON - chr4:105278392-105278412 UUUUUUUAACAAUAAAAAAA 1211 54790_11_1647 TET2 EXON - chr4:105278423-105278443 AGAAAUCAAGUAUUGAAAAA 1212 54790_11_1658 TET2 EXON - chr4:105278470-105278490 CCUGAAUUUUUAGAACAAAA 1213 54790_11_1667 TET2 EXON - chr4:105278513-105278533 ACAGGUGACAUGUUGGCAUA 1214 54790_11_1669 TET2 EXON - chr4:105278514-105278534 CACAGGUGACAUGUUGGCAU 1215 54790_11_1674 TET2 EXON - chr4:105278520-105278540 CAUAAACACAGGUGACAUGU 1216 54790_11_1675 TET2 EXON - chr4:105278531-105278551 CAACAAUUUUACAUAAAACAC 1217 54790_11_1682 TET2 EXON - chr4:105278589-105278609 AGAAGGGAUUCAAAAAUAAAA 1218 54790_11_1683 TET2 EXON - chr4:105278590-105278610 UAGAAGGGAUUCAAAAUAAA 1219 54790_11_1685 TET2 EXON - chr4:105278605-105278625 CAUGUACAAGUAAAAAUAGAA 1220 54790_11_1687 TET2 EXON - chr4:105278606-105278626 ACAUGUACAAGUAAAAUAGA 1221 54790_11_1733 TET2 EXON - chr4:105278813-105278833 GAGAGUUACAAGUAAGUCUC 1222 54790_11_1739 TET2 EXON - chr4:105278855-105278875 UAUGUACCUUCAGUGAUUC 1223 54790_11_1746 TET2 EXON - chr4:105278880-105278900 CUGUUUAAGGGAAGUCCACA 1224 54790_11_1749 TET2 EXON - chr4:105278892-105278912 GUAGGUGUUUGCCUGUUUAA 1225 54790_11_1751 TET2 EXON - chr4:105278893-105278913 UGUAGGUGUUUGCCUGUUUA 1226 54790_11_1754 TET2 EXON - chr4:105278910-105278930 CUGUUGCACACCAUACCUGU 1227 54790_11_1758 TET2 EXON - chr4:105278953-105278973 UAGUAAGCAAAAAUGUAUUU 1228 54790_11_1768 TET2 EXON - chr4:105279007-105279027 AUUUGACCAAUAAAACCCAA 1229 54790_11_1784 TET2 EXON - chr4:105279084-105279104 UUUUGGAAAUGUUUGCAAAU 1230 54790_11_1789 TET2 EXON - chr4:105279101-105279121 GUAAGCAAAGCAAACAUUUU 1231 54790_11_1792 TET2 EXON - chr4:105279127-105279147 CAAAAAACAUUAAAAUCAUG 1232 54790_11_1796 TET2 EXON - chr4:105279154-105279174 AUGUUUGGGGCUAGAUAUUA 1233 54790_11_1797 TET2 EXON - chr4:105279167-105279187 AUGUAGUAAUCAAAUGUUUG 1234 54790_11_1798 TET2 EXON - chr4:105279168-105279188 CAUGUAGUAAUCAAAUGUUU 1235 54790_11_1800 TET2 EXON - chr4:105279169-105279189 ACAUGUAGUAAUCAAAUGUU 1236 54790_11_1803 TET2 EXON - chr4:105279212-105279232 CAGAAAUCAAAUAUUAAGAA 1237 54790_11_1809 TET2 EXON - chr4:105279240-105279260 GCAGUUUAACAAAUGACAGU 1238 54790_11_1814 TET2 EXON - chr4:105279266-105279286 ACUAUACUUCCUGUUCUUGU 1239 54790_11_1832 TET2 EXON - chr4:105279376-105279396 CCAUUCAUUCUAACUGAGGC 1240 54790_11_1833 TET2 EXON - chr4:105279380-105279400 CUUUCCAUUCAUUCUAACUG 1241 54790_11_1841 TET2 EXON - chr4:105279449-105279469 CUCAGAAGCAUUUUCAAGUA 1242 54790_11_1877 TET2 EXON - chr4:105279748-105279768 AACACUCACAUAGCAUUAUC 1243

TALEN Gene Editing Systems

TALEN is produced artificially by fusing the effector DNA-binding domain of TAL with a DNA cleavage domain. Transcription activator effects (TALE) can be engineered to bind to any desired DNA sequence, including part of the HLA or TCR gene. By combining engineered TALEs with a DNA cleavage domain, a restriction enzyme can be generated that is specific for any desired DNA sequence, including an HLA or TCR sequence. They can then be introduced into a cell where they can be used to edit the genome. Boch (2011) Nature Biotech . 29:135-6; and Boch et al. (2009) Science 326: 1509-12; Moscow et al. (2009) Science 326: 3501.

TALE are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a repetitive, highly conserved sequence of 33-34 amino acids, except for the 12th and 13th amino acids. These two positions are highly variable, showing strong correlation with specific nucleotide recognition. Thus, they can be engineered to bind to the desired DNA sequence.

To obtain TALEN, the TALE protein is fused to a nuclease (N), which is a wild-type FokI endonuclease or a mutant FokI endonuclease. Several mutations have been made to FokI for its application to TALEN; they, for example, increase cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39:e82; Miller et al. (2011) Nature Biotech . 29:143-8; Hockemeyer et al. (2011) Nature Biotech . 29:731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25:786-793; and Guo et al. (2010) J. Mol. Biol. 200:96.

The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains for regions in the target genome with proper orientations and spacings. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech. 29:143-8.

Anti-Tet TALEN, eg Tet1, Tet2 and/or Tet3, eg Tet2, can be used intracellularly to provide a double strand break (DSB). A mutation can be introduced at the site of the break if the repair mechanisms do not properly repair the break through non-homologous end joining. For example, improper repair may introduce a frameshift mutation. Alternatively, foreign DNA can be introduced into the cell along with TALEN, eg DNA encoding CAR, eg as described herein; depending on the foreign DNA sequence and chromosomal sequence, this process can be used to insert DNA encoding CAR, for example, as described herein, at or near the site targeted by TALEN. As shown in the present description in the examples, but without being bound by theory, such an insertion can lead to the expression of CARs, as well as disruption of the Tet gene, for example, Tet1, Tet2 and/or Tet3, for example Tet2. Such a foreign DNA molecule is referred to herein as "messenger DNA". In some embodiments, the template DNA further comprises homology arms on the 5' side, 3' side, or both 5' and 3' sides of the nucleic acid of the template DNA that encodes the molecule or molecules of interest (e.g. , which encodes the CAR described in the present description), where the specified homology arms are complementary to the genomic DNA sequence flanking the target sequence.

TALENs specific to sequences in Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, can be constructed using any method known in the art, including various schemes using modular components. Zhang et al. (2011) Nature Biotech. 29:149-53; Geibler et al. (2011) PLoS ONE 6: e19509; US 8420782; US 8470973, the contents of which are incorporated herein by reference in their entirety.

Zinc finger nuclease for Tet inhibition, eg Tet1, Tet2 and/or Tet3, eg Tet2

"ZFN" or "zinc finger nuclease" refers to a zinc finger nuclease, which is an artificial nuclease that can be used to modify, for example, delete, one or more nucleic acids with a desired nucleic acid sequence, for example, Tet, for example, Tet1, Tet2 and/or Tet3, such as Tet2.

Like TALEN, ZFN contains a FokI nuclease domain (or a derivative thereof) fused to a DNA binding domain. In the case of ZFN, the DNA binding domain contains one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. sci. USA 93: 1156-1160.

The zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger may contain, for example, Cys2His2, and may recognize a sequence of approximately 3 bp. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18 bp in size. Various selection and modular assembly techniques are available to produce zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast monohybrid systems, bacterial monohybrid and two-hybrid systems, and mammalian cells.

Like TALEN, ZFN must dimerize in order to cleave DNA. Thus, a ZFN pair is required to target non-palindromic DNA regions. The two individual ZFNs must bind opposite strands of DNA, with proper space between their nucleases. Bitinaite et al. (1998) Proc. Natl. Acad. sci. USA 95: 10570-5.

Also like TALEN, ZFN can introduce a double-strand break in DNA, which can lead to a frameshift mutation upon improper repair, causing a decrease in the expression and amount of Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, in the cell. ZFNs can also be used with homologous recombination to mutate a Tet gene, eg Tet1, Tet2 and/or Tet3, eg Tet2, or to introduce a CAR-encoding nucleic acid at or near a target sequence. As described above, a nucleic acid encoding a CAR may be introduced as part of the template DNA. In some embodiments, the template DNA further comprises homology arms on the 5' side, 3' side, or both 5' and 3' sides of the nucleic acid of the template DNA that encodes the molecule or molecules of interest (e.g. , which encodes the CAR described in the present description), where the specified homology arms are complementary to the genomic DNA sequence flanking the target sequence.

ZFNs specific for sequences in the Tet gene, eg Tet1, Tet2 and/or Tet3, eg Tet2, can be constructed using any method known in the art. See, for example, Provasi (2011) Nature Med. 18:807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16:1200-7; and Guo et al. (2010) J. Mol. Biol. 400:96; US Patent Publication 2011/0158957; and US Patent Publication 2012/0060230, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the ZFN gene editing system may also comprise a nucleic acid encoding one or more components of a ZFN gene editing system, such as a ZFN gene editing system targeting Tet, such as Tet1, Tet2, and/or Tet3, such as Tet2.

Without wishing to be bound by theory, it is believed that the use of gene editing systems (e.g., CRISPR/Cas gene editing systems) that target Tet, e.g., Tet1, Tet2, and/or Tet3, e.g. , Tet1, Tet2, and/or Tet3, such as Tet2, for example, providing an edit event that results in the expression of a shortened Tet, such as Tet1, Tet2, and/or Tet3, such as Tet2. Again, without wishing to be bound by theory, such truncated Tet proteins, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, may retain one or more Tet functions, e.g. Tet1, Tet2 and/or Tet3, e.g. ) while inhibiting one or more other functions, eg Tet1, Tet2 and/or Tet3, eg Tet2 (eg catalytic function), and therefore may be preferred. Gene editing systems that target the late exon or intron of a Tet gene, eg the Tet1, Tet2 and/or Tet3 gene, eg the Tet2 gene, may be particularly preferred in this regard. In one aspect, a Tet inhibitor, eg a Tet1, Tet2 and/or Tet3 inhibitor, eg a Tet2 inhibitor, of the gene editing system of the invention targets the late exon or intron of the tet gene. In one aspect, a Tet inhibitor, such as a Tet1, Tet2 and/or Tet3 inhibitor, such as a Tet2 inhibitor, the gene editing system of the invention targets an exon or intron below exon 8. In one aspect, a Tet inhibitor, such as a Tet1, Tet2 and/or Tet3, for example, an inhibitor of Tet2, a gene editing system, targets exon 8 or exon 9, such as exon 9, of the tet2 gene.

Without wishing to be bound by theory, it is believed that in other embodiments, it may also be preferable to the early exon or intron of the Tet gene, e.g., the Tet1, Tet2 and/or Tet3 gene, e.g. target, resulting in the absence of a gene product or the expression of a completely non-functional gene product. Gene editing systems that target the early exon or intron of a Tet gene, eg the Tet1, Tet2 and/or Tet3 gene, eg the Tet2 gene, may be particularly preferred in this regard. In one aspect, a Tet inhibitor, eg a Tet1, Tet2 and/or Tet3 inhibitor, eg a Tet2 inhibitor, of the gene editing system of the invention targets an early exon or intron of the tet gene. In one aspect, a Tet inhibitor, such as a Tet1, Tet2 and/or Tet3 inhibitor, such as a Tet2 inhibitor, the gene editing system of the invention targets an exon or intron above exon 4. In some embodiments, a Tet inhibitor, such as a Tet1, Tet2 and/or or Tet3, eg an inhibitor of the Tet2 gene editing system, targets exon 1, exon 2, or exon 3, eg exon 3, of the tet2 gene.

Without wishing to be bound by theory, in other embodiments, it may also be preferable to target a Tet gene sequence, e.g., a Tet1, Tet2, and/or Tet3 gene, e.g., a Tet2 gene, that is specific for one or more tet isoforms (e.g., the tet2 gene) , but does not affect one or more other tet isoforms (eg Tet2). In some embodiments, specific targeting of tet isoforms (eg, Tet2) that contain a catalytic domain may be preferred.

Tet inhibitors, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, based on dsRNA, e.g. siRNA or shRNA

As inhibitors of Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, double-stranded RNA ("dsRNA"), eg siRNA or shRNA, can be used in accordance with the present invention. Also within the scope of the present invention is the use of a nucleic acid encoding said dsRNA-based Tet inhibitors, eg Tet1, Tet2 and/or Tet3, eg Tet2.

In one embodiment, the Tet inhibitor, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, is a nucleic acid, e.g. dsRNA, e.g. siRNA or shRNA, specific for a nucleic acid encoding Tet, e.g. or Tet3, eg Tet2, eg genomic DNA or mRNA encoding Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2.

One aspect of the invention provides a composition containing dsRNA, e.g., siRNA or shRNA, containing at least 15 consecutive nucleotides, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides e.g., 21 consecutive nucleotides that are complementary (e.g., 100% complementary) to a Tet nucleic acid sequence, e.g., Tet1, Tet2, and/or Tet3, e.g. Tet2 (e.g., genomic DNA or mRNA encoding Tet, e.g. Tet2 and/or Tet3, such as Tet2). In some embodiments, at least 15 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides, such as 21 consecutive nucleotides, includes consecutive nucleotides shRNA target sequences or nucleic acid encoding Tet2 shRNA shown in Table 4. It is understood that some of the target sequences and/or shRNA molecules are present as DNA, but dsRNA-based tools that target or contain these sequences, may be RNA, or any nucleotide, modified nucleotide, or substituent thereof described herein and/or known in the art, provided that the molecule can still mediate RNA interference.

In one embodiment, a nucleic acid molecule that encodes a dsRNA molecule that inhibits the expression of Tet, such as Tet1, Tet2, and/or Tet3, such as Tet2, is operably linked to a promoter, such as a promoter derived from H1 or U6, such that the dsRNA molecule , which inhibits the expression of Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, is expressed in a CAR expressing cell. See, for example, Tiscornia G. , "Development of Lentiviral Vectors Expressing siRNA", Chapter 3, Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007; Brummelkamp TR, et al. (2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat. Biotechnol. 19:497-500. In one embodiment, a nucleic acid molecule that encodes a dsRNA molecule that inhibits Tet, such as Tet1, Tet2, and/or Tet3, such as Tet2, is on the same vector, such as a lentiviral vector, that contains a nucleic acid molecule that encodes a component eg all components, CAR. In such an embodiment, a nucleic acid molecule that encodes a dsRNA molecule that inhibits Tet, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, is located on the vector, e.g. a nucleic acid that encodes a component, eg all components of a CAR. A nucleic acid molecule that encodes a dsRNA molecule that inhibits the expression of Tet, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, may be transcribed at the same or different position relative to the nucleic acid that encodes a component, e.g. all components, of CAR. In one embodiment, a nucleic acid molecule that encodes a dsRNA molecule that inhibits the expression of Tet, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, is on a vector other than a vector that contains a nucleic acid molecule that encodes a component, e.g. , all components, CAR. In one embodiment, a nucleic acid molecule that encodes a dsRNA molecule that inhibits the expression of Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, is transiently expressed in a CAR expressing cell. In one embodiment, a nucleic acid molecule that encodes a dsRNA molecule that inhibits the expression of Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, is stably integrated into the genome of a CAR expressing cell.

Examples of nucleic acid sequences that encode shRNA sequences are provided below. The target sequence refers to a sequence in the Tet2 genomic DNA (or surrounding DNA). The nucleic acid encoding the shRNA against Tet2 encodes shRNA molecules useful in the present invention. In one embodiment, the Tet2 inhibitor is an siRNA or shRNA specific for the target sequence below or specific for its complementary mRNA. In some embodiments, the Tet2 inhibitor is a shRNA encoded by a nucleic acid encoding an anti-Tet2 shRNA according to Table 4 below. In some embodiments, the Tet2 inhibitor is a nucleic acid comprising a nucleic acid encoding an anti-Tet2 shRNA according to Table 4 below, for example, which is under the control of a U6 or H1 promoter such that an anti-Tet2 shRNA is produced. In some embodiments, the invention relates to an siRNA or shRNA containing a sequence that is an RNA analog (i.e., all T nucleic acid residues are replaced by U nucleic acid residues) of a shRNA target sequence, for example, a shRNA target sequence of any of the shRNAs shown in table 4.

Table 4

NAME shRNA shRNA target sequence Nucleic acid encoding anti-Tet2 shRNA TET2 TET2-3838_76472_insert
(shRNA vs TET2 #1) CACATGGCGTTTATCCAGAAT (SEQ ID NO: 1244) CACATGGCGTTTATCCAGAAT CTCGAGATTCTGGATAAACGCCATGTGTTTTTTGAATTCGCACCAGCACGCTACGCACACACAGTACACACACTGACGTTTCGCCGTCTTC (SEQ ID NO: 1253) TET2 TET2_NM_017628.4_25616_concept
(shRNA vs TET2 #2) CAGATGCACAGGCCAATTAAG (SEQ ID NO: 1245) GAAGACGCACCGGCAGATGTACAGGCTAATTAAGGTTAATATTCATAGCCTTAATTGGCCTGTGCATCTGTTTTTTGAATTCGCACCAGCACGCTACGCAACACGTCAACCAGTGTCAGTGTTTCGCCGT (SEQ ID NO: 1254) TET2 TET2_NM_017628.4_25625_concept
(shRNA vs TET2 #3) GAGCTGCTGAATTCAACTAGA (SEQ ID NO: 1246) GAAGACGCACCGGGAGCTGCTGAATTCAATTAGAGTTAATATTCATAGCTCTAGTTGAATTCAGCAGCTCTTTTTTGAATTCGCACCAGCACGCTACGCATGCAGTCAACCAGTGTCAACCATTCGCCGT (SEQ ID NO: 1255) TET2 TET2-6571_76471_target
(shRNA vs TET2 #4) CAGATCGCCATAACATAAATA (SEQ ID NO: 1247) CAGATCGCCATAACATAAATACTCGAGTATTTATGTTATGGCGATCTGTTTTTTGAATTCGCACCAGCACGCTACGCATGACCAGTACACACACTGCATGTTCGCCGTCTTC (SEQ ID NO: 1256) TET2 TET2_NM_017628.4_25619_target
(shRNA vs TET2 #5) GACCATGGAGCAGCATCTGAA (SEQ ID NO: 1248) GAAGACGCACCGGGACCATGGAGTAGCATTTGAAGTTAATATTCATAGCTTCAGATGCTGCTCCATGGTCTTTTTTGAATTCGCACCAGCACGCTACGCATGGTGTCAACCAGTGTCAGTTGTTCGCCGT (SEQ ID NO: 1257) TET2 shRNA vs TET2 #6 GCCAAGTCATTATTTGACCAT (SEQ ID NO: 1249) GCCAAGTCATTATTTGACCATCTCGAGATGGTCAAATAATGACTTGGCTTTTTTGA (SEQ ID NO: 1258) TET2 shRNA vs TET2 #7 CCTCAGAGATATTGTGGGTTT (SEQ ID NO: 1250) CCTCAGAGATATTGTGGGTTTCTCGAGAAACCCACAATATCTCTGAGGTTTTTTGA (SEQ ID NO: 1259) TET2 shRNA vs TET2 #8 GGGTAAGCCAAGAAAGAAA (SEQ ID NO: 1251) GGGTAAGCCAAGAAAGAAACTCGAGTTTCTTTCTTGGCTTACCCTTTTTTGA (SEQ ID NO: 1260) TET2 TET2 8 long
(shRNA vs TET2 #9) GGGTAAGCCAAGAAAGAAA (SEQ ID NO: 1252) GAAGACGCACCGGGGGTAAGCCAAGAAAGAAAGTTAATATTCATAGCTTTCTTTCTTGGCTTACCCTTTTTTGAATTCGCACCAGCACGCTACGCAACACGTCAACCAGTGTCAGTGTTTCGCCGT (SEQ ID NO: 1261)

An additional Tet2 inhibitor based on dsRNA molecules, such as shRNA and siRNA, can be designed and tested using methods known in the art and described herein. In some embodiments, a dsRNA-based Tet2 inhibitor, such as shRNA or siRNA, targets SEQ ID NO: 1358. In some embodiments, a dsRNA-based Tet2 inhibitor, such as shRNA or siRNA, targets SEQ ID NO: 1359. In some embodiments, a dsRNA-based Tet2 inhibitor, such as shRNA or siRNA, targets SEQ ID NO: 1360. In some embodiments, a dsRNA-based Tet2 inhibitor, such as shRNA or siRNA, targets SEQ ID NO: 1361. In some embodiments, a dsRNA-based Tet2 inhibitor, such as shRNA or siRNA, targets SEQ ID NO: 1362. In some embodiments, a dsRNA-based Tet2 inhibitor, such as shRNA or siRNA, targets SEQ ID NO: 1363. In some embodiments, a dsRNA-based Tet2 inhibitor, such as shRNA or siRNA, targets an mRNA sequence encoding Tet2.

In some embodiments, the inhibitor is a nucleic acid, such as DNA, encoding a dsRNA-based Tet2 inhibitor, such as shRNA or siRNA, according to any of the embodiments described above. In some embodiments, the nucleic acid, eg, DNA, is in a vector, eg, any conventional expression system, eg, as described herein, eg, in a lentiviral vector.

Without wishing to be bound by theory, a dsRNA (e.g., siRNA or shRNA) TET inhibitor targeting a Tet mRNA sequence, e.g., Tet1, Tet2, and/or Tet3, e.g. Tet2) but does not affect one or more other tet isoforms (e.g., Tet2) (e.g., by targeting a unique splice point or targeting a domain that is present in one or more isoforms of tet, such as Tet2, but not present in one or more other isoforms of tet, such as Tet2). In some embodiments, specific targeting of tet isoforms (eg, Tet2) that contain a catalytic domain may be preferred.

Low molecular weight compounds

Tet inhibitors

In some embodiments, the Tet inhibitor is a small molecule compound that inhibits the expression and/or function of Tet, such as Tet1, Tet2, and/or Tet3, such as Tet2.

Tet2 inhibitors

In some embodiments, the Tet2 inhibitor is a small molecule compound that inhibits the expression and/or function of Tet2. For example, the Tet2 inhibitor of the present invention is 2-hydroxyglutarate (CAS #2889-31-8).

In another example, a Tet2 inhibitor according to the present invention has the following structure:

Pluripotin SC1

In another example, the Tet2 inhibitor of the present invention is N-[3-[7-(2,5-dimethyl-2H-pyrazol-3-ylamino)-1-methyl-2-oxo-1,4-dihydro- 2H-pyrimido[4,5-d]pyrimidin-3-yl]-4-methylphenyl]-3-trifluoromethylbenzamide (CAS #839707-37-8) and has the following structure:

In another example, the Tet2 inhibitor of the present invention is 2-[(2,6-dichloro-3-methylphenyl)amino]benzoic acid (CAS # 644-62-2) and has the following structure:

meclofenamic acid

In some embodiments, the Tet2 inhibitor of the present invention is a pharmaceutically acceptable salt of any of the above.

HDAC inhibitors

Any known HDAC inhibitors can be used in accordance with the present invention. Non-limiting examples of HDAC inhibitors include Voninostat (Zolinza®); Romidepsin (Istodax®); Trichostatin A (TSA); Oxamflatine; Vorinostat (Zolinza®, suberoylanilide hydroxamic acid); Pyroxamide (suberoyl-3-aminopyridinamide hydroxamic acid); Trapoxin A (RF-1023A); Trapoxin B (RF-10238); cyclo[(αS,2S)-α-amino-η-oxo-2-oxiranoctanoyl-O-methyl-D-tyrosyl-L-isoleucyl-L-prolyl] (Cyl-1); cyclo[(αS,2S)-α-amino-η-oxo-2-oxiranoctanoyl-O-methyl-D-tyrosyl-L-isoleucyl-(2S)-2-piperidinecarbonyl] (Cyl-2); cyclo[L-alanyl-D-alanyl-(2S)-η-oxo-L-α-aminooxyranoctanoyl-D-prolyl] (HC-toxin); cyclo[(αS,2S)-α-amino-η-oxo-2-oxiranoctanoyl-D-phenylalanyl-L-leucyl-(2S)-2-piperidinecarbonyl] (WF-3161); Chlamydocin ((S)-cyclo(2-methylalanyl-L-phenylalanyl-D-prolyl-η-oxo-L-α-aminooxyranoctanoyl); Apidicin (cyclo(8-oxo-L-2-aminodecanoyl-1-methoxy-L -tryptophyl-L-isoleucyl-D-2-piperidinecarbonyl); romidepsin (Istodax®, FR-901228); 4-phenylbutyrate; Spiruchostatin A; Milproin (valproic acid); Entinostat (MS-275, N-(2-aminophenyl) -4-[N-(pyridin-3-ylmethoxycarbonyl)-amino-methyl]-benzamide); D-ido-undeca-1,6-dienitol); 4-(acetylamino)-N-(2-aminophenyl)-benzamide (also known as CI-994); N1-(2-aminophenyl)-N8-phenyloctandiamide (also known as BML-210); 4-(dimethylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide (also known as M344); (E)-3-(4-(((2-(1H -indol-3-yl)ethyl)(2-hydroxyethyl)amino)-methyl)phenyl)-N-hydroxyacrylamide (NVP-LAQ824); Panobinostat (Farydak®); Mocetinostat and Belinostat.

Squirrels

Dominant-negative Tet2

In accordance with the present invention, dominant negative Tet2 isoforms and a nucleic acid encoding dominant negative Tet2 can be used as Tet2 inhibitors. In some embodiments, a dominant negative Tet2 lacks the catalytic function of Tet2. An example of a dominant negative Tet2 is a protein containing or consisting of SEQ ID NO: 1357 with the mutation R1261G, according to SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein containing or consisting of SEQ ID NO: 1357 with the mutation R1262A, according to SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein containing or consisting of SEQ ID NO: 1357 with the mutation S1290A, according to SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein , containing or consisting of SEQ ID NO: 1357 with the WSMYYN (amino acids 1291-1296 SEQ ID NO: 1357) mutation on GGSGGS (SEQ ID NNO: 67), according to SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein containing or consisting of SEQ ID NO: 1357 with mutation M1293A and Y1294A, according to SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein containing or consisting of SEQ ID NO: 1357 with mutation Y1295A, in according to SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein containing or consisting of SEQ ID NO: 1357 with the S1303N mutation, according to SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein containing or consisting of SEQ ID NO: 1357 with mutation H1382Y, according to SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein containing or consisting of SEQ ID NO: 1357 with mutation D1384A, according to SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein containing or consisting of SEQ ID NO: 1357 with the mutation D1384V, according to SEQ ID NO: 1357. In some embodiments, a dominant negative Tet2 may include combinations of any of the above mutations. Such mutations are further described in, for example, Chen et al., Nature, 493:561-564 (2013); Hu et al, Cell, 155:1545-1555 (2013), the contents of which are incorporated herein by reference in their entirety.

Dominant-negative Tet2 binding partners

Without wishing to be bound by theory, it is believed that Tet2 interacts with, e.g., binds to, one or more HDACs, e.g., one or more HDACs expressed in immune effector cells, e.g., T cells, and that such Tet2:HDAC complexes may contribute to Tet2 activity in the cell. In some embodiments, the Tet2 inhibitor of the invention is a dominant negative Tet2 binding partner, such as a dominant negative Tet2 binding HDAC. In other embodiments, the Tet2 inhibitor of the invention comprises a nucleic acid encoding a dominant negative Tet2 binding partner, such as a dominant negative Tet2 binding HDAC.

Vectors encoding Tet2 inhibitors

As described herein, the invention relates to vectors, e.g. as described herein, which encode Tet inhibitors, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, such as gene editing systems, shRNA or siRNA based inhibitors, or dominant negative inhibitors of Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2 (eg as described herein).

In some embodiments, further including, for example, a CAR, the nucleic acid may further comprise a sequence encoding a CAR, for example, as described herein. In some embodiments, the invention relates to a vector containing a nucleic acid sequence encoding a Tet inhibitor, such as Tet1, Tet2 and/or Tet3, such as Tet2 described herein, and containing a nucleic acid sequence encoding a CAR molecule described herein. . In some embodiments, the nucleic acid sequences are on separate vectors. In other embodiments, two or more nucleic acid sequences are encoded by a single nucleic acid molecule in the same reading frame and as a single polypeptide chain. In this aspect, the two or more CARs, for example, may be one or more peptide cleavage sites (eg, an auto-cleavage site or a substrate for an intracellular protease). Examples of peptide cleavage sites of a peptide include the following, where GSG residues are optional:

T2A: (GSG) E G R G S L L T C G D V E E N P G P (SEQ ID NO: 68)

P2A: (GSG) A T N F S L L K Q A G D V E E N P G P (SEQ ID NO: 69)

E2A: (GSG) Q C T N Y A L L K L A G D V E S N P G P (SEQ ID NO: 70)

F2A: (GSG) V K Q T L N F D L L K L A G D V E S N P G P (SEQ ID NO: 71).

These peptide cleavage sites are collectively referred to herein as "2A sites". In some embodiments, the vector comprises a nucleic acid sequence encoding a CAR as described herein and a nucleic acid sequence encoding a Tet inhibitor such as Tet1, Tet2 and/or Tet3 such as Tet2 based on shRNA or siRNA as described herein. . In some embodiments, the vector comprises a nucleic acid sequence encoding a CAR as described herein and a nucleic acid sequence encoding a Tet inhibitor such as Tet1, Tet2 and/or Tet3 such as Tet2 based on a genome editing system (e.g. CRISPR/ Cas) described in the present description.

Methods of administration of Tet inhibitors, eg Tet1, Tet2 and/or Tet3, eg Tet2

The invention relates to methods for increasing the therapeutic efficacy of a CAR expressing cell, e.g., a CAR expressing cell as described herein, e.g., a CAR19 expressing cell (e.g., CTL019), comprising the step of reducing the level of 5-hydroxymethylcytosine in said cell. In some embodiments, the implementation of the method includes reducing or eliminating the function or expression of Tet, such as Tet1, Tet2 and/or Tet3, such as Tet2. In some embodiments, the method includes contacting said cells with a Tet inhibitor, eg Tet1, Tet2 and/or Tet3, eg Tet2, as described herein.

The invention further relates to methods for producing a CAR expressing cell, e.g., a CAR expressing cell, having improved function (e.g., having increased efficiency, e.g., tumor targeting or proliferation), comprising the step of reducing or eliminating Tet expression or function, e.g., Tet1 , Tet2 and/or Tet3, such as Tet2, in the specified cell. In some embodiments, the method includes contacting said cells with a Tet inhibitor, eg Tet1, Tet2 and/or Tet3, eg Tet2, as described herein. In some embodiments, the implementation of bringing into contact is carried out ex vivo. In some embodiments, the implementation of bringing into contact is carried out in vivo . In some embodiments, the implementation of bringing into contact is carried out before, simultaneously with or after the modification of these cells to express CAR, for example, CAR, as described in the present description.

In some embodiments, the invention relates to a method for inhibiting the function or expression of Tet, e.g., Tet1, Tet2, and/or Tet3, e.g., Tet2, in a CAR expressing cell, e.g., a CAR expressing cell as described herein, e.g., a CAR19 expressing cell (eg, expressing CTL019 cell), and the method includes the step of reducing the level of 5-hydroxymethylcytosine in the specified cell. In some embodiments, the implementation of the method includes reducing or eliminating the function or expression of Tet, such as Tet1, Tet2 and/or Tet3, such as Tet2. In some embodiments, the method includes contacting said cells with a Tet inhibitor, eg Tet1, Tet2 and/or Tet3, eg Tet2, as described herein.

In one embodiment, the invention relates to a method, e.g., the method described above, which comprises introducing a nucleic acid encoding a CAR into a cell, e.g., an immune effector cell, e.g., a T cell, at a site in the Tet gene or its regulatory elements. so that the expression of Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2, is disrupted. Insertion at a site in a Tet gene, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, can be carried out, e.g., using a Tet-targeted, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2, gene editing system as described above. .

In one embodiment, the invention relates to a method, for example, the method described above, comprising the step of introducing into the cell a gene editing system, for example, a CRISPR/Cas gene editing system, which targets Tet, for example, Tet1, Tet2 and/or Tet3, eg Tet2, eg, a CRISPR/Cas system containing an gRNA that has a targeting sequence complementary to the Tet gene target sequence, eg Tet1, Tet2 and/or Tet3, eg Tet2. In some embodiments, the implementation of the CRISPR/Cas system is introduced into the specified cell as a ribonucleoprotein complex of gRNA and enzyme, for example, administered by electroporation. In one embodiment, the method includes introducing a nucleic acid encoding one or more components of the CRISPR/Cas system into said cell. In one embodiment, the specified nucleic acid is on a vector encoding a CAR, for example, CAR, as described in the present description.

In one embodiment, the invention relates to a method, for example, the method described above, comprising the step of introducing into the cell an inhibitory dsRNA, e.g., shRNA or siRNA, that targets Tet, e.g. Tet1, Tet2 and/or Tet3, e.g. Tet2. In one embodiment, the method comprises introducing into said cell a nucleic acid encoding an inhibitory dsRNA, eg shRNA or siRNA, that targets Tet, eg Tet1, Tet2 and/or Tet3, eg Tet2. In one embodiment, the specified nucleic acid is on a vector encoding a CAR, for example, CAR, as described in the present description.

Additional components of CAR and CART cells and methods related to the invention are described below.

Within the scope of the present invention, compositions and methods of use are provided for the treatment of a disease, such as cancer, using immune effector cells (eg, T cells, NK cells) constructed with the CARs of the invention.

In one aspect, the invention relates to a series of chimeric antigen receptors (CARs) containing an antigen-binding domain (e.g., an antibody or antibody fragment, TCR or TCR fragment) designed to specifically bind to a tumor antigen, e.g., the tumor antigen described herein. In one aspect, the invention relates to an immune effector cell (eg, T cell, NK cell) engineered to express CAR, wherein the modified immune effector cell exhibits an anti-cancer property. In one aspect, the cell is transformed with CAR and the CAR is expressed on the surface of the cell. In some embodiments, the cell (eg, T cell, NK cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express CAR. In another embodiment, the cell (eg, T cell, NK cell) is transfected with a nucleic acid, eg, mRNA, cDNA, DNA encoding a CAR. In some such embodiments, the cell may transiently express a CAR.

In one aspect, the antigen-binding domain of the CAR described herein is an scFv antibody fragment. In one aspect, such antibody fragments are functional because they retain an equivalent binding affinity, eg, they bind the same antigen with an affinity comparable to the IgG antibody from which they are derived. In other embodiments, an antibody fragment has a lower binding affinity, e.g., it binds the same antigen with a lower binding affinity than the antibody from which it is derived, yet is functional in that it provides the biological response described herein. In one embodiment, the CAR molecule contains an antibody fragment that has a KD binding affinity of 10 ^-4 M to 10 ^-8 M, such as 10 ^-5 M to 10 ^-7 M, such as 10 ^-6 M or 10 ^-7 M , in relation to the target antigen. In one embodiment, the antibody fragment has a binding affinity that is at least five-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or 1000-fold less than that of a reference antibody, e.g., antibodies described in the present description.

In one aspect, such antibody fragments are functional in that they provide a biological response, which may include, but is not limited to, activation of an immune response, inhibition of signal transduction from a target antigen, inhibition of kinase activity, and the like, as will be appreciated. a specialist in this field.

In one aspect, a CAR antigen binding domain is an antibody scFv fragment that is humanized relative to the mouse scFv sequence from which it is derived.

In one aspect, a CAR antigen-binding domain of the invention (eg, scFv) is encoded by a nucleic acid molecule whose sequence has been codon-optimized for expression in a mammalian cell. In one aspect, the entire CAR construct of the invention is encoded by a nucleic acid molecule whose entire sequence has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (ie, codons that code for the same amino acid) in coding DNA is biased across species. This codon degeneracy allows identical polypeptides to be encoded by different nucleotide sequences. Various methods for optimizing codons are known in the art and include, for example, those described in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

In one aspect, in the CAR of the invention, the antigen-binding domain of a specific antibody is combined with an intracellular signaling molecule. For example, in some aspects, the intracellular signaling molecule includes, but is not limited to, the CD3-zeta, 4-1BB, and CD28 chain signaling modules, and combinations thereof.

Moreover, the present invention relates to CAR and CAR expressing cells and their use in drugs and methods of treatment, among other diseases, cancer or any malignancy, or autoimmune diseases involving cells or tissues that express a tumor antigen, as described in the present description.

In one aspect, the CAR of the invention can be used to eliminate normal cells that express a tumor antigen as described herein, thereby being used as cell conditioning prior to cell transplantation. In one aspect, the normal cell that expresses the tumor antigen as described herein is a normal stem cell and the cell transplant is a stem cell transplant.

In one aspect, the invention relates to an immune effector cell (eg, T cell, NK cell) engineered to express a chimeric antigen receptor (CAR), wherein the engineered immune effector cell exhibits an antitumor property. A preferred antigen is a cancer-associated antigen (ie, a tumor antigen) as described herein. In one aspect, the CAR antigen binding domain comprises a partially humanized antibody fragment. In one aspect, the CAR antigen binding domain contains a partially humanized scFv. Thus, the invention relates to a CAR that contains a humanized antigen-binding domain and is introduced by techniques into a cell, such as a T cell or NK cell, and methods of using it for adoptive therapy.

In one aspect, the CARs of the invention comprise at least one intracellular domain selected from the group of CD137 signaling domain (4-1BB), CD28 signaling domain, CD27 signaling domain, CD3-zeta signaling domain, and any combination thereof. In one aspect, the CAR of the invention, comprising at least one intracellular signaling domain, is derived from one or more co-stimulatory molecule(s) other than CD137 (4-1BB) or CD28.

The sequences of some examples of various CAR components of the present invention are shown in Table 1, where a.k. means amino acids, and n.k. means nucleic acids that encode the corresponding peptide.

Table 1 . Sequences of various CAR components (a.k. - amino acids, n.k. - nucleic acids that encode the corresponding protein)

SEQID NO Description Sequence Compliant with huCD19 1 EF-1 promoter CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGG TTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTTCCATTTCAGGTGTCGTGA one hundred 2 Leader sequence (a.k.) MALPVTALLLPLALLLHAARP 13 3 Leader sequence (n.c.) ATGGCCCTGCCTGTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCC 54 four Hinge area CD8 (a.k.) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD fourteen five Hinge region CD8 (n.c.) ACCAGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT 55 6 Hinge region Ig4 (a.k.) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM 102 7 Hinge region Ig4 (n.c.) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG 103 8 Hinge region IgD (a.k.) RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH 47 nine Hinge region IgD (n.c.) AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT 48 10 Hinge area GS /linker (a.k.) GGGGSGGGGS 49 eleven Hinge area GS /linker (n.c.) GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 50 12 CD8TM (a.k.) IYIWAPLAGTCGVLLLSLVITLYC 15 13 CD8 TM (n.c.) ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC 56 fourteen Intracellular domain 4-1BB (a.k.) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL sixteen 15 Intracellular domain 4-1BB (n.c.) AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG 60 sixteen CD27 (a.k.) QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP 51 17 CD27 (n.c.) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGACTTCGCAGCCTATCGCTCC 52 eighteen CD3-zeta (a.k.) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 17 nineteen CD3-zeta (n.c.) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 101 20 CD3-zeta (a.k.) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 43 21 CD3-zeta (n.c.) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAG
AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT
TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA
AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGG
AGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC
ACGATGGCCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC
CCTTCACATGCAGGCCCTGCCCCCTCGC 44 22 Linker GGGGS eighteen 23 Linker GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 50 24 Extracellular domain of PD-1 (a.k.) Pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv 25 Extracellular domain of PD-1 (n.c.) Cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtc 26 PD-1 CAR (a.k.) with signal sequence Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr 27 PD-1 CAR (n.c.) Atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagaccacccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaa agaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggcccttccccctcgc 28 Linker (Gly-Gly-Gly-Ser)n, where n=1-10 105 29 Linker (Gly4 Ser)4 106 thirty Linker (Gly4 Ser)3 107 31 linker (Gly3Ser) 108 32 Poly-A aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa a aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 118 33 Poly-A aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 104 34 Poly-A aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa a aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa aa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa aaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa aaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 109 35 Poly-A tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 110 36 Poly-A tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttt t tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttt tt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttt ttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttt tttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 111 37 Poly-A aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 113 39 PD1 CAR (a.k.) Pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv tttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr

Cancer-associated antigens

The present invention relates to immune effector cells (eg, T cells, NK cells) that are engineered to contain one or more CARs that target immune effector cells to cancer. This is achieved through an antigen-binding domain on CAR that is specific for an antigen associated with cancer. There are two classes of cancer-associated antigens (tumor antigens) to which the CARs of the present invention can be targeted: (1) cancer-associated antigens that are expressed on the surface of cancer cells; and (2) cancer-associated antigens that are themselves intracellular, but a fragment of such an antigen (peptide) is presented on the surface of cancer cells by MHC (major histocompatibility complex).

Thus, the present invention relates to CARs that target the following cancer-associated antigens (tumor antigens): CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, VEGFR2, LewisY, CD24, PDGFR-beta , PRSS21, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, prostasis, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, 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-1a, legumain, HPV E6,E7, MAGE-A1, MAGE A1 , ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, protein, survivin and telomerase, PCTA-1/galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC , TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1.

Tumor-supporting antigens

The CAR described herein may comprise an antigen-binding domain (eg, an antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (eg, a tumor-supporting antigen as described herein). In some embodiments, the tumor-supporting antigen is an antigen present on a stromal cell or myeloid-derived suppressor cell (MDSC). Stromal cells can secrete growth factors to stimulate cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation. Without being bound by theory, in some embodiments, CAR-expressing cells destroy tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival.

In some embodiments, the stromal cell antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), and tenascinv. In one embodiment, the FAP-specific antibody is, competes for binding to, or has the same CDRs as sibrotuzumab. In some embodiments, the MDSC antigen is selected from one or more of: CD33, CD11b, C14, CD15, and CD66b. Thus, in some embodiments, the tumor supporting antigen is selected from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), or tenascin, CD33, CD11b, C14, CD15, and CD66b.

Chimeric antigen receptor (CAR)

The present invention provides a recombinant DNA construct comprising sequences encoding a CAR, wherein the CAR contains an antigen-binding domain (e.g., an antibody or antibody fragment, TCR or TCR fragment) that specifically binds to a cancer-associated antigen described herein, wherein the sequence the antigen-binding fragment is adjacent to the nucleic acid sequence encoding the intracellular signaling domain, and is in the same reading frame with it. The intracellular signaling domain may comprise a co-stimulatory signaling domain and/or a primary signaling domain, such as the zeta chain. A costimulatory signaling domain refers to a portion of a CAR containing at least a portion of the intracellular domain of a costimulatory molecule.

In specific aspects, the CAR construct of the invention comprises an scFv domain, where the scFv may be preceded by an optional leader sequence, such as the sequence shown in SEQ ID NO: 2, and may be followed by an optional hinge sequence, such as the sequence shown in SEQ ID NO. : 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10, transmembrane region such as the sequence shown in SEQ ID NO: 12, intracellular signaling domain which includes SEQ ID NO: 14 or SEQ ID NO: 16, and a CD3-zeta sequence that includes SEQ ID NO: 18 or SEQ ID NO: 20, where the domains are adjacent and in the same reading frame to form a fusion protein.

In one aspect, exemplary CAR constructs comprise an optional leader sequence (e.g., the leader described herein), an extracellular antigen-binding domain (e.g., the antigen-binding domain described herein), a hinge region (e.g., the hinge region described herein) , a transmembrane domain (eg, the transmembrane domain described herein) and an intracellular stimulatory domain (eg, the intracellular stimulatory domain described herein). In one aspect, an exemplary CAR construct comprises an optional leader sequence (e.g., the leader described herein), an extracellular antigen-binding domain (e.g., the antigen-binding domain described herein), a hinge region (e.g., the hinge region described herein) , a transmembrane domain (e.g., the transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein), and/or an intracellular primary signaling domain (e.g., a primary signaling domain described herein) .

An exemplary leader sequence is provided as SEQ ID NO: 2. An exemplary hinge/spacer sequence is provided as SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10. An exemplary transmembrane domain sequence is shown as SEQ ID NO: 12. An exemplary intracellular signaling domain sequence of the 4-1BB protein is shown as SEQ ID NO: 14. An exemplary CD27 intracellular signaling domain sequence is shown as SEQ ID NO: 16. An exemplary CD3-zeta domain sequence is shown in as SEQ ID NO: 18 or SEQ ID NO: 20.

In one aspect, the present invention relates to a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen-binding domain, e.g., as described herein, which is adjacent and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain.

In one aspect, the present invention provides a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen-binding domain, where the sequence is adjacent to a nucleic acid sequence encoding an intracellular signaling domain and is located in same reading frame with it. An exemplary intracellular signaling domain that can be used in CAR includes, but is not limited to, one or more intracellular signaling domains from, for example, CD3-zeta, CD28, CD27, 4-1BB, and the like. In some cases, a CAR may contain any combination of CD3-zeta, CD28, 4-1BB, and the like.

Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as screening libraries of cells expressing the nucleic acid molecule, obtaining a nucleic acid molecule from a vector known to include it, or isolation directly from cells and tissues containing it using standard methods. Alternatively, the nucleic acid of interest can be obtained synthetically rather than cloned.

The present invention includes retroviral and lentiviral vector constructs expressing CARs that can be directly transduced into a cell.

The present invention also includes an RNA construct that can be directly transfected into a cell. A method for producing mRNA for use in transfection involves in vitro transcription (IVT) of the template using specially designed primers, followed by poly-A attachment to produce a construct containing a 3' and 5' untranslated sequence ("UTR") (e.g., 3'- and/or 5'-UTR as described herein), 5'-cap (e.g., 5'-cap as described herein) and/or internal ribosome entry site (IRES) (e.g., IRES as described herein), a nucleic acid to be expressed, and a poly-A tail, typically 50-2000 bases long (SEQ ID NO: 32). The RNA thus obtained can be efficiently transfected into various cell types. In one embodiment, the template includes CAR sequences. In one embodiment, the CAR vector RNA is transduced into a cell, such as a T cell or NK cell, by electroporation.

Antigen binding domain

In one aspect, a CAR of the invention comprises a target-specific binding element, otherwise referred to as an antigen-binding domain. The choice of part depends on the type and amount of ligands that define the surface of the target cell. For example, an antigen binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CARs of the invention include markers associated with viral, bacterial and parasitic infections, autoimmune disease, and malignant cells.

In one aspect, a CAR-mediated T cell response can be directed to an antigen of interest by inserting an antigen-binding domain that specifically binds the desired antigen into the CAR by means of engineering.

In one aspect, the antigen-binding domain portion of the CAR contains an antigen-binding domain that targets a tumor antigen, such as the tumor antigen described herein.

An antigen binding domain can be any domain that binds to an antigen, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including, but not limited to, a single domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of a nanoantibody derived from an animal of the camelid family, and an alternative framework known in the art to function as an antigen-binding domain, such as a recombinant fibronectin domain, T cell receptor (TCR), or a fragment thereof, eg, single chain TCR, and the like. In some cases, it is useful that the antigen-binding domain be delivered to the same species in which the CAR will ultimately be used. For example, for use in humans, it may be useful for the CAR antigen-binding domain to contain human or humanized residues of the antigen-binding domain of an antibody or antibody fragment.

In one embodiment, the anti-CD19 CAR is the anti-CD19 CAR described in US Pat. No. 8,399,645; US patent No. 7446190; Xu et al., Leuk Lymphoma. 2013 54(2):255-260(2012); Cruz et al., Blood 122(17):2965-2973 (2013); Brentjens et al., Blood, 118(18):4817-4828 (2011); Kochenderfer et al., Blood 116(20):4099-102 (2010); Kochenderfer et al., Blood 122(25):4129-39(2013); or 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May 15-18, Salt Lake City) 2013, Abst 10 (each of which is incorporated herein by reference in its entirety). In one embodiment, an anti-CD19 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from a CAR, an antibody, or an antigen-binding fragment thereof, as described, for example, in PCT publication WO2012/079000 (incorporated herein by reference in its entirety). In one embodiment, the antigen-binding domain against CD19 is an antigen-binding portion, eg, a CDR, from a CAR, an antibody, or an antigen-binding fragment thereof, as described, for example, in PCT publication WO2014/153270; Kochenderfer, J.N. et al., J. Immunother. 32 (7), 689-702 (2009); Kochenderfer, J.N., et al., Blood, 116 (20), 4099-4102 (2010); PCT publications WO2014/031687; Bejcek, Cancer Research, 55, 2346-2351, 1995; or US patent No. 7446190 (each of which is incorporated into the present description by reference in its entirety).

In one embodiment, the anti-mesothelin antigen-binding domain is or may be derived from an antigen-binding domain, e.g., CDR, scFv, or VH and VL, an antibody, an antigen-binding fragment, or CAR, as described in, e.g., PCT Publication WO2015/090230 (in one embodiment, CAR is the CAR described in WO2015/090230, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the anti-mesothelin antigen-binding domain is or is derived from an antigen-binding portion, e.g., a CDR, scFv, or VH and VL, an antibody, an antigen-binding fragment, or a CAR, as described, for example, in PCT Publications WO1997/025068, WO1999/028471, WO2005/ 014652, WO2006/099141, WO2009/045957, WO2009/068204, WO2013/142034, WO2013/040557 or WO2013/063419 (each of which is incorporated herein by reference in its entirety).

In one embodiment, the anti-CD123 antigen-binding domain is or is derived from an antigen-binding portion, e.g., a CDR, scFv, or VH and VL, an antibody, an antigen-binding fragment, or a CAR, as described, for example, in PCT Publication WO2014/130635 (incorporated herein by reference in full). In one embodiment, the anti-CD123 antigen-binding domain is or is derived from an antigen-binding portion, e.g., a CDR, scFv, or VH and VL, an antibody, an antigen-binding fragment, or a CAR, as described, for example, in PCT Publication WO2016/028896 (incorporated by reference in its entirety). ); in some embodiments, the CAR is the CAR described in WO2016/028896. In one embodiment, the anti-CD123 antigen-binding domain is or is derived from an antigen-binding portion, e.g., a CDR, scFv, or VL and VH, an antibody, an antigen-binding fragment, or a CAR, as described in, e.g., PCT Publication WO1997/024373, WO2008/127735 (e.g., CD123 binding domain of 26292, 32701, 37716 or 32703), WO2014/138805 (e.g., CD123 binding domain of CSL362), WO2014/138819, WO2013/173820, WO2014/144622, WO2001/66139, WO223, domain from any of Old4, Old5, Old17, Old19, New102, or Old6), WO2014/144622, or US2009/0252742 (each incorporated by reference in its entirety).

In one embodiment, the antigen-binding domain against CD22 is an antigen-binding portion, e.g., a CDR, from an antibody described, for example, in Haso et al., Blood, 121(7): 1165-1174 (2013); Wayne et al., Clin Cancer Res 16(6): 1894-1903 (2010); Kato et al., Leuk Res 37(1):83-88 (2013); Creative BioMart (creativebiomart.net): MOM-18047-S(P).

In one embodiment, the antigen-binding domain against CS-1 is an antigen-binding portion, eg, CDR, from elotuzumab (BMS), see, eg, Tai et al., 2008, Blood 112(4):1329-37; Tai et al., 2007, Blood. 110(5):1656-63.

In one embodiment, an anti-CLL-1 antigen-binding domain is an antigen-binding portion, e.g., the CDR or VH and VL, of an antibody, antigen-binding fragment, or CAR as described, for example, in PCT publication WO2016/014535, the contents of which are incorporated herein by reference in in full. In one embodiment, the anti-CLL-1 antigen-binding domain is an antigen-binding portion, eg, CDR, from an antibody available from R&D, ebiosciences, Abcam, eg, PE-CLL1-hu, Cat # 353604 (BioLegend); and PE-CLL1 (CLEC12A), catalog number 562566 (BD).

In one embodiment, an anti-CD33 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody described in e.g. 6), Caron et al., Cancer Res 52(24):6761-6767 (1992) (Lintuzumab, HuM195), Lapusan et al., Invest New Drugs 30(3):1121-1131 (2012) (AVE9633), Aigner et al., Leukemia 27(5): 1107-1115 (2013) (AMG330, CD33 BiTE), Dutour et al., Adv hematol 2012:683065 (2012) and Pizzitola et al., Leukemia doi:10,1038/ Lue.2014,62 (2014). Exemplary CAR molecules that target CD33 are described herein and presented in WO2016/014576, for example, Table 2 of WO2016/014576 (included herein in its entirety).

In one embodiment, the antigen-binding domain against GD2 is an antigen-binding portion, eg, a CDR, from an antibody as described in, eg, Mujoo et al., Cancer Res. 47(4):1098-1104 (1987); Cheung et al., Cancer Res 45(6):2642-2649 (1985), Cheung et al., J Clin Oncol 5(9):1430-1440 (1987), Cheung et al., J Clin Oncol 16(9 ):3053-3060 (1998), Handgretinger et al., Cancer Immunol Immunother 35(3):199-204 (1992). In some embodiments, the antigen-binding domain against GD2 is the antigen-binding portion of an antibody selected from mAb 14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9, see for example, WO2012033885, WO2013040371, WO2013192294, WO2013061273, WO2013123061, WO2013074916 and WO201385552. In some embodiments, the antigen-binding domain against GD2 is the antigen-binding portion of an antibody described in US Publication No. 20100150910 or PCT Publication No. WO 2011160119.

In one embodiment, the antigen-binding domain against BCMA is an antigen-binding portion, for example, a CDR, from an antibody described in, for example, WO2012163805, WO200112812 and WO2003062401. In some embodiments, additional exemplary anti-BCMA CAR constructs are generated using an antigen-binding domain, e.g., the CDR, scFv or VH and VL sequences of PCT Publication WO2012/0163805 (the contents of which are hereby incorporated by reference in their entirety). In some embodiments, additional exemplary anti-BCMA CAR constructs are generated using an antigen-binding domain, e.g., the CDRs, scFv or VH and VL sequences of PCT Publication WO2016/014565 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, additional exemplary anti-BCMA CAR constructs are generated using an antigen binding domain, e.g., the CDR, scFv or VH and VL sequences from PCT Publication WO2014/122144 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, additional exemplary anti-BCMA CAR constructs are generated using CAR molecules and/or BCMA binding domains (e.g., CDR, scFv, or VH and VL sequences) from PCT Publication WO2016/014789 (the contents of which are incorporated herein by reference in its entirety). volume). In some embodiments, additional exemplary anti-BCMA CAR constructs are generated using CAR molecules and/or BCMA binding domains (e.g., CDR, scFv, or VH and VL sequences) from PCT Publication WO2014/089335 (the contents of which are incorporated herein by reference in its entirety). volume). In some embodiments, additional exemplary anti-BCMA CAR constructs are generated using CAR molecules and/or BCMA binding domains (e.g., CDR, scFv, or VH and VL sequences) from PCT Publication WO2014/140248 (the contents of which are incorporated herein by reference in its entirety). volume).

In one embodiment, the antigen-binding domain against a Tn antigen is an antigen-binding portion, e.g., a CDR, from an antibody described in, e.g., US 2014/0178365, US8440798, Brooks et al., PNAS 107(22):10056-10061 (2010) , and Stone et al., OncoImmunology 1(6):863-873(2012).

In one embodiment, the antigen-binding domain against PSMA is an antigen-binding portion, e.g. a CDR, from an antibody described in e.g. Parker et al., Protein Expr Purif 89(2):136-145 (2013), US 20110268656 (J591 ScFv ); Frigerio et al, European J Cancer 49(9):2223-2232 (2013) (scFvD2B); WO 2006125481 (mAb 3/A12, 3/E7 and 3/F11) and single chain antibody fragments (scFv A5 and D7).

In one embodiment, the anti-ROR1 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody as described in, e.g., Hudecek et al., Clin Cancer Res 19(12):3153-3164 (2013); WO 2011159847 and US20130101607.

In one embodiment, an anti-FLT3 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody described in e.g.

In one embodiment, the anti-TAG72 antigen-binding domain is an antigen-binding portion, eg a CDR, from an antibody as described in, eg, Hombach et al., Gastroenterology 113(4):1163-1170 (1997); and Abcam ab691.

In one embodiment, the anti-FAP antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody described in, e.g., Ostermann et al., Clinical Cancer Research 14:4584-4592 (2008) (FAP5), U.S. Patent Publication No. 2009 /0304718; sibrotuzumab (see, for example, Hofheinz et al., Oncology Research and Treatment 26(1), 2003); and Tran et al., J Exp Med 210(6):1125-1135 (2013).

In one embodiment, the anti-CD38 antigen-binding domain is an antigen-binding portion, e.g., CDR, from daratumumab (see, e.g., Groen et al., Blood 116(21):1261-1262 (2010); MOR202 (see, e.g., US8263746); or antibodies described in US8362211.

In one embodiment, the anti-CD44v6 antigen-binding domain is an antigen-binding portion, eg a CDR, from an antibody as described in, eg, Casucci et al., Blood 122(20):3461-3472 (2013).

In one embodiment, the antigen-binding domain against CEA is an antigen-binding portion, eg a CDR, from an antibody as described in, eg, Chmielewski et al., Gastoenterology 143(4):1095-1107 (2012).

In one embodiment, the anti-EPCAM antigen-binding domain is an antigen-binding portion, eg, CDR, from an antibody selected from MT110, an anti-EpCAM-CD3 bispecific Ab (see, eg, clinicaltrials.gov/ct2/show/NCT00635596); edrecolomab; 3622W94; ING-1 and adecatumumab (MT201).

In one embodiment, the anti-PRSS21 antigen-binding domain is an antigen-binding portion, eg, a CDR, from an antibody described in US Pat. No. 8,080,650.

In one embodiment, the antigen-binding domain against B7H3 is an antigen-binding portion, eg, a CDR, from an antibody MGA271 (Macrogenics).

In one embodiment, the antigen-binding domain against KIT is an antigen-binding portion, eg, a CDR, from an antibody described in, eg, US7915391, US20120288506, and several commercial catalog antibodies.

In one embodiment, the anti-IL-13Ra2 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody described in e.g.

In one embodiment, the antigen-binding domain against CD30 is an antigen-binding portion, eg, a CDR, from an antibody as described in, eg, US7090843 B1 and EP0805871.

In one embodiment, the antigen-binding domain against GD3 is an antigen-binding portion, eg, a CDR, from an antibody described in, eg, US7253263; US 8207308; US20120276046; EP1013761; WO2005035577 and US6437098.

In one embodiment, the antigen-binding domain against CD171 is an antigen-binding portion, e.g., a CDR, from an antibody described in, e.g., Hong et al., J Immunother 37(2):93-104 (2014).

In one embodiment, the anti-IL-11Ra antigen-binding domain is an antigen-binding portion, eg, a CDR, from an antibody available from Abcam (catalog # ab55262) or Novus Biologicals (catalog # EPR5446). In another embodiment, the anti-IL-11Ra antigen-binding domain is a peptide, see, for example, Huang et al., Cancer Res 72(1):271-281 (2012).

In one embodiment, the antigen-binding domain against PSCA is an antigen-binding portion, eg, a CDR, from an antibody as described in, eg, Morgenroth et al., Prostate 67(10):1121-1131 (2007) (scFv 7F5); Nejatollahi et al., J of Oncology 2013(2013), article ID 839831 (scFv C5-II); and in US Patent Publication No. 20090311181.

In one embodiment, the antigen-binding domain against VEGFR2 is an antigen-binding portion, e.g., a CDR, from an antibody described, for example, in Chinnasamy et al., J Clin Invest 120(11):3953-3968 (2010).

In one embodiment, the antigen-binding domain against LewisY is an antigen-binding portion, e.g., a CDR, from an antibody described in, e.g., Kelly et al., Cancer Biother Radiopharm 23(4):411-423 (2008) (hu3S193 Ab (scFvs) ); Dolezal et al., Protein Engineering 16(1):47-56 (2003) (NC10 scFv).

In one embodiment, the antigen-binding domain against CD24 is an antigen-binding portion, e.g., a CDR, from an antibody described, for example, in Maliar et al., Gastroenterology 143(5):1375-1384 (2012).

In one embodiment, the anti-PDGFR-beta antigen-binding domain is an antigen-binding portion, eg, a CDR, from an Abcam ab32570 antibody.

In one embodiment, the antigen-binding domain against SSEA-4 is an antigen-binding portion, eg, a CDR, from the MC813 antibody (Cell Signaling), or from other commercially available antibodies.

In one embodiment, the antigen-binding domain against CD20 is an antigen-binding portion, e.g., a CDR, from the antibody rituximab, ofatumumab, ocrelizumab, veltuzumab, or GA101.

In one embodiment, the antigen-binding domain against folate receptor-alpha is an antigen-binding portion, e.g., a CDR, from an IMGN853 antibody or an antibody described in US20120009181; US4851332, LK26: US5952484.

In one embodiment, the anti-ERBB2 antigen-binding domain (Her2/neu) is an antigen-binding portion, eg, a CDR, from a trastuzumab or pertuzumab antibody.

In one embodiment, the anti-MUC1 antigen-binding domain is an antigen-binding portion, eg, a CDR, from the SAR566658 antibody.

In one embodiment, the anti-EGFR antigen-binding domain is an antigen-binding portion, eg, a CDR, from a cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab antibody. In one embodiment, the anti-EGFRvIII antigen-binding domain is or may be derived from an antigen-binding domain, e.g., a CDR, scFv, or VH and VL of an antibody, an antigen-binding fragment, or a CAR as described, for example, in PCT publication WO2014/130657 (in one embodiment, the CAR is is the CAR described in WO2014/130657, the contents of which are incorporated herein by reference in their entirety).

In one embodiment, the anti-NCAM antigen-binding domain is an antigen-binding portion, e.g., a CDR, from antibody clone 2-2B: MAB5324 (EMD Millipore)

In one embodiment, the anti-ephrin B2 antigen-binding domain is an antigen-binding portion, eg a CDR, from an antibody as described in, eg, Abengozar et al., Blood 119(19):4565-4576 (2012).

In one embodiment, the antigen-binding domain against the IGF-I receptor is an antigen-binding portion, eg, a CDR, from an antibody described in, eg, US8344112 B2; EP2322550A1; WO 2006/138315 or PCT/US2006/022995.

In one embodiment, the anti-CAIX antigen-binding domain is an antigen-binding portion, eg, CDR, from antibody clone 303123 (R&D Systems).

In one embodiment, the antigen-binding domain against LMP2 is an antigen-binding portion, for example, a CDR, from an antibody described in, for example, US7410640 or US20050129701.

In one embodiment, the antigen-binding domain against gp100 is an antigen-binding portion, e.g., a CDR, from an HMB45 antibody, NKIbetaB, or an antibody described in WO2013165940 or US20130295007

In one embodiment, the anti-tyrosinase antigen-binding domain is an antigen-binding portion, such as a CDR, from an antibody described in, for example, US5843674 or US19950504048.

In one embodiment, the antigen-binding domain against EphA2 is an antigen-binding portion, eg, a CDR, from an antibody as described in, eg, Yu et al., Mol Ther 22(1):102-111 (2014).

In one embodiment, the antigen-binding domain against GD3 is an antigen-binding portion, eg, a CDR, from an antibody described in, eg, US7253263; US 8207308; US20120276046; EP1013761A3; 20120276046; WO2005035577 or US6437098.

In one embodiment, the anti-fucosyl-GM1 antigen-binding domain is an antigen-binding portion, eg a CDR, from an antibody described in, eg, US20100297138 or WO2007/067992.

In one embodiment, the antigen-binding domain against sLe is an antigen-binding portion, e.g., CDR, from antibody G193 (for lewis Y), see Scott AM et al, Cancer Res 60: 3254-61 (2000), as also described in Neeson et al, J Immunol May 2013 190 (Meeting Abstract Supplement) 177.10.

In one embodiment, the antigen-binding domain against GM3 is an antigen-binding portion, eg, CDR, from antibody CA 2523449 (mAb 14F7).

In one embodiment, the anti-HMWMAA antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody described in, e.g., Kmiecik et al., Oncoimmunology 3(1):e27185 (2014) (PMID: 24575382) (mAb9.2.27) ; US6528481; WO2010033866 or US20140004124.

In one embodiment, the anti-o-acetyl-GD2 antigen-binding domain is an antigen-binding portion, eg, a CDR, from an 8B6 antibody.

In one embodiment, the anti-TEM1/CD248 antigen-binding domain is an antigen-binding portion, eg, a CDR, from an antibody as described in, eg, Marty et al., Cancer Lett 235(2):298-308 (2006); Zhao et al., J Immunol Methods 363(2):221-232 (2011).

In one embodiment, the anti-CLDN6 antigen-binding domain is an antigen-binding portion, eg, CDR, from the IMAB027 antibody (Ganymed Pharmaceuticals), see, eg, clinicaltrial.gov/show/NCT02054351.

In one embodiment, the anti-TSHR antigen-binding domain is an antigen-binding portion, eg, a CDR, from an antibody described in, eg, US8603466; US8501415 or US8309693.

In one embodiment, the antigen-binding domain against GPRC5D is an antigen-binding portion, such as a CDR, from antibody FAB6300A (R&D Systems) or LS-A4180 (Lifespan Biosciences).

In one embodiment, the antigen-binding domain against CD97 is an antigen-binding portion, eg, a CDR, from an antibody described in, eg, US6846911; de Groot et al., J Immunol 183(6):4127-4134 (2009); or antibodies from R&D:MAB3734.

In one embodiment, the anti-ALK antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody as described in, e.g., Mino-Kenudson et al., Clin Cancer Res 16(5):1561-1571 (2010).

In one embodiment, the polysialic acid antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody described, for example, in Nagae et al., J Biol Chem 288(47):33784-33796 (2013).

In one embodiment, the anti-PLAC1 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody described, for example, in Ghods et al., Biotechnol Appl Biochem 2013 doi:10.1002/bab.1177.

In one embodiment, the antigen-binding domain against GloboH is the antigen-binding portion of a VK9 antibody or an antibody as described in, for example, Kudryashov V et al, Glycoconj J.15(3):243-9 (1998), Lou et al., Proc Natl Acad. Sci USA 111(7):2482-2487 (2014); MBr1: Bremer E-G et al. J Biol Chem 259:14773-14777 (1984).

In one embodiment, the anti-NY-BR-1 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody as described in, e.g., Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007).

In one embodiment, the antigen-binding domain against WT-1 is an antigen-binding portion, e.g., a CDR, from an antibody described, for example, in Dao et al., Sci Transl Med 5(176):176ra33 (2013) or WO2012/135854.

In one embodiment, the anti-MAGE-A1 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody described in, e.g., Willemsen et al., J Immunol 174(12):7853-7858 (2005) (TCR-like scFv ).

In one embodiment, the antigen-binding domain against sperm protein 17 is an antigen-binding portion, e.g., a CDR, from an antibody described in, e.g., Song et al., Target Oncol 2013 Aug 14 (PMID: 23943313); Song et al., Med Oncol 29(4):2923-2931 (2012).

In one embodiment, the antigen-binding domain against Tie 2 is an antigen-binding portion, eg, a CDR, from an AB33 antibody (Cell Signaling Technology).

In one embodiment, the antigen-binding domain against MAD-CT-2 is an antigen-binding portion, eg, a CDR, from an antibody described in, eg, PMID: 2450952; US7635753.

In one embodiment, the antigen-binding domain against Fos-related antigen 1 is an antigen-binding portion, eg CDR, from the 12F9 antibody (Novus Biologicals).

In one embodiment, the anti-MelanA/MART1 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an antibody described in EP2514766 A2 or US 7,749,719.

In one embodiment, the antigen-binding domain against sarcoma translocation breakpoints is an antigen-binding portion, eg, a CDR, from an antibody as described in, eg, Luo et al, EMBO Mol. Med. 4(6):453-461 (2012).

In one embodiment, the anti-TRP-2 antigen-binding domain is an antigen-binding portion, eg, a CDR, from an antibody as described in, eg, Wang et al, J Exp Med. 184(6):2207-16 (1996).

In one embodiment, the anti-CYP1B1 antigen-binding domain is an antigen-binding portion, eg a CDR, from an antibody as described in, eg, Maecker et al, Blood 102 (9): 3287-3294 (2003).

In one embodiment, the anti-RAGE-1 antigen-binding domain is an antigen-binding portion, eg, a CDR, from the MAB5328 antibody (EMD Millipore).

In one embodiment, the antigen-binding domain against human telomerase reverse transcriptase is an antigen-binding portion, e.g., a CDR, from an antibody having catalog number LS-B95-100 (Lifespan Biosciences)

In one embodiment, the anti-intestinal carboxylesterase antigen-binding domain is the antigen-binding portion, eg, CDR, from antibody 4F12, catalog number: LS-B6190-50 (Lifespan Biosciences).

In one embodiment, the anti-mut hsp70-2 antigen-binding domain is the antigen-binding portion, eg, CDR, from a Lifespan Biosciences antibody, monoclonal, catalog number: LS-C133261-100 (Lifespan Biosciences).

In one embodiment, the anti-CD79a antigen-binding domain is an antigen-binding portion, eg, CDR, from an anti-CD79a antibody [HM47/A9] (ab3121) available from Abcam; CD79A Antibody #3351, available from Cell Signaling Technology; or antibody HPA017748, an antibody against CD79A produced in rabbit, available from Sigma Aldrich.

In one embodiment, an anti-CD79b antigen-binding domain is an antigen-binding portion, e.g., a CDR, from the polatuzumab vedotin antibody, an anti-CD79b antibody described in Dornan et al., "Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b- vc-MMAE, for the treatment of non-Hodgkin lymphoma" Blood. 2009 Sep 24;114(13):2721-9. doi:10.1182/blood-2009-02-205500. Epub 2009 Jul 24, or the anti-CD79b/CD3 bispecific antibody described in "4507 Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a Potential Therapy for B Cell Malignancies" Abstracts of 56 ^th ASH Annual Meeting and Exposition, San Francisco, CA December 6-9 2014.

In one embodiment, the anti-CD72 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from the J3-109 antibody described in Myers and Uckun, "An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia". Leuk Lymphoma. 1995 Jun;18(1-2):119-22, or an anti-CD72 (10D6.8.1, mIgG1) antibody described in Polson et al., "Antibody-Drug Conjugates for the Treatment of Non-Hodgkin's lymphoma: Target and Linker -Drug Selection" Cancer Res March 15, 2009 69; 2358.

In one embodiment, the antigen-binding domain against LAIR1 is the antigen-binding portion, eg, CDR, from the ANT-301 LAIR1 antibody available from ProSpec; or an anti-human CD305 antibody (LAIR1) available from BioLegend.

In one embodiment, the anti-FCAR antigen-binding domain is an antigen-binding portion, e.g., a CDR, from the CD89/FCARAntibody antibody (catalog number #10414-H08H) available from Sino Biological Inc.

In one embodiment, an anti-LILRA2 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an anti-LILRA2 monoclonal antibody (M17), clone 3C7, available from Abnova, or a mouse anti-LILRA2 antibody, monoclonal (2D7) available from Lifespan Biosciences.

In one embodiment, the anti-CD300LF antigen-binding domain is an antigen-binding portion, e.g., a CDR, from a mouse anti-CMRF35-like molecule 1 monoclonal [UP-D2] available from BioLegend or a rat anti-CMRF35-like molecule 1 monoclonal [234903 ], available from R&D Systems.

In one embodiment, an anti-CLEC12A antigen-binding domain is an antigen-binding portion, e.g., a CDR, from a bispecific scFv antibody anti-T cell recruiter antibody (BiTE) and ADC described in Noordhuis et al., "Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1xCD3 BiTE Antibody" 53rd ASH Annual Meeting and Exposition, December 10-13, 2011, and MCLA-117 (Merus).

In one embodiment, the anti-BST2 (also referred to as CD317) antigen-binding domain is the antigen-binding portion, e.g., CDR, from mouse anti-CD317 monoclonal [3H4], available from Antibody-Online, or mouse anti-CD317 monoclonal [696739], available from R&D Systems.

In one embodiment, the anti-EMR2 (also referred to as CD312) antigen-binding domain is the antigen-binding portion, e.g., CDR, from mouse anti-CD312 monoclonal [LS-B8033] available from Lifespan Biosciences or mouse anti-CD312 monoclonal [494025] available from R&D Systems.

In one embodiment, the anti-LY75 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from a mouse anti-lymphocyte antigen 75 monoclonal [HD30] available from EMD Millipore or a mouse anti-lymphocyte antigen 75 monoclonal [A15797] available from life technologies.

In one embodiment, the antigen-binding domain against GPC3 is an antigen-binding portion, eg, CDR, from the hGC33 antibody described in Nakano K, Ishiguro T, Konishi H, et al. Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization. Anticancer Drugs. Nov 2010; 21(10):907-916, or MDX-1414, HN3, or YP7, all three of which are described in Feng et al., "Glypican-3 antibodies: a new therapeutic target for liver cancer". FEBS Lett. 2014 Jan 21;588(2):377-82.

In one embodiment, an anti-FCRL5 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from an anti-FcRL5 antibody described in Elkins et al., "FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma" by Mol Cancer Ther. 2012 Oct;11(10):2222-32.

In one embodiment, the anti-IGLL1 antigen-binding domain is an antigen-binding portion, e.g., a CDR, from a mouse anti-IgL-like polypeptide 1 monoclonal antibody [AT1G4] available from Lifespan Biosciences, an anti-IgL lambda-like polypeptide 1 antibody, monoclonal [HSL11] , available from BioLegend.

In one embodiment, the antigen binding domain comprises one, two, three (e.g., all three) of the heavy chain CDRs, CDR1 HC, CDR2 HC, and CDR3 HC, from the antibody above and/or one, two, three (e.g., all three ) light chain CDRs, CDR1 LC, CDR2 LC and CDR3 LC, from the antibody above. In one embodiment, the antigen-binding domain comprises a heavy chain variable region and/or a light chain variable region of an antibody as defined above.

In another aspect, the antigen binding domain comprises a humanized antibody or antibody fragment. In some aspects, a non-human antibody is humanized, where certain sequences or regions of the antibody have been modified to resemble an antibody naturally produced in humans, or a fragment thereof. In one aspect, the antigen binding domain is humanized.

Humanized antibody can be obtained using various methods known in the art, including, but not limited to, CDR transplantation (see, for example, European patent No. EP 239400; international publication No. WO 91/09967 and US patents No. 5225539, 5530101 and 5585089, each of which is incorporated into the present description by reference in its entirety), veneering or surface modification, see, for example, European patents No. EP 592106 and EP 519596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973, each of which is incorporated herein by reference in its entirety), chain shuffling (see, for example, US Pat. No. 5,565,332, which is incorporated herein by reference in its entirety) and the methods described, for example, in US Patent Application Publication No. US2005/0042664, US Patent Application Publication No. US2005/0048617, US Patent No. 6407213, US Patent No. 5766886, International Publication No. WO 9317105, Tan et al. , J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267- 79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp) :5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al. ., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. Often, framework residues in framework regions are replaced with the corresponding residue from the CDR-donor antibody to alter, eg, increase, antigen binding. These framework region substitutions are identified by methods well known in the art, e.g., modeling interactions between CDRs and framework residues to identify framework residues important for antigen binding, sequence comparisons to identify unusual framework residues at specific positions (See, for example, Queen et al. al., US patent No. 5585089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety).

A humanized antibody or antibody fragment has one or more amino acid residues left over from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. As described herein, humanized antibodies or antibody fragments comprise one or more CDRs of non-human immunoglobulin molecules and framework regions, wherein the amino acid residues containing the framework region are derived wholly or in large part from a human embryonic-type sequence. Many methods for humanizing antibodies or antibody fragments are well known in the art and can be carried out essentially according to the method of Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing CDRs or rodent CDR sequences with corresponding human antibody sequences, i.e. CDR transplants (EP 239400; PCT Publication No. WO 91/09967; and US Pat. Nos. 4,816,567; 6,331,415; 5,225,539; In such humanized antibodies and fragments, substantially less than an intact human variable domain is replaced by a corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which some of the CDRs and possibly some of the framework (FR) residues are replaced by residues from analogous regions of rodent antibodies. Humanization of antibodies and antibody fragments can also be accomplished by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6) :805-814 (1994), and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (US Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

The choice of human variable domains, both light and heavy, for use in the preparation of humanized antibodies is made so as to reduce antigenicity. According to the so-called "best fit" method, a rodent antibody variable domain sequence is screened against an entire library of known human variable domain sequences. The human sequence that is most similar to the rodent sequence is then adopted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol Biol., 196:901 (1987, the contents of which are incorporated herein by reference in their entirety). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework region can be used for several different humanized antibodies (see, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the framework region, eg all four framework regions, of the heavy chain variable region is derived from the VH4_4-59 germline sequence. In one embodiment, the framework region may contain one, two, three, four, or five modifications, such as substitutions, for example, with an amino acid of the corresponding mouse sequence. In one embodiment, the framework region, eg, all four variable light chain framework regions, is derived from the VK3_1.25 germline sequence. In one embodiment, the framework region may contain one, two, three, four, or five modifications, such as substitutions, per amino acid of the corresponding mouse sequence.

In some aspects, the portion of the CAR composition of the invention that contains the antibody fragment is humanized while retaining high affinity for the target antigen and other beneficial biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are generated through a process for analyzing parental sequences and various conceptual humanized products using 3D models of the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these images allows analysis of the likely role of residues in the functioning of the candidate immunoglobulin sequence, for example, analysis of residues that affect the ability of the candidate immunoglobulin to bind the target antigen. Thus, FR residues can be selected and combined from recipient and import sequences so as to achieve desired characteristics of the antibody or antibody fragment, such as increased affinity for the target antigen. Typically, CDR residues are directly and most significantly involved in influencing antigen binding.

A humanized antibody or antibody fragment may retain antigenic specificity similar to the parent antibody, for example, within the scope of the present invention, the ability to bind a human cancer-associated antigen as described herein. In some embodiments, a humanized antibody or antibody fragment may have increased binding affinity and/or specificity for a human cancer-associated antigen, as described herein.

In one aspect, the antigen-binding domain of the invention is characterized by specific functional features or properties of an antibody or antibody fragment. For example, in one aspect, a portion of a CAR composition of the invention that contains an antigen-binding domain specifically binds a tumor antigen as described herein.

In one aspect, a cancer-associated antigen-binding domain as described herein is a fragment, eg, a single chain variable fragment (scFv). In one aspect, a cancer-associated antigen-binding domain as described herein is a Fv, Fab, (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the invention bind a cancer-associated antigen as described herein with wild-type or enhanced affinity.

In some cases, scFv can be obtained in accordance with the method known in this field (see, for example, Birdet al., (1988) Science 242:423-426 and Hustonet al., (1988) Proc. Natl. Acad. sci. USA 85:5879-5883). ScFv molecules can be generated by linking the VH and VL regions together using flexible polypeptide linkers. The scFv molecules contain a linker (eg, a Ser-Gly linker) with an optimized length and/or amino acid composition. The length of the linker can greatly influence how the scFv variable regions fold and interact. In fact, if a short polypeptide linker (eg, 5-10 amino acids) is used, this prevents intrachain folding. Intrachain folding is also required to bring two variable regions closer together to form a functional epitope binding site. For an example of linker orientation and size, see e.g. Hollingeret al. 1993 Proc Natl Acad. sci. U.S.A. 90:6444-6448, US Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publication Nos. WO2006/020258 and WO2007/024715, which are incorporated herein by reference.

scFv may contain a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence may contain any naturally occurring amino acid. In some embodiments, the implementation of the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains sets of glycine and serine repeats such as (Gly ₄ Ser) _n , where n is a positive integer equal to or greater than 1 (SEQ ID NO: 22). In one embodiment, the linker may be (Gly ₄ Ser) ₄ (SEQ ID NO: 29) or (Gly ₄ Ser) ₃ (SEQ ID NO: 30). Varying the length of the linker may maintain or enhance activity, providing improved performance in activity assays.

In another aspect, the antigen binding domain is a T cell receptor ("TCR") or a fragment thereof, such as a single chain TCR (scTCR). Methods for producing such TCRs are known in the art. See, for example, Willemsen RA et al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther 11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) (references incorporated herein in their entirety). For example, an scTCR can be constructed that contains the Vα and Vβ genes from a T cell clone linked by a linker (eg, a flexible peptide). This approach is very useful for a cancer-associated target that is itself intracellular, but a fragment of such an antigen (peptide) is presented on the cell surface by MHC.

Bispecific CARs

In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding affinity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, eg, on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. . In one embodiment, the bispecific antibody molecule comprises a half-antibody having binding specificity for a first epitope and a half-antibody having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half-antibody or fragment thereof having a binding specificity for a first epitope and a half-antibody or fragment thereof having a binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises an scFv or fragment thereof that has a binding specificity for a first epitope and an scFv or fragment that has a binding specificity for a second epitope.

In certain embodiments, the antibody molecule is a polyspecific (eg, bispecific or trispecific) antibody molecule. Protocols for obtaining bispecific or heterodimeric antibody molecules are known in the art; including, but not limited to, for example, the "protrusion in the cavity" approach described, for example, in US 5731168; formation of Fc pairs by electrostatic induction as described, for example, in WO 09/089004, WO 06/106905 and WO 2010/129304; formation of heterodimers via chain exchange engineered domains (SEED) as described. for example in WO 07/110205; Fab arm exchange as described, for example, in WO 08/119353, WO 2011/131746 and WO 2013/060867; a double antibody conjugate, for example by crosslinking antibodies to form a bispecific structure using a heterobifunctional reagent having an amine reactive group and a reactive sulfhydryl group, as described, for example, in US 4,433,059; bispecific antibody determinants obtained by recombination of half antibodies (heavy-light chain pairs or Fab) from different antibodies through a cycle of reduction and oxidation of disulfide bonds between the two heavy chains, as described, for example, in US 4444878; trifunctional antibodies, eg three Fab' fragments cross-linked via sulfhydryl reactive groups, as described, eg, in US5273743; biosynthetic binding proteins, eg a pair of scFvs, fused at the C-terminus, preferably by disulfide or amine-reactive chemical fusion, as described, eg, in US5534254; bifunctional antibodies, eg Fab fragments, with differing binding specificities dimerized through leucine zippers (eg c-fos and c-jun) that have replaced the constant domain, as described in eg US5582996; bispecific and oligospecific mono- and oligovalent receptors, e.g., the VH-CH1 regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CH1 region of one antibody and the VH region of another antibody, usually with their associated light chains , as described, for example, in US5591828; bispecific DNA conjugates, eg antibody cross-linking or Fab fragments via a double-stranded DNA fragment, as described, for example, in US5635602; bispecific fusion proteins, for example, an expression construct containing two scFvs with a hydrophilic helical peptide linker between them and a complete constant region, as described, for example , in US5637481; polyvalent and polyspecific binding proteins, for example, a dimer of polypeptides having a first domain with an Ig heavy chain variable region binding region and a second domain with an Ig light chain variable region binding region, commonly referred to as diabodies (higher order structures forming bispecific, trispecific or tetraspecific molecules as described in, for example, US5837242; mini-antibody constructs with linked VL and VH chains further connected by peptide spacers to the antibody hinge region and CH3 region, which can dimerize to form bispecific/polyvalent molecules, as described, for example in US5837821: VH and VL domains linked by a short peptide linker (eg 5 or 10 amino acids) or no linker at all in any orientation, which can form dimers to form bispecific diantibodies, trimers and tetramers, as described, for example, in US5844094; a chain of VH domains (or VL domains in members of the family) linked by peptide bonds with crosslinkable groups at the C-terminus, furthermore linked to the VL domains to form a series of FVs (or scFvs) as described, for example, in US5864019; and single-stranded binding polypeptides with both VH and VL domain linked through a peptide linker, combined into multivalent structures by non-covalent or chemical crosslinking to form, for example, homobivalent, heterobivalent, trivalent and tetravalent structures using both the scFV format and and diabody format as described, for example, in US5869620. Дополнительные иллюстративные полиспецифические и биспецифические молекулы и способы их получения могут быть найдены, например, в US5910573, US5932448, US5959083, US5989830, US6005079, US6239259, US6294353, US6333396, US6476198, US6511663, US6670453, US6743896, US6809185, US6833441, US7129330, US7183076, US7521056 , US7527787, US7534866, US7612181, US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1, US2003211078A1, US2004219643A1, US2004220388A1, US2004242847A1, US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1, US2005100543A1, US2005136049A1, US2005136051A1, US2005163782A1, US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1, US2006263367A1, US2007004909A1 , US2007087381A1, US2007128150A1, US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1, US2008069820A1, US2008152645A1, US2008171855A1, US2008241884A1, US2008254512A1, US2008260738A1, US2009130106A1, US2009148905A1, US2009155275A1, US2009162359A1, US2009162360A1, US2009175851A1, US20091758 67A1, US2009232811A1, US2009234105A1, US2009263392A1, US2009274649A1, EP346087A2, WO0006605A2, WO02072635A2, WO04081051A1, WO06020258A2, WO2007044887A2, WO2007095338A2, WO2007137760A2, WO2008119353A1, WO2009021754A2, WO2009068630A1, WO9103493A1, WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1, WO9637621A2, WO9964460A1. The contents of the aforementioned applications are incorporated herein by reference in their entirety.

In each antibody or antibody fragment (eg, scFv) of a bispecific antibody molecule, the VH may be above or below the VL. In some embodiments, the upstream antibody or antibody fragment (eg, scFv) is organized such that its VH (VH ₁ ) is above its VL (VL ₁ ), and the downstream antibody or antibody fragment (eg, scFv) is organized such that its VL (VL ₂ ) is above their VH (VH ₂ ), so that the whole bispecific antibody molecule is of the order VH ₁ -VL ₁ -VL ₂ -VH ₂ . In other embodiments, the upstream antibody or antibody fragment (eg, scFv) is organized such that its VL (VL ₁ ) is upstream of its VH (VH ₁ ) and the downstream antibody or antibody fragment (eg, scFv) is organized such that its VH ( VH ₂ ) is above their VL (VL ₂ ), so that the whole bispecific antibody molecule is of the order VL ₁ -VH ₁ -VH ₂ -VL ₂ . Optionally, the linker is located between two antibodies or antibody fragments (eg, scFv), for example, between VL ₁ and VL ₂ if the design is VH ₁ -VL ₁ -VL ₂ -VH ₂ , or between VH ₁ and VH ₂ if the design has the order VL ₁ -VH ₁ -VH ₂ -VL ₂ . The linker may be a linker as described herein, for example a (Gly ₄ -Ser)n linker where n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 72). Typically, the linker between two scFvs should be long enough to avoid mismatching between the domains of the two scFvs. Optionally, the linker is located between the VL and VH of the first scFv. Optionally, the linker is located between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more of the linkers may be the same or may be different. Thus, in some embodiments, a bispecific CAR contains VL, VH, and optionally one or more linkers, in the order described herein.

Stability and Mutations

The stability of an antigen-binding domain against a cancer-associated antigen as described herein, e.g., scFv molecules (e.g., soluble scFv), can be assessed in comparison to the biophysical properties (e.g., thermal stability) of a conventional scFv control molecule or a full-length antibody. In one embodiment, the humanized scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1 .5, approximately 1.75, approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, approximately 5, approximately 5.5, approximately 6, approximately 6.5, approximately 7, approximately 7.5, approximately 8, approximately 8.5, approximately 9, approximately 9.5, approximately 10 degrees, approximately 11 degrees, approximately 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius improved relative to a control binding molecule (eg, a conventional scFv molecule) in the assays described.

Thus, improved thermal stability of the antigen-binding domain against the cancer-associated antigen described herein, eg scFv, is imparted to the entire CAR construct, resulting in improved therapeutic properties of the CAR construct. The thermal stability of an antigen-binding domain against a cancer-associated antigen described herein, eg scFv, can be improved by at least about 2°C or 3°C compared to a conventional antibody. In one embodiment, an antigen-binding domain against a cancer-associated antigen described herein, eg, scFv, has a 1°C improved thermal stability compared to a conventional antibody. In another embodiment, an antigen-binding domain against a cancer-associated antigen described herein, eg, scFv, has a 2° C. improved thermal stability compared to a conventional antibody. In another embodiment, the scFv has 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15° C. improved thermal stability compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules described herein and the scFv molecules or Fab fragments of the antibody from which the scFv VH and VL are derived. Thermal stability can be quantified using methods known in the art. For example, in one embodiment, Tm can be quantified. Methods for measuring Tm and other methods for determining protein stability are described in more detail below.

Mutations in scFv (due to humanization or direct mutagenesis of soluble scFv) can measure scFv stability and improve overall scFv and CAR construct stability. The stability of the humanized scFv is compared to the mouse scFv using Tm, denaturation temperature, and aggregation temperature.

The binding capacity of mutant scFvs can be determined using assay methods known in the art and described herein.

In one embodiment, the antigen-binding domain against a cancer-associated antigen described herein, eg, scFv, contains at least one mutation due to a humanization process such that the mutant scFv confers improved stability of the CAR construct. In another embodiment, the antigen-binding domain against a cancer-associated antigen, e.g., scFv, contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations due to the humanization process such that the mutated scFv reports improved stability of the CAR design.

Methods for Assessing Protein Stability

The stability of the antigen binding domain can be assessed using, for example, the methods described below. Such methods allow the definition of multiple thermal unfold transitions where the least stable domain either unfolds first or limits the overall stability threshold of a multi-domain element that unfolds cooperatively (eg, a multi-domain protein that exhibits a single unfold transition). The least stable domain can be identified in a number of additional ways. Mutagenesis can be performed to study which domain limits overall stability. In addition, protease resistance studies of a multidomain protein can be carried out under conditions known to naturally unfold by DSC or other spectroscopic methods (Fontana, et al., (1997) Fold. Des., 2: R17-26 Dimasi et al (2009) J Mol Biol 393: 672-692). Once the least stable domain has been identified, the sequence encoding that domain (or a portion thereof) can be used as the sequence of interest in the methods.

a) Thermal stability

The thermal stability of compositions can be analyzed using a variety of non-limiting biophysical or biochemical methods known in the art. In certain embodiments, thermal stability is assessed by analytical spectroscopy.

An exemplary analytical spectroscopy method is differential scanning calorimetry (DSC). DSC uses a calorimeter that is sensitive to heat absorption accompanying the unfolding of most proteins or protein domains (see, for example, Sanchez-Ruiz, et al., Biochemistry, 27: 1648-52, 1988). To determine the thermal stability of a protein, a protein sample is placed in the calorimeter and the temperature is raised until the Fab or scFv unfolds. The temperature at which the protein unfolds indicates the overall stability of the protein.

Another exemplary analytical spectroscopy technique is circular dichroism (CD) spectroscopy. CD spectroscopy measures the optical activity of a composition as a function of temperature rise. Circular dichroism (CD) spectroscopy measures the differences in absorption of left hand polarized left hand polarized light, which is the result of structural asymmetry. A broken or unfolded structure results in a CD spectrum that is significantly different from that of an ordered or folded structure. The CD spectrum reflects the sensitivity of proteins to the denaturing effects of temperature increase and thus is indicative of the thermal stability of the protein (see van Mierlo and Steemsma, J. Biotechnol., 79(3):281-98, 2000).

Another exemplary analytical spectroscopy method for measuring thermal stability is fluorescence emission spectroscopy (see van Mierlo and Steemsma, supra). Another exemplary analytical spectroscopy method for measuring thermal stability is nuclear magnetic resonance (NMR) spectroscopy (see, for example, van Mierlo and Steemsma, supra).

The thermal stability of the composition can be measured biochemically. An exemplary biochemical method for assessing thermal stability is thermal stress analysis. In a "thermal loading assay", the composition is subjected to a range of elevated temperatures for a predetermined period of time. For example, in one embodiment, the scFv molecules to be tested, or molecules containing scFv molecules, are subjected to a range of increasing temperatures, eg, for 1-1.5 hours. The activity of the protein is then analyzed by a suitable biochemical assay. For example, if the protein is a binding protein (eg, scFv or scFv-containing polypeptide), the binding activity of the binding protein can be determined by functional or quantitative ELISA.

Such assays can be performed in high throughput format and in the formats described in the Examples section using E. coli and high throughput screening. You can create a library of antigen-binding domains, for example, which includes an antigen-binding domain against a cancer-associated antigen described herein, for example, scFv variants, using methods known in the art. An antigen-binding domain can be induced, e.g., against a cancer-associated antigen described herein, e.g., scFv, and an antigen-binding domain, e.g., against a cancer-associated antigen described herein, e.g., scFv, can be thermally stressed . Test samples after loading can be analyzed for binding, and those antigen-binding domains against a cancer-associated antigen described herein, eg, scFvs, that are stable can be obtained in greater numbers and further characterized.

Thermal stability is assessed by measuring the melting point (Tm) of the composition using any of the methods described above (eg, analytical spectroscopy methods). The melting point is the melting temperature at the midpoint of the temperature transition curve, where 50% of the molecules of the composition are in a collapsed state (see, for example, Dimasi et al. (2009) J. Mol Biol. 393: 672-692). In one embodiment, the Tm values for an antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv, are approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C , 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58 °C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C , 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83 °C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C , 96°C, 97°C, 98°C, 99°C, 100°C. In one embodiment, the Tm values for IgG are approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62° C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87° C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C. In one embodiment, the Tm values for the polyvalent antibody are approximately 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C , 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62 °C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C , 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87 °C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C , 100°C.

Thermal stability is also evaluated by measuring the specific heat or heat capacity (Cp) of the composition using an analytical calorimetric method (eg DSC). The specific heat of the composition is the energy (eg, in kcal/mol) required to raise the temperature of 1 mol of water by 1°C. A large Cp is indicative of a denatured or inactive protein composition. The change in heat capacity (ΔCp) of the composition is measured by determining the specific heat of the composition before and after its thermal transition. Thermal stability can also be assessed by measuring or determining other parameters of thermodynamic stability, including Gibbs free energy for unfolding (ΔG), enthalpy of unfolding (ΔH), or entropy of unfolding (ΔS). One or more of the biochemical assays described above (eg, thermal stress analysis) are used to determine the temperature (ie, T _C value) at which 50% of the composition retains its activity (eg, binding activity).

In addition, mutations of the antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv, can be introduced to change the thermal stability of the antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv. When a humanized antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv, is included in a CAR construct, the antigen-binding domain against a cancer-associated antigen described herein, e.g., a humanized scFv, imparts thermal stability to the entire CAR over the present invention. In one embodiment, the antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv, contains a single mutation that confers thermal stability to the antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv. In another embodiment, the antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv, contains a plurality of mutations that confer thermal stability to the antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv. In one embodiment, multiple mutations in an antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv, have an additive effect on the thermal stability of the antigen-binding domain against a cancer-associated antigen described herein, e.g., scFv.

b) % Aggregation

The stability of a composition can be determined by determining its tendency to aggregate. Aggregation can be quantified using a variety of non-limiting biochemical or biophysical methods. For example, composition aggregation can be assessed using chromatography, such as size exclusion chromatography (SEC). SEC separates molecules by size. The column is filled with semi-solid granules of a polymer gel that allows ions and low molecular weight compounds to pass inside, but does not pass large ones. When a protein composition is applied to a column, compact, folded proteins (ie, non-aggregated proteins) are distributed in a larger volume of solvent than is available for large protein aggregates. Consequently, large aggregates move faster through the column and thus the mixture can be separated or fractionated into its components. Each fraction can be individually quantified (eg, by light scattering) as it elutes from the gel. Thus, the % aggregation of the composition can be determined by comparing the concentration of the fraction with the total concentration of the protein applied to the gel. Stable compositions elute from the column essentially as a single fraction and have essentially one peak in the elution profile or chromatogram.

c) Binding affinity

The stability of a composition can be assessed by determining its binding affinity for the target. A wide variety of methods are known in the art for determining binding affinity. An exemplary method for determining binding affinity uses surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows real-time analysis of biospecific reactions by detecting changes in protein concentrations on a biosensor array, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For a more detailed description, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B., et al . (1995) J. Mol. Recognize. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

In one aspect, a CAR antigen-binding domain comprises an amino acid sequence that is homologous to the amino acid sequence of an antigen-binding domain described herein, and the antigen-binding domain retains the desired functionality of the antigen-binding domain described herein.

In one specific aspect, the CAR composition of the invention comprises an antibody fragment. In the following aspect, the antibody fragment contains scFv.

In various aspects, a CAR antigen binding domain is constructed by modifying one or more amino acids in one or both variable regions (e.g., VH and/or VL), for example, in one or more CDR regions and/or in one or more framework regions. In one specific aspect, the CAR composition of the invention comprises an antibody fragment. In the following aspect, the antibody fragment contains scFv.

One of skill in the art will appreciate that an antibody or antibody fragment of the invention can be further modified such that its amino acid sequence differs (eg, relative to the wild type), but not with respect to the desired activity. For example, additional nucleotide substitutions can be made to a protein, leading to amino acid substitutions at "non-basic" amino acid residues. For example, a non-basic amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family. In another embodiment, a chain of amino acids may be replaced with a structurally similar chain that differs in the order and/or composition of members of the side chain family, for example, a conservative substitution may be made in which an amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains are defined in the art, including 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), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).

Percent identity in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same. Two sequences are "substantially identical" if two sequences have a specific percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71%. 72%. 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over the specified region or, when not specified, throughout the sequence), when compared and aligned for best fit over the comparison window or specified areas as measured using one of the following sequence comparison algorithms or by manual alignment or visual inspection. Optionally, identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) long, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids).

For sequence comparisons, typically one sequence acts as a reference sequence against which the test sequences are compared. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, the coordinates of the subsequences are indicated, if necessary, and the parameters of the program with the sequence comparison algorithm are assigned. You can use default program settings, or you can alternatively assign settings. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences relative to the reference sequence based on the program parameters. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, using the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443 using the similarity search method Pearson and Lipman, (1988) Proc. Nat'l. Acad. sci. USA 85:2444, using computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. The BLAST analysis software is publicly available through the National Center for Biotechnology Information.

Percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988), which is included in the ALIGN program (version 2.0), using a weight table residues of PAM120, a gap continuation penalty of 12, and a gap penalty of 4. In addition, percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970), which is included with GAP program in the GCG software package (available at http://www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and a gap penalty of 16, 14, 12, 10, 8, 6, or 4 and a gap penalty for continuing to skip 1, 2, 3, 4, 5 or 6.

In one aspect, the present invention provides for modifications to the original amino acid sequence of an antibody or fragment (eg, scFv) that result in functionally equivalent molecules. For example, the VH or VL of an antigen-binding domain against a cancer-associated antigen, eg, scFv, contained in a CAR can be modified to conserve at least about 70%, 71%, 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the original VH or VL framework region of the antigen-binding domain against a cancer-associated antigen, e.g., scFv. The present invention contemplates modifications to the entire CAR construct, eg, modification of one or more amino acid sequences of the various domains of the CAR construct, to obtain functionally equivalent molecules. The CAR design can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % identity with the original CAR construct.

transmembrane domain

With regard to the transmembrane domain, in various embodiments, the implementation of the CAR can be designed to contain a transmembrane domain that is associated with the extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 extracellular amino acids) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is a domain that is associated with one of the other domains used in CAR, for example, in one embodiment, the transmembrane domain can be derived from the same protein as the signal domain, costimulatory domain, or hinge domain. In another aspect, the transmembrane domain does not originate from the same protein as any other CAR domain. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerizing with another CAR on the cell surface of a CAR-expressing cell. In another aspect, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interactions with binding domains of a native binding partner present in the same CAR expressing cell.

The transmembrane domain may be derived from either a natural or recombinant source. When the source is natural, the domain may be from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain(s) when the CAR binds to a target. A transmembrane domain of particular use within the scope of the present invention may include at least the transmembrane region(s), e.g. T cell receptor alpha, beta or zeta chains, CD28, CD27, CD3epsilon, CD45, CD4, CD5, CD8 , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain may include at least the transmembrane region(s), e.g., from KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR , CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R-beta, IL2R-gamma, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6 , VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226 ), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.

In some instances, the transmembrane domain may be linked to an extracellular region of a CAR, eg, a CAR antigen-binding domain, via a hinge region, eg, a hinge region from a human protein. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., IgG4 hinge, IgD hinge), GS linker (e.g., GS linker described herein), KIR2DS2 hinge, or CD8a. In one embodiment, the hinge region or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 4. In one aspect, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO: 12.

In one aspect, the hinge or spacer comprises an IgG4 hinge. Например, в одном варианте осуществления шарнирная область или спейсер содержат шарнирную область с аминокислотной последовательностью: ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO: 6). В некоторых вариантах осуществления шарнирная область или спейсер содержат шарнирную область, кодируемую нуклеотидной последовательностью: GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO: 7).

In one aspect, the hinge or spacer comprises an IgD hinge. Например, в одном варианте осуществления шарнирная область или спейсер содержат шарнирную область с аминокислотной последовательностью RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO: 8). В некоторых вариантах осуществления шарнирная область или спейсер содержат шарнирную область, кодируемую нуклеотидной последовательностью AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT (SEQ ID NO: 9) .

In one aspect, the transmembrane domain may be recombinant, in which case it contains primarily hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan and valine may be present at each end of the recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker of 2 to 10 amino acids in length can form a link between the transmembrane domain and the cytoplasmic region of the CAR. The glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker contains the amino acid sequence GGGGSGGGGS (SEQ ID NO: 10). In some embodiments, the linker is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 11).

In one aspect, the hinge region or spacer comprises a KIR2DS2 hinge region.

Cytoplasmic domain

The cytoplasmic domain or region of the CAR includes an intracellular signaling domain. The intracellular signaling domain is usually responsible for the activation of at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell, for example, may be a cytolytic activity or a helper activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that signals an effector function and directs the cell to perform a specialized function. Although it is usually possible to use the entire intracellular signaling domain, in many cases it is not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the intact strand, provided that it signals the effector function. Thus, the term "intracellular signaling domain" is intended to include any truncated portion of an intracellular signaling domain sufficient to signal an effector function.

Examples of intracellular signaling domains for use in the CARs of the invention include cytoplasmic T cell receptor (TCR) and co-receptor sequences that act together to initiate signal transduction upon antigen receptor binding, as well as any derivative or variant of these sequences, and any recombinant sequence that has the same functionality.

It is known that the signals induced by the TCR alone are not sufficient for full T cell activation, and that a secondary and/or co-stimulatory signal is also required. Thus, T cell activation can be considered to be mediated by two distinct classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via TCRs (primary intracellular signaling domains), and sequences that act in an antigen-independent manner, providing a secondary or costimulatory ( secondary cytoplasmic domain, e.g. costimulatory domain).

The primary signaling domain regulates the primary activation of the TCR complex either stimulatory or inhibitory. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs that are known as immunoreceptor tyrosine activating motifs, or ITAMs.

Examples of ITAM-containing primary intracellular signaling domains that are particularly useful in the context of the invention include the CD3-zeta, total FcR-gamma (FCER1G), Fc-gamma RIIa, FcR-beta (Fc-epsilon R1b), CD3-gamma, CD3 signaling domains. -delta, CD3-epsilon, CD79a, CD79b, DAP10 and DAP12. In one embodiment, the CAR of the invention contains an intracellular signaling domain, such as the primary signaling domain of CD3-zeta.

In one embodiment, the primary signaling domain contains a modified ITAM domain, eg, a mutant ITAM domain, that has altered (eg, increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, such as an optimized and/or truncated ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain contains one, two, three, four or more ITAM motifs.

The CAR intracellular signaling domain may comprise the CD3-zeta signaling domain alone or in combination with any other desired intracellular signaling domain(s) useful in the context of the CAR of the invention. For example, a CAR intracellular signaling domain may contain a CD3-zeta chain portion and a co-stimulatory signaling domain. A cost-stimulatory signaling domain refers to the portion of a CAR containing the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule, other than an antigen receptor or ligands thereof, that is required for an effective lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 , and a ligand that specifically binds to CD83, and the like. For example, costimulation of CD27 has been shown to increase the expansion, effector function, and survival of human CART cells in vitro and enhance the viability and antitumor activity of human T cells in vivo (Song et al. Blood. 2012; 119(3):696-706) . Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8-alpha, CD8-beta, IL2R- beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp and CD19.

The intracellular signal sequences in the cytoplasmic portion of the CARs of the invention may be linked to each other in a random or specific order. Optionally, a short oligo or polypeptide linker, eg 2 to 10 amino acids long (eg 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids), can form a link between intracellular signal sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, eg alanine, glycine, can be used as a suitable linker.

In one aspect, an intracellular signaling domain is designed to contain two or more, eg 2, 3, 4, 5 or more, costimulatory signaling domains. In one embodiment, two or more, eg 2, 3, 4, 5 or more co-stimulatory signaling domains are separated by a linker molecule, such as the linker molecule described herein. In one embodiment, the intracellular signaling domain contains two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed to contain the CD3-zeta signaling domain and the CD28 signaling domain. In one aspect, the intracellular signaling domain is designed to contain the CD3-zeta signaling domain and the 4-1BB signaling domain. In one aspect, the 4-1BB signaling domain is the signal domain of SEQ ID NO: 14. In one aspect, the CD3-zeta signal domain is the signal domain of SEQ ID NO: 18.

In one aspect, the intracellular signaling domain is designed to contain the CD3-zeta signaling domain and the CD27 signaling domain. In one aspect, the CD27 signaling domain contains the amino acid sequence QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 16). In one aspect, the CD27 signaling domain is encoded by the nucleic acid sequence AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 17).

In one aspect, the CAR-expressing cell described herein may further comprise a second CAR, e.g., a second CAR that includes a different antigen-binding domain, e.g., to the same target or a different target (e.g., to a target other than the one associated with cancer antigen described herein, or to a different cancer-associated antigen described herein). In one embodiment, the second CAR comprises an antigen-binding domain to a target expressed by the same cancer cell type as the cancer-associated antigen. In one embodiment, a CAR-expressing cell comprises a first CAR that targets a first antigen and includes an intracellular signaling domain having a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different antigen and includes an intracellular signaling domain, having a primary signaling domain but not a costimulatory signaling domain. Without wishing to be bound by theory, placing a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27, or OX-40, in the first CAR, and a primary signaling domain, e.g., CD3-zeta, in the first CAR may limit CAR activity to cells in which both targets are expressed. In one embodiment, a CAR-expressing cell comprises a first CAR against a cancer-associated antigen that includes an antigen-binding domain that binds a target antigen described herein, a transmembrane domain, and a costimulatory domain, and a second CAR that targets a different target antigen. (eg, an antigen expressed on the same cancer cell type as the first target antigen) and includes an antigen-binding domain, a transmembrane domain, and a primary signaling domain. In another embodiment, a CAR-expressing cell contains a first CAR that includes an antigen-binding domain that binds the target antigen described herein, a transmembrane domain, and a primary signaling domain, and a second CAR that targets an antigen other than the first target antigen ( for example, an antigen expressed by the same cancer cell type as the first target antigen) and includes an antigen-binding domain to the antigen, a transmembrane domain, and a co-stimulatory signaling domain.

In one embodiment, the CAR-expressing cell contains an XCAR as described herein and an inhibitory CAR. In one embodiment, the inhibitory CAR contains an antigen-binding domain that binds an antigen found on normal cells but not on cancerous cells, eg, on normal cells that also express the CLL antigen. In one embodiment, an inhibitory CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of an inhibitory CAR may be the intracellular domain of PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGF-beta.

In one embodiment, when a CAR-expressing cell contains two or more different CARs, the antigen-binding domains of the different CARs may be such that the antigen-binding domains do not interact with each other. For example, a cell expressing the first and second CARs may have the antigen-binding domain of the first CAR, eg, as a fragment, eg scFv, that does not form an association with the antigen-binding domain of the second CAR, eg, the antigen-binding domain of the second CAR is VHH.

In some embodiments, the antigen-binding domain includes single-domain antigen-binding (SDAB) molecules, which include molecules whose complementarity-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy chain variable domains binding molecules naturally devoid of light chains, single domains derived from conventional 4-chain antibodies, engineered domains, and single domain scaffolds other than those derived from antibodies. The SDAB molecules can be any of the prior art molecules or any future single domain molecules. SDAB molecules can be from any species, including, but not limited to, mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bull. The term also includes naturally occurring single domain antibody molecules from a species other than Camelidae and sharks.

In one aspect, the SDAB molecule can be derived from an immunoglobulin variable region found in fish, such as, for example, a variable region that is derived from an immunoglobulin isotype known as a novel antigen receptor (NAR) found in shark serum. Methods for producing single domain molecules derived from the NAR variable region ("IgNAR") are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

According to another aspect, the SDAB molecule is a naturally occurring single domain antigen binding molecule known as a light chain-free heavy chain. Such single domain molecules are described, for example, in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For clarity, this variable domain, derived from a naturally free light chain heavy chain molecule, is known herein as VHH or nanoantibodies to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule may be derived from a Camelidae species such as camel, llama, dromedary, alpaca and guanaco. Species other than the Camelidae can produce heavy chain molecules naturally lacking a light chain; such VHHs are within the scope of the invention.

SDAB molecules can be recombinant, CDR-translated, humanized, camelized, deimmunized, and/or produced in vitro (eg, selected by phage display).

It has also been found that in cells having a plurality of chimeric membrane-immersed receptors containing an antigen binding domain, interactions between the antigen binding domains of the receptors may be undesirable, for example, because it inhibits the ability of one or more antigen binding domains to bind their antigen. Thus, the present disclosure describes cells having first and second non-naturally occurring chimeric membrane-immersed receptors containing antigen-binding domains that minimize such interactions. Also described herein are nucleic acids encoding first and second non-naturally occurring chimeric membrane-immersed receptors containing antigen-binding domains that minimize such interactions, as well as methods for making and using such cells and nucleic acids. In one embodiment, the antigen-binding domain of one of said first and said second non-naturally occurring chimeric membrane-embedded receptors contains scFv and the other contains a single VH domain, e.g., one VH domain of camelids, sharks or lampreys, or one VH domain, derived from a human or mouse sequence.

In some embodiments, the invention comprises a first and a second CAR, wherein the antigen-binding domain of one of said first CAR and said second CAR does not contain a light chain variable domain and a heavy chain variable domain. In some embodiments, the antigen binding domain of one of said first CAR and said second CAR is scFv and the other is not scFv. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR contains a single VH domain, such as a camelid, shark, or lamprey, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR contains a nanoantibody. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR comprises a camelid VHH domain.

In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR contains scFv and the other contains a single VH domain, e.g., a single VH domain of a camelid, shark, or lamprey animal, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR contains scFv and the other contains a nanoantibody. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR contains an scFv and the other contains a Camelid VHH.

In some embodiments, when present on the cell surface, the binding of the antigen-binding domain of said first CAR to its antigen is not substantially reduced by the presence of said second CAR. In some embodiments, the binding of the antigen-binding domain of said first CAR to its antigen in the presence of said second CAR is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding of the antigen-binding domain of said first CAR to its antigen in absence of said second CAR.

In some embodiments, when present on the cell surface, the antigen-binding domains of said first CAR and said second CAR bind to each other to a lesser extent than if they were both scFv antigen-binding domains. In some embodiments, the antigen-binding domains of said first CAR and said second CAR bind to each other 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than if they were both scFv antigen-binding domains. .

In another aspect, the CAR-expressing cell described herein may additionally express another agent, for example, an agent that increases the activity of the CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, such as PD1, in some embodiments, may reduce the ability of a CAR-expressing cell to induce an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGF-beta . In one embodiment, an agent that inhibits an inhibitory molecule, for example, is a molecule described herein, for example, an agent that contains a first polypeptide, for example, an inhibitory molecule associated with a second polypeptide, which gives a positive signal to the cell, for example, intracellular signal domain described in the present description. In one embodiment, the agent comprises a first polypeptide, e.g., from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGF-beta, or a fragment of any of them (for example, at least part of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain described in the present description (for example, containing a co-stimulatory domain (eg, 41BB, CD27 or CD28, eg, as described herein) and/or a primary signaling domain (eg, the CD3-zeta signaling domain described herein)). In one embodiment, the agent comprises a first polypeptide from PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second intracellular signaling domain polypeptide described herein (e.g., the CD28 signaling domain described herein, and/ or the CD3-zeta signaling domain described herein). PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to suppress T cell activation when bound to PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2: 261-8 Carter et al 2002 Eur J Immunol 32:634-43). PD-L1 is prevalent in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094) . Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

In one embodiment, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, such as Programmed Death 1 (PD1), fused to a transmembrane domain and intracellular signaling domains such as 41BB and CD3-zeta (also referred to herein as PD1 CAR). In one embodiment, the PD1 CAR, when used in combination with the XCAR described herein, enhances T cell viability. In one embodiment, the CAR is a PD1 CAR containing the PD1 extracellular domain indicated by underline in SEQ ID NO: 26. In one embodiment, the PD1 CAR contains the amino acid sequence of SEQ ID NO: 26.

Malpvtalllplalllhaarp pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv tttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 26).

In one embodiment, the PD1 CAR contains the amino acid sequence shown below (SEQ ID NO: 39).

pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv tttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 39).

In one embodiment, the agent contains a nucleic acid sequence encoding a PD1 CAR, such as the PD1 CAR described herein. In one embodiment, the nucleic acid sequence for PD1 CAR is shown below with PD1 ECD underlined in SEQ ID NO: 27.

atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagacca cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtc (SEQ ID NO: 27).

In another aspect, the present invention relates to populations of CAR expressing cells, eg, CART cells. In some embodiments, the population of CAR-expressing cells contains a mixture of cells expressing different CARs. For example, in one embodiment, a population of CART cells may include a first cell expressing a CAR having an antigen-binding domain against a cancer-associated antigen described herein, and a second cell expressing a CAR having a different antigen-binding domain, e.g., an antigen-binding domain against a different cancer-associated antigen as described herein, e.g., an antigen-binding domain against a cancer-associated antigen as described herein, which is different from a cancer-associated antigen bound by a CAR antigen-binding domain expressed by the first cell. As another example, a population of CAR-expressing cells may include a first cell expressing a CAR that includes an antigen-binding domain against a cancer-associated antigen described herein, and a second cell expressing a CAR that includes an antigen-binding domain against a target other than that associated with cancer antigen as described herein. In one embodiment, the population of CAR-expressing cells includes, for example, a first cell expressing a CAR that includes a primary intracellular signaling domain and a second cell that expresses a CAR that includes a secondary signaling domain.

In another aspect, the present invention relates to a population of cells, where at least one cell in the population expresses a CAR having an antigen-binding domain against a cancer-associated antigen described herein, and the second cell expresses another agent, for example, an agent that increases activity or the function of the CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. In some embodiments, inhibitory molecules, such as PD-1, can reduce the ability of a CAR-expressing cell to induce an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGF -beta. In one embodiment, an agent that inhibits an inhibitory molecule, for example, is a molecule described herein, for example, an agent that contains a first polypeptide, for example, an inhibitory molecule associated with a second polypeptide, which gives a signal to the cell, for example, an intracellular signaling the domain described in the present description. In one embodiment, the agent comprises a first polypeptide, e.g., from an inhibitory molecule such as PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGF-beta, or a fragment of any of them, and a second polypeptide that is an intracellular signaling domain as described herein (e.g., containing a co-stimulatory domain (e.g., 41BB, CD27, OX40 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., the CD3-zeta signaling domain described herein).In one embodiment, the agent comprises a first PD-1 polypeptide or fragment thereof, and a second an intracellular signaling domain polypeptide as described herein (eg, the CD28 signaling domain as described herein and/or the CD3-zeta signaling domain as described herein).

In one aspect, the present invention relates to methods comprising administering a population of CAR expressing cells, e.g., CART cells, e.g., a mixture of cells expressing different CARs, in combination with another agent, e.g., a kinase inhibitor, such as the kinase inhibitor described in the present description. In another aspect, the present invention relates to methods comprising administering a population of cells, wherein at least one cell in the population expresses a CAR having an antigen-binding domain against a cancer-associated antigen described herein, and the second cell expresses another agent, e.g. , which enhances the activity of a CAR-expressing cell, in combination with another agent, for example, a kinase inhibitor such as the kinase inhibitor described herein.

Regulated chimeric antigen receptors

In some embodiments, a regulated CAR (RCAR) is desirable, where CAR activity can be controlled to optimize the safety and efficacy of CAR therapy. There are many ways to regulate CAR activity. For example, inducible apoptosis using, for example, a caspase fused to a dimerization domain (see, for example, Di et al., N Engl. J. Med. 2011 Nov. 3; 365(18):1673-1683) can be used as a safety switch in CAR therapy of the present invention.

In one aspect, an RCAR comprises a set of polypeptides, typically two in the simplest embodiments, wherein the components of a standard CAR as described herein, such as an antigen-binding domain and an intracellular signaling domain, are separated into separate polypeptides or representatives. In some embodiments, the set of polypeptides includes a dimerization switch that, in the presence of a dimerization molecule, can bind polypeptides to each other, for example, can bind an antigen-binding domain to an intracellular signaling domain.

In one aspect, an RCAR comprises two polypeptides or members: 1) an intracellular signaling member comprising an intracellular signaling domain, such as the primary intracellular signaling domain described herein, and a first switch domain; 2) an antigen-binding member comprising an antigen-binding domain, for example, which targets the tumor antigen described herein as described herein, and a second switch domain. Optionally, RCAR contains a transmembrane domain described in the present description. In one embodiment, the transmembrane domain may be located on an intracellular signaling representative, on an antigen-binding representative, or on both. (Unless otherwise indicated, when representatives or elements of an RCAR are described herein, the order may be as shown, but other orders are also included. In other words, in one embodiment, the order is as indicated in the text, and in other embodiments the order may be different in implementation, for example, the order of elements on one side of the transmembrane region may be different from that given as an example, for example, the location of the switch domain relative to the intracellular signaling domain may be different (eg, may be reversed).

In one embodiment, the first and second switch domains may form an intracellular or extracellular dimerization switch. In one embodiment, the dimerization switch may be a homodimerization switch, eg, where the first and second switch domains are the same, or a heterodimerization switch, eg, where the first and second switch domains are different from each other.

In embodiments, an RCAR may comprise a "multiple switch". The multiple switch may comprise heterodimerization switch domains or homodimerization switch domains. A multiple switch comprises a plurality, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 switch domains, independently on a first member, such as an antigen-binding member, and a second member, such as an intracellular signaling member . In one embodiment, the first representative may comprise a plurality of first switch domains, such as FKBP-based switch domains, and the second representative may comprise a plurality of second switch domains, such as FRB-based switch domains. In one embodiment, the first representative may comprise first and second switch domains, such as an FKBP-based switch domain and an FRB-based switch domain, and the second representative may comprise first and second switch domains, such as an FKBP-based switch domain and an FKBP-based switch domain. FRB basis.

In one embodiment, an intracellular signaling representative comprises one or more intracellular signaling domains, such as a primary intracellular signaling domain and one or more co-stimulatory signaling domains.

In one embodiment, the antigen-binding representative may contain one or more intracellular signaling domains, such as one or more co-stimulatory signaling domains. In one embodiment, the antigen binding member comprises a plurality, e.g., 2 or 3, of the co-stimulatory signaling domains described herein, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40, and in embodiments does not contain a primary intracellular signaling domain. In one embodiment, the antigen-binding representative contains the following extracellular to intracellular co-stimulatory signaling domains: 41BB-CD27; 41BB-CD27; CD27-41BB; 41BB-CD28; CD28-41BB; OX40-CD28; CD28-OX40; CD28-41BB or 41BB-CD28. In such embodiments, the intracellular binding member comprises a CD3-zeta domain. In one such embodiment, the RCAR comprises (1) an antigen-binding member comprising an antigen-binding domain, a transmembrane domain, and two co-stimulatory domains and a first switch domain; and (2) an intracellular signaling domain comprising a transmembrane domain or a membrane-binding domain and at least one primary intracellular signaling domain and a second switch domain.

An embodiment relates to an RCAR where the antigen-binding member is not bound to the cell surface of the CAR. This allows a cell having an intracellular signaling representative to conveniently pair with one or more antigen-binding domains without transforming the cell with a sequence that encodes the antigen-binding representative. In such embodiments, the RCAR comprises: 1) an intracellular signaling representative comprising: a first switching domain, a transmembrane domain, an intracellular signaling domain, eg, a primary intracellular signaling domain, and a first switching domain; and 2) an antigen-binding member comprising: an antigen-binding domain and a second switch domain, wherein the antigen-binding member does not contain a transmembrane domain or a membrane-binding domain, and optionally does not contain an intracellular signaling domain. In some embodiments, an RCAR may further comprise 3) a second antigen-binding member comprising: a second antigen-binding domain, eg, a second antigen-binding domain that binds a different antigen than that of the antigen-binding domain; and a second switching domain.

Also contemplated within the scope of the present invention are RCARs wherein the antigen-binding member has the ability to bispecifically activate and target. In this embodiment, the antigen-binding member may comprise a plurality, such as 2, 3, 4, or 5 antigen-binding domains, such as scFv, where each antigen-binding domain binds to a target antigen, such as different antigens or a single antigen, such as the same or different epitopes on the same antigen. In one embodiment, a plurality of antigen-binding domains are arranged in tandem, and optionally there is a linker or hinge region between the antigen-binding domains. Suitable linkers and hinge regions are described in the present description.

An embodiment relates to RCARs having a configuration that allows proliferation switching. In this embodiment, the RCAR contains: 1) an intracellular signaling representative comprising: optionally a transmembrane domain or a membrane-binding domain; one or more costimulatory signaling domains, eg, selected from 41BB, CD28, CD27, ICOS, and OX40, and a switch domain; and 2) an antigen-binding member comprising: an antigen-binding domain, a transmembrane domain, and a primary intracellular signaling domain, e.g., a CD3-zeta domain, wherein the antigen-binding member does not contain a switch domain or does not contain a switch domain that dimerizes with the switch domain on the intracellular signal member. In one embodiment, the antigen-binding member does not contain a costimulatory signaling domain. In one embodiment, the intracellular signaling representative contains a switch domain from a homodimerization switch. In one embodiment, the intracellular signaling member comprises a first heterodimerization switch switching domain and RCAR comprises a second intracellular signaling representative that comprises a second heterodimerization switch switching domain. In such embodiments, the implementation of the second intracellular signaling representative contains the same intracellular signaling domains as the intracellular signaling representative. In one embodiment, the dimerization switch is intracellular. In one embodiment, the dimerization switch is extracellular.

In any of the RCAR configurations described herein, the first and second switch domains comprise an FKBP/FRB based switch as described herein.

Also described herein are cells containing the RCAR described herein. Any cell that has been engineered to express RCAR can be used as an RCARX cell. In one embodiment, the RCARX cell is a T cell and is referred to as a RCART cell. In one embodiment, the RCARX cell is a NK cell and is referred to as an RCARN cell.

Also within the scope of the present invention are nucleic acids and vectors containing RCAR coding sequences. The sequence encoding the various RCAR elements may be located on a single nucleic acid molecule, eg on a single plasmid or vector, eg a viral vector, eg a lentiviral vector. In one embodiment, (i) the sequence encoding the antigen-binding member and (ii) the sequence encoding the intracellular signal member may be present on a single nucleic acid, such as a vector. The production of the respective proteins can be achieved, for example, using separate promoters or using a bicistronic transcription product (which can produce two proteins by cleavage of a single translation product or by translation of two separate protein products). In one embodiment, between (i) and (ii) is a sequence encoding a cleavable peptide, such as a P2A or F2A sequence. In one embodiment, between (i) and (ii) is the sequence encoding the IRES, for example, IRES EMCV or EV71. In these embodiments, (i) and (ii) are transcribed as a single RNA. In one embodiment, (i) is operably linked to a first promoter and (ii) is operably linked to a second promoter such that (i) and (ii) are transcribed as separate mRNAs. Examples of peptide cleavage sites include the following, where GSG residues are optional:

T2A: (GSG) E G R G S L L T C G D V E E N P G P (SEQ ID NO: 68)

P2A: (GSG) A T N F S L L K Q A G D V E E N P G P (SEQ ID NO: 69)

E2A: (GSG) Q C T N Y A L L K L A G D V E S N P G P (SEQ ID NO: 70)

F2A: (GSG) V K Q T L N F D L L K L A G D V E S N P G P (SEQ ID NO: 71)

In one embodiment, the sequence encoding the IRES, eg, EMCV or EV71 IRES, is between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In one embodiment, the first promoter is operably linked to (i) and the second promoter is operably linked to (ii) such that (i) and (ii) are transcribed as separate mRNAs.

Alternatively, the sequence encoding different RCAR elements can be located on different nucleic acid molecules, eg different plasmids or vectors, eg viral vectors, eg lentiviral vectors. For example, sequence (i) encoding an antigen-binding member may be present on a first nucleic acid, such as a first vector, and sequence (ii) encoding an intracellular signal member may be present on a second nucleic acid, such as a second vector.

Dimerization switches

Dimerization switches can be non-covalent or covalent. In a non-covalent dimerization switch, the dimerization molecule provides a non-covalent interaction between the switch domains. In a covalent dimerization switch, the dimerization molecule provides a covalent interaction between the switch domains.

In one embodiment, the RCAR comprises a dimerization switch based on FKBP/FRAP or FKBP/FRB. FKBP12 (FKBP, or FK506 binding protein) is a ubiquitous cytoplasmic protein that serves as the initial intracellular target for the natural immunosuppressive drug rapamycin. Rapamycin binds to FKBP and to the large PI3K homologue FRAP (RAFT, mTOR). FRB is a 93 amino acid portion of FRAP that is sufficient to bind the FKBP-rapamycin complex (Chen, J., Zheng, XF, Brown, EJ & Schreiber, SL (1995) Identification of an 11-kDa FKBP12- rapamycin-binding domain within the 289-kDa FKBP12- rapamycin-associated protein and characterization of a critical serine residue ( Proc Natl Acad Sci USA 92: 4947-51.)

In embodiments, a dimerization molecule, such as rapamycin or a rapamycin analog, may be used in a FKBP/FRAP-based switch, eg FKBP/FRB.

The amino acid sequence of FKBP is as follows:

ETS Y (SEQ ID NO: 54)

In embodiments, the FKBP switch domain may comprise an FKBP fragment having the ability to bind FRB, or a fragment or analogue thereof in the presence of rapamycin or analogue, for example, the underlined portion of SEQ ID NO: 54, which is:

(SEQ ID NO: 55)

The amino acid sequence of FRB is as follows:

ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMER GPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK (SEQ ID NO: 56)

"FKBP/FRAP based switch, e.g. FKBP/FRB" as used herein, refers to a dimerization switch comprising: a first switch domain that contains a FKBP moiety or analogue thereof capable of binding to FRB, or fragment or analogue, in the presence of rapamycin or rapalogue, for example RAD001, and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with or differs by no more than 30%, 25%, 20, 15, 10, 5, 4, 3, 2 or 1 amino acid residue from the FKBP sequence SEQ ID NO: 54 or 55; and a second switch domain that contains a FRB fragment or analogs thereof having the ability to bind to FRB, or a fragment or analog thereof, in the presence of rapamycin or rapalog, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from the FRB sequence of SEQ ID NO: 56. In one embodiment, RCAR, described herein contains one switch domain that contains the amino acid residues described in SEQ ID NO: 54 (or SEQ ID NO: 55) and one switch domain that contains the amino acid residues described in SEQ ID NO: 56.

In embodiments, the FKBP/FRB dimerization switch comprises a modified FRB switch domain that exhibits altered, e.g. enhanced, complex formation between an FRB-based switch domain, e.g., a modified FRB switch domain, an FKBP-based switch domain, and a dimerization molecule, e.g., rapamycin or rapalogue, for example RAD001. In one embodiment, the modified FRB switch domain contains one or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, selected from mutations in amino acid position(s) L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105 and F2108, wherein the wild-type amino acid is replaced by any other naturally occurring amino acid. In one embodiment, the mutant FRB contains a mutation at position E2032, where E2032 is replaced with phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g. SEQ ID NO : 57, or leucine (E2032L), e.g. SEQ ID NO: 58. In one embodiment, the mutant FRB contains the mutation T2098, where T2098 is replaced with phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO: 59. In one embodiment, in an embodiment, the mutant FRB contains a mutation in E2032 and in T2098, where E2032 is replaced with any amino acid and where T2098 is replaced with any amino acid, e.g., SEQ ID NO: 60. NO: 61. In one embodiment, the mutant FRB contains the E2032L and T2098L mutation, e.g., SEQ ID NO: 62.

Table 13 Exemplary FRB mutant having increased affinity for the dimerization molecule.

Mutant FRB Amino acid sequence SEQID NO: Mutant E2032I ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS 57 Mutant E2032L ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS 58 Mutant T2098L ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 59 Mutant E2032, T2098 ILWHEMWHEGL X EASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDL X QAWDLYYHVFRRISKTS 60 Mutant E2032I, T2098L ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 161 Mutant E2032L, T2098L ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 62

Other suitable dimerization switches include a GyrB-GyrB dimerization switch, a gibberelin-based dimerization switch, a tag/linker dimerization switch, a halo-tag/snap-tag dimerization switch. According to the guidance provided in the present description, such switches and the corresponding dimerization molecules will be obvious to a person skilled in the art.

Dimerization molecule

The association between the switching domains is provided by the dimerization molecule. In the presence of a dimerization molecule, interaction or association between such domains provides for signal transduction between a polypeptide associated, eg fused, with the first switch domain and a polypeptide associated with, eg fused, the second switch domain. In the presence of non-limiting levels of the dimerization molecule, signal transduction increases by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 times, for example, when measured in the system described in the present description.

Rapamycin and rapamycin analogs (sometimes referred to as rapalogues), such as RAD001, can be used as dimerization molecules in the FKBP/FRB-based dimerization switch described herein. In one embodiment, the dimerization molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotaprolimus, temsirolimus, AP-23573 (ridaphorolimus), biolimus, and AP21967. Additional rapamycin analogs suitable for use with FKBP/FRB-based dimerization switches are described below in the "Combination Therapy" section, or in the subsection titled "Exemplary mTOR Inhibitors".

Cleaved CARs

In some embodiments, cleaved CAR is used in CAR-expressing cells. The split CAR approach is described in more detail in WO2014/055442 and WO2014/055657. Briefly, a cleaved CAR system includes a cell expressing a first CAR having a first antigen-binding domain and a costimulatory domain (e.g., 41BB), and the cell also expressing a second CAR having a second antigen-binding domain and an intracellular signaling domain (e.g., CD3-zeta). When the cell encounters the first antigen, the co-stimulatory domain is activated and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell killing activity begins. Thus, a CAR-expressing cell is fully activated in the presence of both antigens.

RNA transfection

The present disclosure describes methods for producing in vitro transcribed CAR RNA. The present invention also includes an RNA coding construct for a CAR that can be directly transfected into a cell. A method for producing mRNA for use in transfection may involve in vitro transcription (IVT) of the template using specially designed primers followed by poly-A attachment to produce a construct containing a 3' and 5' untranslated sequence ("UTR"), 5' -cap and/or internal ribosome entry site (IRES), nucleic acid to be expressed, and poly-A tail, typically 50-2000 bases long (SEQ ID NO: 32). RNA thus obtained can be efficiently transfected into various cell types. In one aspect, the matrix includes sequences for CARs.

In one aspect, the CAR of the present invention is encoded by messenger RNA (mRNA). In one aspect, mRNA encoding a CAR as described herein is introduced into an immune effector cell, such as a T cell or NK cell, to produce a CAR expressing cell, such as a CART cell or CAR NK cell.

In one embodiment, the in vitro transcribed CAR RNA can be introduced into a cell via transient transfection. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using suitable primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, a synthetic DNA sequence, or any other suitable DNA source. The desired template for in vitro transcription is CAR as described herein. For example, a CAR RNA template contains an extracellular region containing a single chain variable domain of an antibody to a tumor associated antigen described herein; a hinge region (eg, the hinge region described herein), a transmembrane domain (eg, a transmembrane domain described herein, such as the transmembrane domain of CD8a); and a cytoplasmic region that includes an intracellular signaling domain, eg, an intracellular csignaling domain as described herein, eg, comprising a CD3-zeta signaling domain and a 4-1BB signaling domain.

In one embodiment, the DNA for use in PCR contains an open reading frame. The DNA may be derived from a naturally occurring DNA sequence of the organism's genome. In one embodiment, the nucleic acid may include some or all of the 5' and/or 3' untranslated regions (UTRs). Nucleic acid may include exons and introns. In one embodiment, the DNA for use in PCR is a human nucleic acid sequence. In another embodiment, the DNA for use in PCR is a human nucleic acid sequence comprising 5' and 3' UTRs. Alternatively, the DNA may be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is a DNA sequence that contains portions of genes that are ligated to form an open reading frame that encodes a fusion protein. The pieces of DNA that are ligated together may be from the same organism or from more than one organism.

PCR is used to generate a template for in vitro transcription of mRNA, which is used for transfection. PCR methods are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of DNA used as a template for PCR. "Substantially complementary" as used herein refers to nucleotide sequences where most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. Substantially complementary sequences are capable of annealing or hybridizing to the intended target DNA under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, primers can be designed to amplify the portion of the nucleic acid that is normally transcribed in cells (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify the portion of the nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the human cDNA coding region, including all or parts of the 5' and 3' UTRs. Primers suitable for PCR can be prepared by synthetic methods that are well known in the art. "Forward primers" are primers that contain a region of nucleotides that is substantially complementary to nucleotides on the DNA template upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to the position 5' of the DNA sequence to be amplified relative to the coding strand. "Reverse primers" are primers that contain a region of nucleotides substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to the position 3' of the DNA sequence to be amplified relative to the coding strand.

Any DNA polymerase suitable for PCR can be used in the methods described herein. The reagents and polymerase are commercially available from a number of sources.

You can also use chemical structures that have the ability to increase the stability and/or efficiency of translation. Preferably the RNA has a 5' and a 3' UTR. In one embodiment, the 5'-UTR is from one to 3000 nucleotides in length. The length of the 5'- and 3'-UTR sequences for attachment to the coding region can be varied in a variety of ways, including, but not limited to, designing PCR primers that hybridize to different regions of the UTR. Using this approach, a person skilled in the art can modify the length of the 5'- and 3'-UTR required to achieve optimal translation efficiency after transfection of the transcribed RNA.

The 5' and 3' UTRs may be the naturally occurring endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are endogenous to the nucleic acid of interest can be added by including UTR sequences in the forward and reverse primers, or through any other modification of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest may be useful in modifying the stability and/or efficiency of RNA translation. For example, it is known that AU-rich elements in 3'-UTR sequences can reduce mRNA stability. Thus, the 3'-UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of the UTR, which are well known in the art.

In one embodiment, the 5'-UTR may contain a Kozak sequence from an endogenous nucleic acid. Alternatively, when a 5'-UTR that is not endogenous to the nucleic acid of interest is added by the PCR method as described above, the Kozak consensus sequence can be redesigned by adding the 5'-UTR sequence. Kozak sequences may increase the efficiency of translation of some RNA transcripts, but do not appear to be required to ensure efficient translation of all RNAs. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5'UTR may be the 5'UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to prevent degradation of the mRNA by exonucleases.

To allow synthesis of RNA from a DNA template without the need for gene cloning, a transcription promoter must be linked to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter is included in the PCR product upstream of the open reading frame to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter as described herein. Other suitable promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3 and SP6 promoters are known in the art.

In a preferred embodiment, the mRNA has both a 5' cap and a 3' poly(A) tail that determines ribosome binding, translation initiation, and cellular stability of the mRNA. On a circular DNA template, such as plasmid DNA, RNA polymerase produces a long concatemeric product that is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3'-UTR end results in a normal-sized mRNA that is not efficient in eukaryotic transfection even though it is polyadenylated after transcription.

On a linear DNA template, T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J Biochem., 270:1485-65 (2003).

A common method for inserting poly-A/T regions into a DNA template is molecular cloning. However, the poly-A/T sequence inserted into plasmid DNA can lead to plasmid instability, so plasmid DNA templates derived from bacterial cells are often highly contaminated with deletions and other aberrations. This makes the cloning procedure not only laborious and time-consuming, but often unreliable. Therefore, a method is highly desirable that will allow the construction of DNA templates with poly-A/T 3' regions without cloning.

A poly-A/T segment in a DNA transcription template can be produced by a PCR process using a reverse primer containing a poly-T tail such as a 100T tail (SEQ ID NO: 35) (size can be 50-5000 T (SEQ ID NO: 36)), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly-(A)-tails also provide RNA stability and reduce their degradation. Generally, the length of the poly-(A)-tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail has 100 to 5000 adenosine residues (SEQ ID NO: 37).

Poly(A) tail RNAs can also be extended after in vitro transcription using a poly(A) polymerase such as E. coli poly A polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 38) results in an approximately two-fold increase in RNA translation efficiency. In addition, the attachment of different chemical groups to the 3' end can increase the stability of the mRNA. Such attachment may include modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be inserted into the poly(A) tail using a poly(A) polymerase. In addition, ATP analogues can increase the stability of RNA.

5' caps also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods described herein include a 5' cap. The 5' cap is provided using methods known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7 :1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

RNA produced by the methods described herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or engineered sequence that initiates cap-independent binding of the ribosome to mRNA and promotes translation initiation. Any solute suitable for cell electroporation may be included, which may contain factors that promote cell permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

RNA can be incorporated into target cells using any of a number of different methods, such as commercially available methods that include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic systems. particle delivery, such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

Non-viral delivery methods

In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR as described herein into a cell or tissue or individual.

In some embodiments, the non-viral method includes the use of a transposon (also referred to as a transposable genetic element). In some embodiments, a transposon is a DNA fragment that can be inserted at a position in the genome, such as a DNA fragment that is capable of self-replicating and inserting a copy of it into the genome, or a DNA fragment that can be cut by splicing from a longer nucleic acid and inserted to a different position in the genome. For example, a transposon contains a DNA sequence consisting of inverted repeats flanking genes for transposition.

Exemplary transposon nucleic acid delivery methods include the Sleeping Beauty transposon system (SBTS) and the piggyBac transposon system (PB). See, for example, Aronovich et al. Hum. Mol. Genet. 20.R1(2011): R14-20; Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al. Mol. Ther. 16(2008):580-589; Grabundzija et al. Mol. Ther. 18(2010):1200-1209; Kebriaei et al. Blood. 122.21(2013):166; Williams. Molecular Therapy 16.9(2008):1515-16; Bell et al. Nat. Protocol. 2.12(2007):3153-65; and Ding et al. cell. 122.3(2005):473-83, all of which are incorporated herein by reference.

SBTS includes two components: 1) a transposon containing the transgene, and 2) a source of the transposase enzyme. A transposase can transfer a transposon from a carrier plasmid (or other donor DNA) to a target DNA, such as the host cell's chromosome/genome. For example, a transposase binds to the DNA of a host/donor plasmid, cuts the transposon (including the transgene(s)) from the plasmid, and inserts it into the genome of the host cell. See, for example, Aronovich et al., supra .

Illustrative transposons include the pT2 based transposon. See, for example, Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47; and Singh et al. Cancer Res. 68.8(2008): 2961-2971, all of which are incorporated herein by reference. Exemplary transposases include a Tc1/mariner-type transposase, eg, SB10 transposase or SB11 transposase (an overactive transposase that can be expressed, for example, from a cytomegalovirus promoter). See, for example, Aronovich et al.; Kebriaei et al.; and Grabundzija et al., all of which are incorporated herein by reference.

The use of SBTS allows efficient integration and expression of a transgene, for example, a nucleic acid encoding a CAR as described herein. It is within the scope of the present invention to provide a cell, eg a T cell or NK cell, that stably expresses a CAR as described herein, eg using a transposon system such as SBTS.

According to the methods described herein, in some embodiments, one or more nucleic acids, eg, plasmids, containing SBTS components are delivered into a cell (eg, a T or NK cell). For example, the nucleic acid(s) are delivered by standard nucleic acid delivery methods (eg, plasmid DNA), eg, by the methods described herein, eg, electroporation, transfection, or lipofection. In some embodiments, the implementation of the nucleic acid contains a transposon containing a transgene, for example, a nucleic acid encoding a CAR described in the present description. In some embodiments, the nucleic acid comprises a transposon containing a transgene (eg, a nucleic acid encoding a CAR as described herein) as well as a nucleic acid sequence encoding a transposase enzyme. In other embodiments, a dual nucleic acid system is provided, eg, a dual plasmid system, eg, wherein the first plasmid contains a transposon containing the transgene and the second plasmid contains a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are delivered to the cell together.

In some embodiments, cells, e.g., T or NK cells, are obtained that express the CAR described herein using a combination of gene insertion using SBTS and genetic editing using a nuclease (e.g., zinc finger nuclease (ZFN), effector nucleases like transcription activators (TALEN), the CRISPR/Cas system, or engineered meganucleases - rearranged homing endonucleases).

In some embodiments, the use of a non-viral delivery method allows reprogramming of cells, such as T or NK cells, and direct infusion of the cells into an individual. The advantages of non-viral vectors include, but are not limited to, ease and relatively low cost of obtaining sufficient quantities required to meet the needs of the patient population, storage stability, and lack of immunogenicity.

Nucleic acid constructs encoding CAR

The present invention also relates to nucleic acid molecules encoding one or more of the CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

Thus, in one aspect, the invention provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain that binds to a tumor antigen described herein, a transmembrane domain (e.g., the transmembrane domain described herein ) and an intracellular signaling domain (for example, the intracellular signaling domain described herein) containing a stimulatory domain, for example, a costimulatory signaling domain (for example, a costimulatory signaling domain as described herein) and/or a primary signaling domain (for example, a primary signaling domain). domain as described herein, e.g., the zeta chain as described herein). In one embodiment, the transmembrane domain is a protein transmembrane domain selected from the group consisting of T cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, the transmembrane domain may include at least the transmembrane region(s), e.g., from KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR , CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R-beta, IL2R-gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108) , SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp.

In one embodiment, the transmembrane domain contains the sequence of SEQ ID NO: 12 or a sequence with 95-99% identity with it. In one embodiment, the antigen-binding domain is linked to the transmembrane domain via a hinge region, such as the hinge region described herein. In one embodiment, the hinge region contains SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10, or a sequence with 95-99% identity with them. In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a co-stimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4-1BB (CD137 ). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8-alpha, CD8-beta, IL2R- beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, 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, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76 and PAG/Cbp. In one embodiment, the co-stimulatory domain contains the sequence of SEQ ID NO: 16, or a sequence with 95-99% identity with it. In one embodiment, the intracellular signaling domain comprises a functional 4-1BB signaling domain and a functional CD3-zeta signaling domain. In one embodiment, the intracellular signaling domain contains the sequence SEQ ID NO: 14 or SEQ ID NO: 16, or a sequence with 95-99% identity with it, and the sequence SEQ ID NO: 18 or SEQ ID NO: 20, or a sequence having 95-99% identity with it, where sequences containing an intracellular signal domain are expressed in one reading frame and as one polypeptide chain.

In another aspect, the invention provides an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence of SEQ ID NO: 2, an scFv domain as described herein, a hinge region of SEQ ID NO: 4, or SEQ ID NO: 6, or SEQ ID NO: 8, or SEQ ID NO: 10 (or a sequence with 95-99% identity with it), a transmembrane domain with the sequence of SEQ ID NO: 12 (or a sequence with 95-99% identity with it), a 4-1BB co-stimulatory domain having the sequence of SEQ ID NO: 14, or a CD27 co-stimulatory domain having the sequence of SEQ ID NO: 16 (or a sequence having 95-99% identity with it) and a CD3-zeta stimulatory domain having the sequence of SEQ ID NO: 18 or SEQ ID NO: 20 (or a sequence with 95-99% identity therewith).

In another aspect, the invention relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, which contains an antigen-binding domain, a transmembrane domain and an intracellular signaling domain containing stimulatory, and where the specified antigen-binding domain binds to a tumor antigen selected from the group consisting of : TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR , NCAM, prostasis, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, receptor folate beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR 51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD -CT-2, Fos-related antigen 1, p53, p53 mutant, protein, survivin and telomerase, PCTA-1/galectin 8, MelanA/MART1, Ras mutant, hTERT, breakpoints in sarcoma translocation, ML-IAP, ERG ( fusion gene TMPRSS2 ETS), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, reverse transcriptase human telomerase, RU1, RU2, intestinal carboxyester, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1

In one embodiment, the encoded CAR molecule further comprises a sequence encoding a co-stimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO: 14. In one embodiment, the transmembrane domain is the transmembrane domain of a protein selected from the group consisting of T cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 12. In one embodiment, the intracellular signaling domain comprises a 4-1BB functional signaling domain and a zeta functional signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 14 and the sequence of SEQ ID NO: 18, where the sequences containing the intracellular signaling domain are expressed in one reading frame and as a single polypeptide chain. In one embodiment, the anti-cancer-associated antigen binding domain described herein is linked to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises SEQ ID NO: 4. In one embodiment, the hinge region comprises SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as screening libraries of cells expressing the gene, obtaining the gene from a vector known to contain it, or isolating directly from cells or tissues containing it using standard methods. Alternatively, the gene of interest may be obtained by synthetic means rather than cloned.

The present invention also relates to vectors into which the DNA of the present invention is inserted. Vectors derived from retroviruses, such as lentivirus, are suitable tools for achieving long-term gene transfer because they provide long-term stable transgene insertion and expansion in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity. The retroviral vector may also be, for example, a retroviral gamma vector. The gamma retroviral vector may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (eg, two) long terminal repeats (LTRs), and a transgene of interest, for example, a gene encoding CAR. The gamma retroviral vector may be devoid of viral structural genes such as gag, pol and env. Exemplary gamma-retroviral vectors include murine leukemia virus (MLV), spleen necrosis virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. Jun 2011; 3(6): 677-713.

In another embodiment, the vector containing the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, expression of CAR-encoding nucleic acids can be performed using transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See June et al below. 2009 Nature Reviews Immunology 9,10: 704-716, incorporated herein by reference.

Briefly, expression of natural or synthetic CAR-encoding nucleic acids is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide, or portion thereof, to a promoter and inserting the construct into an expression vector. Vectors may be suitable for replication and insertion in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and a promoter suitable for directing expression of the desired nucleic acid sequence.

The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Gene delivery methods are known in the art. See, for example, US Pat. Nos. 5,399,346; 5,580,859; In another embodiment, the invention relates to a gene therapy vector.

Nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into a vector, including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replicating vectors, probe vectors, and sequencing vectors.

In addition, the expression vector may be provided to the cell in the form of a viral vector. 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 other reference books on virology and molecular biology . Viruses that are suitable as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, restriction endonuclease convenient sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058 and US Pat. No. 6,326,193) .

A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into the vector and retroviral particles packaged using methods known in the art. The recombinant virus can then be isolated and delivered to the individual's cells, either in vivo or ex vivo . A number of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A number of adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

Additional promoter elements, such as enhancers, regulate the frequency of transcription initiations. As a rule, they are located in the area at 30-110 bp. upstream of the initiation site, although a number of promoters have also been shown to contain functional elements downstream of the initiation site. The spacer between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacer between the promoter elements can be extended up to 50 bp. before the activity begins to decline. It turned out that, depending on the promoter, individual elements can function either jointly or independently, activating transcription. Illustrative promoters include the CMV IE, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) gene promoters.

An example of a promoter that is capable of expressing a CAR-encoding nucleic acid molecule in a mammalian T cell is the EF1a promoter. The native EF1a promoter controls the expression of the alpha subunit of the elongation factor 1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to the ribosome. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from nucleic acid molecules cloned into a lentiviral vector. See, for example, Milone et al ., Mol. Ther. 17(8): 1453-1464 (2009). In one aspect, the EF1a promoter contains the sequence shown as SEQ ID NO: 1.

Another example of a promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a potent constitutive promoter sequence capable of providing high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences may also be used, including, but not limited to, the early 40 monkey virus (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, elongation factor 1α promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the invention is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of inducible promoters provides a molecular switch capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, the metalthionine promoter, the glycocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

Also, the vector may include, for example, a signal sequence to facilitate secretion, a polyadenylation signal and a transcription terminator (for example, from the bovine growth hormone (BGH) gene), an element that allows episomal replication and replication in prokaryotes (for example, origin SV40 and ColE1 or others, known in the art) and/or an element allowing selection (eg ampicillin resistance gene and/or zeocin).

In order to evaluate the expression of a CAR polypeptide or parts thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that intend to transfect or infect with viral vectors. In other aspects, the selectable marker may be contained on a separate DNA fragment and used in a co-transfection technique. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences to ensure expression in host cells. Suitable selectable markers include, for example, antibiotic resistance genes such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present or not expressed in the organism or tissue of the recipient and which encodes a polypeptide whose expression exhibits some easily detectable property, such as enzymatic activity. The expression of the reporter gene is analyzed at the appropriate time after the insertion of the DNA into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or a green fluorescent protein gene (eg, Ui-Tei et al ., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known methods or purchased from commercial suppliers. As a rule, a construct with a minimal 5'-flanking region, showing the highest level of reporter gene expression, is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

Methods for introducing and expressing genes in cells are known in the art. In the context of an expression vector, the vector can easily be introduced into a host cell, such as a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, an expression vector can be transferred into a host cell by physical, chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for obtaining cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al ., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for introducing a polynucleotide into a host cell is by transfection with calcium phosphate.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for introducing genes into mammalian, eg human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloid system for use as an in vitro and in vivo delivery vehicle is a liposome (eg, an artificial membrane vesicle). Other prior art methods for targeted delivery of nucleic acids are available, such as delivery of polynucleotides using targeted nanoparticles or other suitable submicron delivery system.

In the case where a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. For the introduction of nucleic acids into a cell ( in vitro , ex vivo or in vivo ) provides for the use of lipid formulations. In another aspect, the nucleic acid can be linked to a lipid. The nucleic acid associated with the lipid can be encapsulated in the aqueous content of the liposome, distributed in the lipid bilayer of the liposome, associated with the liposome through a linker molecule that is associated with both the liposome and the oligonucleotide, enclosed in the liposome, be complexed with the liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed with a micelle, or may otherwise be associated with a lipid. Compositions associated with lipids, lipids/DNA or lipids/expression vectors are not limited to any particular structure in solution. For example, they may be in a bilayer structure as micelles, or may be with a collapsed structure. They may also simply be distributed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fat droplets that occur naturally in the cytoplasm, as well as a class of compounds that contains long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

] Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, A.L.). The original solutions of lipids in chloroform or a mixture of chloroform/methanol can be stored at approximately -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term covering a plurality of unilamellar and multilamellar lipid vesicles formed by the formation of nested lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilayer liposomes have many lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of an aqueous solution. Lipid components undergo self-rearrangement before forming closed structures and trapping water and solutes between lipid layers (Ghosh et al ., 1991 Glycobiology 5: 505-10). However, compositions are also contemplated which have structures in solution that differ from the normal vesicular structure. For example, lipids can take on a micellar structure or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cell to an inhibitor of the present invention, various assays can be performed to confirm the presence of a recombinant DNA sequence in a host cell. Such assays include, for example, "molecular biological" assays well known to those skilled 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, for example, by immunological methods (ELISA and Western blotting) or assays described herein, to identify agents within the scope of the invention.

In addition, the present invention relates to a vector containing a nucleic acid molecule encoding a CAR. In one aspect, the CAR vector can be directly transduced into a cell, such as a T cell or NK cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviral, and lentiviral vector constructs. In one aspect, the vector is capable of expressing the CAR construct in mammalian immune effector cells (eg, T cells, NK cells). In one aspect, the mammalian T cell is a human T cell. In one aspect, the mammalian NK cell is a human NK cell.

Cell Sources

Before the increase in number and genetic modification or other modification, the source of cells, for example, T-cells or natural killer (NK) cells) can be obtained from the individual. The term "individual" is intended to include living organisms in which an immune response can be induced (eg, mammals). Examples of individuals include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascitic fluid, pleural effusion, spleen tissue, and tumors.

In some aspects of the present invention, immune effector cells, such as T cells, can be obtained from a unit of blood taken from an individual using any of a variety of methods known to the skilled person, such as separation in Ficoll ^TM . In one preferred aspect, cells from the individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, apheresis-derived cells can be washed to remove a plasma fraction and optionally place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution is devoid of calcium and may be devoid of magnesium, or may be devoid of many, or even all, divalent cations.

The initial stages of activation in the absence of calcium can lead to increased activation. As will be well understood by those skilled in the art, the washing step can be carried out by methods known in the art, such as using a semi-automated "flow through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) in according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers such as Ca-free, Mg-free PBS, PlasmaLyte A, or other buffered or unbuffered saline. Alternatively, unwanted components of the apheresis sample can be removed and the cells directly resuspended in culture medium.

It is well known that culture media conditions comprising 5% or less, e.g. 2%, human AB serum can be used in methods of use, and known culture media conditions and compositions can be used, e.g., those described in Smith et al. Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement" Clinical & Translational Immunology (2015) 4, e31; doi:10.1038/cti.2014.31.

In one aspect, T cells are isolated from human peripheral blood lymphocytes by lysis of erythrocytes and elimination of monocytes, for example by PERCOLL ^™ gradient centrifugation or countercurrent centrifuge elution.

The methods described herein may include, for example, selecting a particular subset of immune effector cells, e.g., T cells, which is a regulatory T cell depleted population, a CD25+ cell depleted population, using, for example, a negative selection method. , for example, described in the present description. Preferably, the regulatory T cell depleted cell population contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

In one embodiment, regulatory T cells, eg, CD25+ T cells, are eliminated from the population using an anti-CD25 antibody or fragment thereof, or a CD25 binding ligand, IL-2. In one embodiment, the anti-CD25 antibody, or fragment thereof, or CD25 binding ligand, is conjugated to a support, eg a bead, or otherwise applied to a substrate, eg a bead. In one embodiment, the CD25 antibody, or fragment thereof, is conjugated to a support as described herein.

In one embodiment, regulatory T cells, eg, CD25+ T cells, are removed from the population using Miltenyi ^™ CD25 Depletion Reagent. In one embodiment, the ratio of cells to CD25 depleting reagent is 1e7 cells per 20 µl, or 1e7 cells per 15 µl, or 1e7 cells per 10 µl, or 1e7 cells per 5 µl, or 1e7 cells per 2.5 µl, or 1e7 cells per 1.25 µl. In one embodiment, for example, for regulatory T cells, for example, a depletion of CD25+ cells greater than 500 million cells/ml is used. In a further aspect, a cell concentration of 600, 700, 800, or 900 million cells/mL is used.

In one embodiment, the population of immune effector cells to be depleted includes approximately 6×10 ⁹ CD25+ T cells. In other aspects, the population of immune effector cells to be depleted includes from about 1×10 ⁹ to 1×10 ¹⁰ CD25+ T cells and any integer in between. In one embodiment, the resulting regulatory T cell depleted population has 2×10 ⁹ regulatory T cells, e.g., CD25+ cells, or less (e.g., 1×10 ⁹ , 5×10 ⁸ , 1×10 ⁸ , 5×10 ⁷ , 1×10 ⁷ or less CD25+ cells).

In one embodiment, regulatory T cells, eg, CD25+ cells, are removed from the population using a CliniMAC system with a set of depletion tubes, such as 162-01 tubes, for example. In one embodiment, the CliniMAC system is used in depletion settings such as DEPLETION2.1, for example.

Without wishing to be bound by a particular theory, reducing the levels of negative immune cell regulators (e.g., reducing the number of unwanted immune cells, e.g., T _REG cells) in an individual prior to apheresis or during the manufacture of a product with CAR-expressing cells, may reduce the risk of relapse in the individual. For example, methods for depleting T _REG cells are known in the art. Methods for reducing the number of T _REG cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (an anti-GITR antibody described herein), CD25 depletion, and combinations thereof.

In some embodiments, the production methods include reducing (eg, depleting) T _REG cells prior to generating CAR-expressing cells. For example, preparation methods include contacting a sample, such as an apheresis sample, with an anti-GITR antibody and/or an anti-CD25 antibody (or fragment or CD25 binding ligand thereof), for example, to deplete T _REG cells prior to making a product expressing CARs. cells (eg, T cells, NK cells).

In one embodiment, the individual is pre-treated with one or more therapies that reduce T _REG cells prior to harvesting the cells to make a CAR expressing cell product, thereby reducing the individual's risk of relapse following treatment with CAR expressing cells. In one embodiment, methods for reducing T _REG cells include, but are not limited to, administering to the individual one or more of cyclophosphamide, an anti-GITR antibody, a CD25 depleting agent, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibody, CD25 depleting agent, or a combination thereof may occur before, during or after infusion of the product into CAR expressing cells.

In one embodiment, the individual is pre-treated with cyclophosphamide prior to harvesting the cells to make a product with CAR expressing cells, thereby reducing the individual's risk of relapse after treatment with CAR expressing cells. In one embodiment, the individual is pre-treated with an anti-GITR antibody prior to harvesting the cells to make a product with CAR expressing cells, thereby reducing the individual's risk of relapse after treatment with CAR expressing cells.

In one embodiment, the cell population to be depleted is neither regulatory T cells, nor tumor cells, but cells that otherwise adversely affect the expansion and/or function of CART cells, e.g., cells expressing CD14, CD11b, CD33 , CD15, or other markers expressed by potentially immunosuppressive cells. In one embodiment, such cells are removed simultaneously with regulatory T cells and/or tumor cells, or after said depletion, or in another order.

The methods described herein may include more than one selection step, for example more than one depletion step. An increase in the number of a population of T cells through negative selection can be carried out, for example, using a combination of antibodies directed to surface markers unique to cells subjected to negative selection. One way is to sort and/or select cells by negative magnetic immune adhesion or flow cytometry, which use a mixture of monoclonal antibodies directed to cell surface markers present on cells subjected to negative selection. For example, to increase the number of CD4+ cells through negative selection, a mixture of monoclonal antibodies may include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

The methods described herein may further include removing cells from a population that express a tumor antigen, e.g., a tumor antigen that does not contain CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, thereby , resulting in a population of depleted regulatory T cells, eg, CD25+ depleted cells, and tumor antigen depleted cells, which is suitable for expression of a CAR, eg, the CAR described herein. In one embodiment, tumor antigen-expressing cells are removed at the same time as regulatory T cells, such as CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-tumor antibody, or fragment thereof, can be bound to the same substrate, such as beads that can be used to remove cells, or an anti-CD25 antibody, or fragment thereof, or an anti-tumor antibody. the antigen, or fragment thereof, can be associated with individual beads, a mixture of which can be used to remove cells. In other embodiments, the removal of T-regulatory cells, eg, CD25+ cells, and the removal of tumor antigen-expressing cells are sequential and may occur, for example, in any order.

Also contemplated are methods that include eliminating cells from a population that express a checkpoint inhibitor, e.g., the checkpoint inhibitor described herein, e.g., one or more of PD1+ cell types, LAG3+ cells, and TIM3+ cells, thereby providing a population with depleted regulatory T cells, eg CD25+ depleted cells; and checkpoint inhibitor depleted cells, eg PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA and LAIR1. In one embodiment, cells expressing the checkpoint inhibitor are removed at the same time as regulatory T cells, eg, CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-checkpoint inhibitor antibody or fragment thereof can be bound to the same beads that can be used to remove cells, or an anti-CD25 antibody or fragment thereof, or an anti-checkpoint inhibitor antibody or fragment thereof. , can be associated with individual beads, a mixture of which can be used to remove cells. In other embodiments, the removal of T-regulatory cells, eg CD25+ cells, and the removal of cells expressing the checkpoint inhibitor are sequential and may occur, for example, in any order.

The methods described herein may include a positive selection step. For example, T cells can be isolated by incubation with anti-CD3/anti-CD28 antibody conjugated beads (e.g., 3×28) such as DYNABEAD® M-450 CD3/CD28 T for a period of time sufficient to positively select for the desired T cells. In one embodiment, the time period is approximately 30 minutes. In the following embodiment, the time period is in the range of 30 minutes to 36 hours or more, including all integers in between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In another preferred embodiment, the time period is from 10 to 24 hours, such as 24 hours. A longer incubation time can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. In addition, the use of a longer incubation time may increase the efficiency of CD8+ T cell capture. Thus, by simply shortening or lengthening the time, T cells are allowed to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as further described herein), one can advantageously select for or against a subpopulation T cells at culture initiation or at other times in the process. In addition, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, one can preferentially select for or against a subset of T cells at culture initiation or at other desired time points.

In one embodiment, a population of T cells can be selected that express one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13 , granzyme B and perforin, or other suitable molecules such as other cytokines. Methods for screening cell expression can be determined, for example, by the methods described in PCT Publication No. WO 2013/126712.

To highlight the desired population of cells through positive or negative selection, the concentration of cells and surface (eg, particles such as granules) can be varied. In some aspects, it may be desirable to significantly reduce the volume in which the beads and cells are mixed with each other (eg, increase the concentration of cells) to ensure maximum contact of cells and beads. For example, in one aspect, a concentration of 10 billion cells/ml, 9 billion cells/ml, 8 billion cells/ml, 7 billion cells/ml, 6 billion cells/ml, or 5 billion cells/ml is used. In one aspect, a concentration of 1 billion cells/ml is used. In a further aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/ml is used. In the following aspects, concentrations of 125 or 150 million cells/ml can be used.

The use of high concentrations can lead to increased cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or capture from samples where many tumor cells are present (eg, leukemia blood, tumor fabric, etc.). Such populations of cells may be of therapeutic value and it may be desirable to obtain them. For example, the use of a high concentration of cells provides a more efficient selection of CD8+ T cells, which usually have a weaker expression of CD28.

In a similar aspect, it may be desirable to reduce the concentration of cells. By significantly diluting the mixture of T cells and surfaces (eg, particles such as beads), interactions between particles and cells are minimized. This results in the selection of cells that express high amounts of the desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells at dilute concentrations. In one aspect, the concentration of cells used is 5×10 e ⁶ /ml. In other aspects, the concentration used can be from about 1×10 ⁵ /ml to 1×10 ⁶ /ml, including any integer in between.

In other aspects, the cells can be incubated on a rotator for different periods of time at different speeds, either at 2-10°C or at room temperature.

T cells to be stimulated can also be frozen after the washing step. Without being bound by theory, it is believed that freezing followed by thawing provides a more uniform product due to the elimination of granulocytes and to some extent monocytes in the cell population. After a washing step that eliminates plasma and platelets, the cells can be suspended in a freezing solution. While a variety of freezing solutions and parameters are known in the art and useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or a culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7 .5% DMSO or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, the cells are then frozen to -80° C. at a rate of 1° per minute and stored in a liquid nitrogen vapor phase storage vessel. You can use other methods of controlled freezing, as well as uncontrolled freezing directly to -20°C or in liquid nitrogen.

In some aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature before being activated using the methods of the present invention.

It is also within the context of the invention to obtain blood samples or an apheresis product from an individual in the period before the cells described herein may be required. As such, a source of cells to be increased in number can be harvested at any desired time point, and desired cells, such as T cells, can be isolated and frozen for later use in immune effector cell therapy for any of a variety of diseases and conditions in which useful immune effector cell therapy, such as the diseases and conditions described herein. In one aspect, obtaining a blood sample or apheresis product is from a generally healthy individual. In some aspects, a blood sample or apheresis product is obtained from a generally healthy individual who is at risk of developing a disease but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In some aspects, T cells can be expanded, frozen, and used at a later time. In some aspects, sampling is carried out immediately after the diagnosis of a particular disease, as described in the present description, but before any of the treatments. In a further aspect, cells are isolated from a blood sample or apheresis product from an individual prior to any of a variety of appropriate treatments, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation therapy, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunosuppressive agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiotherapy.

In a further aspect of the present invention, T cells are obtained from a patient immediately after treatment that preserves functional T cells in the individual. In this regard, it has been found that in the case of certain treatments for cancer, in particular treatment with drugs that damage the immune system, shortly after treatment during the period when patients normally recover from treatment, the quality of the resulting T cells can be optimal. or improved in terms of their ability to increase in number ex vivo . Likewise, after being manipulated ex vivo using the methods described herein, these cells may be in a preferred state for increased engraftment and expansion in vivo . Thus, it is within the context of the invention to collect blood cells, including T cells, dendritic cells, or other cells of hematopoietic growth, during this recovery step. In addition, in some aspects, mobilization (e.g., GM-CSF mobilization) and conditioning regimens can be used in an individual that promote repopulation, recycling, regeneration, and/or expansion of specific cell types, especially over a period of time following therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

In one embodiment, immune effector cells expressing a CAR molecule, eg, the CAR molecule described herein, are obtained from an individual who has been administered a low immune-boosting dose of an mTOR inhibitor. In one embodiment, a population of immune effector cells, e.g., T cells, to be engineered to express CAR, is harvested after a sufficient period of time, or after sufficient dosing of a low immune-enhancing dose of an mTOR inhibitor, such that the level of PD1-negative immune effector cells, e.g. , T cells, or the ratio of PD-1 negative immune effector cells, e.g., T cells/PD-1 positive immune effector cells, e.g., T cells, in or after collection from the individual is at least temporarily increased.

In other embodiments, a population of immune effector cells, e.g., T cells that are or will be engineered to express CAR, can be treated ex vivo by contacting an amount of an mTOR inhibitor that increases the number of PD1 negative immune effector cells, e.g. , T cells, or increases the ratio of PD-1 negative immune effector cells, eg T cells/PD-1 positive immune effector cells, eg T cells.

In one embodiment, the T cell population is diaglycerol kinase (DGK) deficient. DGK-deficient cells include cells that do not express DGK RNA or protein, or have reduced or inhibited DGK activity. Cells deficient in DGK can be obtained through genetic approaches, for example, the introduction of RNA interference agents, for example, siRNA, shRNA, miRNA, to reduce or prevent the expression of DGK. Alternatively, DGK deficient cells can be obtained by treatment with the DGK inhibitors described herein.

In one embodiment, the T cell population is Ikaros deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity. Ikaros-deficient cells can be generated using genetic approaches such as introducing RNA interference agents, eg siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be obtained by treatment with Ikaros inhibitors, such as lenalidomide.

In embodiments, the T cell population is DGK-deficient and Ikaros-deficient, eg, does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros deficient cells can be obtained by any of the methods described herein.

In one embodiment, NK cells are obtained from an individual. In another embodiment, the NK cells are an NK cell line, such as the NK-92 cell line (Conkwest).

Allogeneic CAR

In the embodiments described herein, the immune effector cell may be an allogeneic effector cell, such as a T cell or NK cell. For example, the cell may be an allogeneic T cell, such as an allogeneic T cell lacking expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA), such as HLA class I and/or HLA class II.

For example, a T cell lacking a functional TCR can be engineered not to express any functional TCR on its surface, can be engineered not to express one or more subunits that contain a functional TCR, or engineered to it produced very little functional TCR on its surface. Alternatively, a T cell may express a substantially disrupted TCR, for example, by expressing mutant or truncated forms of one or more TCR subunits. The term "substantially disrupted TCR" means that the TCR does not induce an adverse immune response in the host.

The T cell described herein can be engineered, for example, to not express functional HLA on its surface. For example, a T cell described herein can be engineered to suppress cell surface expression of HLA, eg, HLA class 1 and/or HLA class II.

In some embodiments, the T cell may lack a functional TCR and a functional HLA, such as HLA class I and/or HLA class II.

Modified T cells that lack functional TCR and/or HLA expression can be generated by any suitable means, including knocking out or knocking out one or more TCR or HLA subunits. For example, a T cell can include TCR and/or HLA knockdown using siRNA, shRNA, regularly clustered short palindromic repeats (CRISPR), transcription activator-like nuclease (TALEN), or zinc finger endonuclease (ZFN).

In some embodiments, the allogeneic cell may be a cell that does not or does not express at low levels an inhibitory molecule, for example, by any of the methods described herein. For example, the cell may be a cell that does not express or expresses at low levels an inhibitory molecule, for example, which can reduce the ability of a CAR-expressing cell to induce an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGF-beta . Inhibition of an inhibitory molecule, for example, through inhibition at the DNA, RNA, or protein level, can optimize the efficiency of a CAR-expressing cell. In embodiments, an inhibitory nucleic acid, e.g., dsRNA, e.g., siRNA or shRNA, regularly clustered short palindromic repeats (CRISPR), transcription activator-like nuclease (TALEN), or zinc finger endonuclease (ZFN), may be used, e.g., as described. in this description.

siRNA and shRNA to inhibit TCR or HLA

In some embodiments, TCR expression and/or HLA expression can be inhibited using siRNAs or shRNAs that target a TCR and/or HLA encoding nucleic acid in a T cell.

Expression of siRNA or shRNA in T cells can be achieved using any conventional expression system such as, for example, a lentiviral expression system.

Exemplary shRNAs that repress the expression of TCR components are described, for example, in US Publication No. 2012/0321667. Exemplary miRNAs and shRNAs that repress the expression of HLA class I and/or HLA class II genes are described, for example, in US Publication No. US 2007/0036773.

CRISPR to inhibit TCR or HLA

"CRISPR" or "CRISPR against TCR and/or HLA" or "CRISPR for TCR and/or HLA inhibition" as used herein refers to a regularly clustered set of short palindromic repeats or a system containing such a set of repeats. . "Cas" as used herein refers to a CRISPR-associated protein. The "CRISPR/Cas" system refers to a system derived from CRISPR and Cas that can be used to silence or mutate the TCR and/or HLA genes.

Naturally occurring CRISPR/Cas systems are found in approximately 40% of sequenced eubacteria and 90% of sequenced archaeal genomes. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.

The CRISPR/Cas system has been modified for use in gene editing (silencing, amplification or modification of specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This was done by introducing into a eukaryotic cell a plasmid containing a specially designed CRISPR and one or more of the corresponding Cas.

The CRISPR sequence, sometimes referred to as the CRISPR locus, contains alternating repeats and spacers. In naturally occurring CRISPR, spacers typically contain sequences foreign to the bacterium, such as a plasmid or phage sequence; in the CRISPR/Cas system against TCR and/or HLA, spacers are derived from TCR or HLA gene sequences.

RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. They contain a spacer flanked by repeat sequences. RNA directs other Cas proteins to suppress exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1:7. Thus, spacers act as templates for RNA molecules, similar to miRNAs. Pennisi (2013) Science 341: 833-836.

Because they occur naturally in many different types of bacteria, the precise organization and structure of CRISPR, the function and number of Cas genes and their products differ to some extent from species to species. Haft et al. (2005) PLoS Comput. Biol. 1:e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mol. Evol. 60:174-182; Bolotin et al. (2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151:653-663; and Stern et al. (2010) Trends. Genet . 28:335-340. For example, Cse proteins (a subtype of Cas, E. coli ) (eg, CasA) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat elements that Cascade retains. Browns et al. (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes the CRISPR transcript. CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2. Cmr proteins (RAMP Cas module) in Pyrococcus furiosus and other prokaryotes form a functional complex with CRISPR small RNAs that recognize and cleave target RNAs. A simpler CRISPR system is based on the Cas9 protein, which is a nuclease with two active cleavage sites, one for each strand of the double helix. The combination of Cas9 and a modified CRISPR RNA locus can be used in a gene editing system. Pennisi (2013) Science 341: 833-836.

Thus, the CRISPR/Cas system can be used to edit the TCR and/or HLA gene (base pair addition or deletion) or introduce a premature stop codon, thus reducing TCR and/or HLA expression. Alternatively, the CRISPR/Cas system can be used similar to RNA interference, turning off the TCR and/or HLA genes in a reversible manner. In a mammalian cell, for example, RNA can direct the Cas protein to the TCR and/or HLA promoter, spatially blocking RNA polymerases.

Artificial CRISPR/Cas systems that inhibit TCR and/or HLA can be made using technology known in the art, such as that described in US Publication No. 20140068797, and Cong (2013) Science 339: 819-823. Other artificial CRISPR/Cas systems known in the art that inhibit TCR and/or HLA can also be made, such as those described in Tsai (2014) Nature Biotechnol. , 32:6 569-576, US Pat. No. 8,871,445; 8865406; 8795965; 8771945 and 8697359.

TALEN to inhibit TCR and/or HLA

"TALEN" or "TALEN against HLA and/or TCR" or "TALEN for inhibition of HLA and/or TCR" refers to an effector nuclease like a transcription activator, which is an artificial nuclease that can be used to edit the HLA and/or TCR gene .

TALEN is produced artificially by fusing the effector DNA-binding domain of TAL with a DNA cleavage domain. Transcription activator effects (TALE) can be engineered to bind to any desired DNA sequence, including part of the HLA or TCR gene. By combining engineered TALEs with a DNA cleavage domain, a restriction enzyme can be generated that is specific for any desired DNA sequence, including an HLA or TCR sequence. They can then be introduced into a cell where they can be used to edit the genome. Boch (2011) Nature Biotech . 29:135-6; and Boch et al. (2009) Science 326: 1509-12; Moscow et al. (2009) Science 326: 3501.

TALE are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a repetitive, highly conserved sequence of 33-34 amino acids, except for the 12th and 13th amino acids. These two positions are highly variable, showing strong correlation with specific nucleotide recognition. Thus, they can be engineered to bind to the desired DNA sequence.

To obtain TALEN, the TALE protein is fused to a nuclease (N), which is a wild-type FokI endonuclease or a mutant FokI endonuclease. Several mutations have been made to FokI for its application to TALEN; they, for example, increase cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39:e82; Miller et al. (2011) Nature Biotech . 29:143-8; Hockemeyer et al. (2011) Nature Biotech . 29:731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25:786-793; and Guo et al. (2010) J. Mol. Biol. 200:96.

The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains for regions in the target genome with proper orientations and spacings. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech. 29:143-8.

TALEN against HLA or TCR can be used intracellularly to provide a double strand break (DSB). A mutation can be introduced at the site of the break if the repair mechanisms do not properly repair the break through non-homologous end joining. For example, improper repair may introduce a frameshift mutation. Alternatively, foreign DNA can be introduced into the cell along with TALEN; depending on the foreign DNA sequences and chromosomal sequence, this process can be used to correct a defect in the HLA or TCR gene, or introduce such a defect in the wt HLA or TCR gene, thereby reducing HLA or TCR expression.

TALENs specific to sequences in HLA or TCR can be constructed using any method known in the art, including various schemes using modular components. Zhang et al. (2011) Nature Biotech. 29:149-53; Geibler et al. (2011) PLoS ONE 6: e19509.

Zinc finger nuclease for HLA and/or TCR inhibition

"ZFN" or "zinc finger nuclease" or "ZFN against HLA and/or TCR" or "ZFN for inhibition of HLA and/or TCR" refers to a zinc finger nuclease, which is an artificial nuclease that can be used to edit a gene HLA and/or TCR.

Like TALEN, ZFN contains a FokI nuclease domain (or a derivative thereof) fused to a DNA binding domain. In the case of ZFN, the DNA binding domain contains one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. sci. USA 93: 1156-1160.

The zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger may contain, for example, Cys2His2, and may recognize a sequence of approximately 3 bp. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18 bp in size. Various selection and modular assembly techniques are available to produce zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast monohybrid systems, bacterial monohybrid and two-hybrid systems, and mammalian cells.

Like TALEN, ZFN must dimerize in order to cleave DNA. Thus, a ZFN pair is required to target non-palindromic DNA regions. The two individual ZFNs must bind opposite strands of DNA, with proper space between their nucleases. Bitinaite et al. (1998) Proc. Natl. Acad. sci. USA 95: 10570-5.

Also like TALEN, ZFN can introduce a double-strand break in DNA, which can create a frameshift mutation in the event of improper repair, resulting in a decrease in the expression and amount of HLA and/or TCR in the cell. ZFNs can also be used with homologous recombination to introduce mutations in the HLA or TCR genes.

ZFNs specific for sequences in HLA and/or TCR can be constructed using any method known in the art. See, for example, Provasi (2011) Nature Med. 18:807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16:1200-7; and Guo et al. (2010) J. Mol. Biol. 400:96; US Patent Publication 2011/0158957; US Patent Publication 2012/0060230.

Telomerase expression

Without wishing to be bound by any particular theory, in some embodiments, the therapeutic T cell has a short stay in the patient due to shortened telomeres in the T cells; thus, transfection of the telomerase gene can lengthen T cell telomeres and increase the lifetime of T cells in a patient. See Carl June, "Adoptive T cell therapy for cancer in the clinic", Journal of Clinical Investigation, 117:1466-1476 (2007). Thus, in one embodiment, an immune effector cell, eg, a T cell, ectopically expresses a telomerase subunit, eg, a telomerase catalytic subunit, eg TERT, eg hTERT. In some aspects, the present invention provides a method for producing a CAR-expressing cell, comprising contacting the cell with a nucleic acid encoding a telomerase subunit, eg a telomerase catalytic subunit, eg TERT, eg hTERT. The cell can be brought into contact with the nucleic acid before, simultaneously with, or after being brought into contact with a construct encoding a CAR.

In one aspect, the present invention relates to a method for obtaining a population of immune effector cells (eg, T cells, NK cells). In one embodiment, the method includes: providing a population of immune effector cells (eg, T cells or NK cells), contacting the population of immune effector cells with a nucleic acid encoding a CAR; and contacting a population of immune effector cells with a nucleic acid encoding a telomerase subunit, eg hTERT, under conditions that allow expression of CAR and telomerase.

In one embodiment, the nucleic acid encoding the telomerase subunit is DNA. In one embodiment, the nucleic acid encoding the telomerase subunit contains a promoter capable of driving the expression of the telomerase subunit.

In one embodiment, hTERT has the amino acid sequence GenBank Protein ID AAC51724.1 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization" Cell Volume 90, Issue 4, 22 August 1997, Pages 785-795), which is as follows:

MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTN IYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD (SEQ ID NO: 63)

In one embodiment, hTERT has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 63. In one embodiment, hTERT has the sequence SEQ ID NO: 63. In one embodiment, hTERT contains a deletion (eg, no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both. In one embodiment, the hTERT contains a transgenic amino acid sequence (e.g., no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both.

In one embodiment, hTERT is encoded by the GenBank nucleic acid sequence Accession No. AF018167 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization" Cell Volume 90, Issue 4, August 22, 1997, Pages 785-795):

1 caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc

61 cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc

121 tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg

181 gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg

241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg

301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg

361 cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc gtgcgcagct

n 421 acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc

481 gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg

541 tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca

601 ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg

661 cctggaacca

721 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg

781 ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga

841 cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag

901 ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc

961 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc

1021 ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc

1081 ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg

1141 agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc

1201 tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc

1261 agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag

1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg

1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt

1441 acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc

1501 acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca

1561 agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca

1621 ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg

1681 ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt

1741 atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag agtgtctgga

1801 gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt

1861 cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc

1921 gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag

1981 ccagaacgtt ccgcagagaa aaggggccg agcgtctcac ctcgagggtg aaggcactgt

2041 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg

2101 gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc

2161 cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc

2221 aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc

2281 gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc

2341 acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg

2401 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca

2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg

2521 gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct

2581 gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacggggc

2641 tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa

2701 ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga

2761 agacagtggt gaacttccct gtagaagacg aggccctgggg tggcacggct tttgttcaga

2821 tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg

2881 tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc

2941 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt

3001 gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc acccaacatct

3061 acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc

3121 atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc

3181 tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg

3241 ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc

3301 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gcccagacgc

3361 agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg

3421 cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg

3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc

3541 cccacccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct

3601 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc

3661 tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc

3721 agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc

3781 cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc

3841 caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt

3901 gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtggggt caaattgggg

3961 ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa

4021 aaaaaaa (SEQ ID NO: 64)

In one embodiment, hTERT is encoded by a nucleic acid having a sequence that is at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 64. In one embodiment, hTERT is encoded by the nucleic acid SEQ ID NO: 64.

Activation and increase in the number of immune effector cells (eg, T cells)

Immune effector cells, such as T cells, can be activated and increased in number, mainly using the methods described, for example, in US patents 6352694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681; 7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514; 6,867,041 and US Patent Application Publication No. 20060121005.

, Франция), которые можно использовать, а также можно использовать и другие способы, широко известные в данной области (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).In general, a population of immune effector cells, such as cells depleted of regulatory T cells, can be increased in number by contact with a surface having an agent associated with it that stimulates the signal associated with the CD3/TCR complex and a ligand that stimulates the costimulatory molecule on the surface of T cells. In particular, T cell populations can be stimulated as described herein, for example, by contact with an anti-CD3 antibody or 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) together with a calcium ionophore. To stimulate a helper molecule on the surface of T cells, a ligand that binds the helper can be used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to promote T cell proliferation. To stimulate the proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone,

, France) which can be used, as well as other methods widely known in the art (Berg et al ., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al ., J. Exp. Med. 190(9):13191328, 1999; Garland et al ., J. Immunol Meth. 227(1-2):53-63, 1999).

In some aspects, the primary stimulatory signal and the co-stimulatory signal for T cells may be provided through different protocols. For example, the means providing each signal may be in solution or may be associated with a surface. When they are surface bound, the agents may be bound to the same surface (ie, in "cis" form) or to different surfaces (ie, in "trans" form). Alternatively, one agent may be associated with the surface and the other agent may be in solution. In one aspect, the agent providing the costimulatory signal is associated with the cell surface and the agent providing the primary activating signal is in solution or associated with the surface. In some aspects, both agents may be in solution. In one aspect, the agents may be in soluble form and then crosslinked to a surface, such as a cell expressing Fc receptors, or an antibody or other binding compound that will bind to the agents. In this regard, see, for example, US Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPC) which are contemplated for use in activating and expanding T cells in the context of the present invention.

In one aspect, the two agents are immobilized on the beads, or on the same beads, ie. "cis", or on separate granules, i. "trance". By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing a costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and both agents are co-immobilized on the same beads in equivalent molecular amounts. In one aspect, a 1:1 ratio is used for each bead-bound antibody to increase CD4+ T cells and increase T cells. In some aspects of the present invention, a ratio of bead-bound CD3:CD28 antibodies is used such that an increase in T cell expansion is observed over that observed using a 1:1 ratio. In one particular aspect, there is an increase of about 1 to about 3 times the expansion observed using a 1:1 ratio. In one aspect, the ratio of anti-CD3:CD28 antibodies associated with the beads is in the range of 100:1 to 1:100, including all whole numbers in between. In one aspect, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i. the CD3:CD28 ratio is less than one. In certain aspects, the ratio of anti-CD28 antibody to bead-bound anti-CD3 antibody is greater than 2:1. In one specific aspect, a 1:100 ratio of anti-CD3:CD28 antibodies associated with the beads is used. In one aspect, a 1:75 ratio of anti-CD3:CD28 antibodies associated with the beads is used. In a further aspect, a 1:50 ratio of anti-CD3:CD28 antibodies associated with beads is used. In one aspect, a 1:30 ratio of anti-CD3:CD28 antibodies bound to the beads is used. In one preferred aspect, a 1:10 ratio of anti-CD3:CD28 antibodies bound to the beads is used. In one aspect, a 1:3 ratio of anti-CD3:CD28 antibodies associated with beads is used. In a further aspect, a 3:1 ratio of anti-CD3:CD28 antibodies associated with beads is used.

Particle-to-cell ratios of 1:500 to 500:1, including any integers in between, can be used to stimulate T cells and other target cells. As will be well understood by those skilled in the art, the ratio of particles to cells may depend on the size of the particles relative to the target cell. For example, small granules may bind only a few cells, while large granules may bind many cells. In some aspects, a ratio of cells to particles that is in the range of 1:100 to 100:1, including any whole numbers in between, can be used to stimulate T cells, and in the following aspects, the ratio includes from 1:9 to 9:1, including any integers in between. The ratio of anti-CD3 antibody to anti-CD28 and T cell particles that results in T cell stimulation can vary as noted above, but certain preferred values include 1:100, 1:50, 1:40, 1:30 , 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3 :1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and 15:1, with one preferred ratio of particles per T cell being at least 1: 1. In one aspect, a particle to cell ratio of 1:1 or less is used. In one particular aspect, the particle:cell ratio is 1:5. In the following aspects, the ratio of particles and cells may vary depending on the day of stimulation. For example, in one aspect, the ratio of particles to cells is from 1:1 to 10:1 on the first day, and additional particles are added to the cells every knock or every other day thereafter for up to 10 days, in final ratios from 1: 1 to 1:10 (based on the number of cells on the day of addition). In one specific aspect, the particle to cell ratio is 1:1 on the first day of stimulation and is adjusted to 1:5 on days three and five of stimulation. In one aspect, the particles are added once a day or every two days to a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In one aspect, the particle to cell ratio is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In one aspect, the particles are added every knock or every two days to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation. A person skilled in the art will understand that many other ratios may be suitable for use in the present invention. In particular, the ratios will vary with particle size and with cell size and type. In one aspect, the most typical ratios for use are about 1:1, 2:1 and 3:1 on the first day.

In the following aspects, cells, such as T cells, are combined with agent-coated beads, then the beads and cells are separated, and then the cells are cultured. In an alternative aspect, before culturing, the coated beads and cells are not separated, but cultured together. In a further aspect, the beads and cells are first concentrated using a force, such as a magnetic force, which results in increased ligation of cell surface markers, thereby inducing cell stimulation.

As an example, cell surface proteins can be ligated by allowing anti-CD3 antibody and anti-CD28 antibody-bound paramagnetic beads (3x28 beads) to contact T cells. In one aspect, cells (eg, 10 ⁴ to 10 ⁹ T cells) and beads (eg, DYNABEAD® M-450 CD3/CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, such as PBS (without divalent cations, such as calcium and magnesium). Again, one of skill in the art will appreciate that any concentration of cells can be used. For example, target cells may be very rare in a sample and may comprise only 0.01% of the sample, or the entire sample (ie, 100%) may be composed of target cells of interest. Thus, any number of cells is contemplated within the context of the present invention. In some aspects, it may be desirable to significantly reduce the volume in which the particles and cells are mixed with each other (ie, increase the concentration of cells) to ensure maximum contact of cells and particles. For example, in one aspect, a concentration of approximately 10 billion cells/ml, 9 billion cells/ml, 8 billion cells/ml, 7 billion cells/ml, 6 billion cells/ml, 5 billion cells/ml, or 2 billion cells/ml is used. In one aspect, more than 100 million cells/ml are used. In a further aspect, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In another aspect, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells/ml is used. In the following aspects, concentrations of 125 or 150 million cells/ml can be used. The use of high concentrations can lead to increased cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28 negative T cells. Such populations of cells may be of therapeutic value, and in some aspects it may be desirable to obtain them. For example, the use of a high concentration of cells allows more efficient selection of CD8+ T cells, which typically have weaker expression of CD28.

In one embodiment, cells transduced with a nucleic acid encoding a CAR, such as the CAR described herein, are increased in number, for example, by the method described herein. In one embodiment, cells are expanded in culture over a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment, the cells are increased in number over a period of 4 to 9 days. In one embodiment, cells are increased in number over a period of 8 days or less, such as 7, 6, or 5 days. In one embodiment, cells, e.g., anti-CD19 CAR cells described herein, are increased in culture for 5 days, and the resulting cells are more potent than the same cells that are increased in culture for 9 days. days under the same cultivation conditions. Efficacy can be determined, for example, by various functions of T cells, for example, proliferation, killing of target cells, production of cytokines, activation, migration, and combinations thereof. In one embodiment, cells, e.g., anti-CD19 CAR cells described herein, that are increased in number for 5 days, show an increase in cell doublings of at least one, two, three, or four times when challenged with antigen compared to the same cells that were increased in number for 9 days under the same cultivation conditions. In one embodiment, cells, e.g., cells expressing the anti-CD19 CARs described herein, are increased in culture for 5 days and the resulting cells show higher production of pro-inflammatory cytokines, e.g., IFN-γ and/or GM levels. -CSF, compared to the same cells that were increased in culture for 9 days under the same culture conditions. In one embodiment, cells, e.g., anti-CD19 CAR cells described herein, which are increased in number over 5 days, show an increase in pg/ml production of pro-inflammatory cytokines, e.g., levels of IFN-γ and/or GM-CSF , at least one, two, three, four, five, ten times or more compared to the same cells that were increased in culture for 9 days under the same culture conditions.

It may also be desirable to have multiple cycles of stimulation such that the T cell culture time is 60 days or more. Conditions suitable for culturing T cells include appropriate media (e.g., minimal maintenance or RPMI 1640 or, X-vivo 15, (Lonza)), which may contain factors necessary for proliferation and viability, including serum (e.g., human fetal serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF -α or any other cell growth supplements known to the person skilled in the art. Other cell growth additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate and vitamins, either serum-free or supplemented with an appropriate amount serum (or plasma) or a certain set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, such as penicillin and streptomycin, are only included in experimental cultures, not in cell cultures that are to be infused into an individual. The target cells are maintained under conditions necessary to support growth, eg at the appropriate temperature (eg 37° C.) and in the appropriate atmosphere (eg air plus 5% CO ₂ ).

In one embodiment, cells are expanded in a suitable medium (e.g., the media described herein) that includes one or more interleukins that result in an increase in cell numbers of at least 200-fold (e.g., 200-fold, 250 times, 300 times, 350 times) during an expansion period of 14 days, for example, when measured by the method described in the present description, such as flow cytometry. In one embodiment, the cells are expanded in the presence of IL-15 and/or IL-7 (eg IL-15 and IL-7).

In embodiments, the methods described herein, e.g., methods for obtaining CAR-expressing cells, comprise removing regulatory T cells, e.g., CD25+ T cells, from a population, e.g., using an anti-CD25 antibody or fragment thereof, or a CD25-binding ligand, IL-2. Methods for removing regulatory T cells, eg, CD25+ T cells, from a population of cells are described herein. In embodiments, methods, e.g., production methods, further comprise contacting a population of cells (e.g., a population of cells depleted in regulatory T cells, such as CD25+ T cells; or a population of cells that has previously been contacted with an antibody against CD25, fragment thereof, or CD25 binding ligand) with IL-15 and/or IL-7. For example, a cell population (eg, which has previously been contacted with an anti-CD25 antibody, fragment thereof, or CD25 binding ligand) is increased in number in the presence of IL-15 and/or IL-7.

In some embodiments, a CAR-expressing cell described herein is contacted with a composition comprising an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide. , and an IL-15Ra polypeptide, eg hetIL-15, during the production of a CAR-expressing cell, eg ex vivo . In embodiments, a CAR-expressing cell described herein is contacted with a composition containing an IL-15 polypeptide during the production of a CAR-expressing cell, for example, ex vivo . In embodiments, a CAR-expressing cell described herein is contacted with a composition containing a combination of both an IL-15 polypeptide and an IL-15 Ra polypeptide during the production of a CAR-expressing cell, e.g., ex vivo . In embodiments, a CAR expressing cell as described herein is contacted with a composition containing hetIL-15 during the production of a CAR expressing cell, eg ex vivo .

In one embodiment, the CAR-expressing cell described herein is contacted with a composition containing hetIL-15 in an ex vivo expansion process. In one embodiment, a CAR-expressing cell described herein is contacted with a composition containing an IL-15 polypeptide in an ex vivo expansion process. In one embodiment, a CAR expressing cell described herein is contacted with a composition containing both an IL-15 polypeptide and an IL-15Ra polypeptide in an ex vivo expansion process. In one embodiment, contact leads to the survival and proliferation of a subpopulation of lymphocytes, eg, CD8+ T cells.

T cells exposed to different durations of stimulation periods may exhibit different characteristics. For example, typical blood or mononuclear cell products of apheresis peripheral blood have a population of helper T cells (TH, CD4+) that outnumbers that of cytotoxic or suppressor T cells (TC, CD8+). Ex vivo expansion of T cells via stimulation of the CD3 and CD28 receptors produces a T cell population that up to about 8-9 days consists primarily of TH cells, while after 8-9 days, the T cell population contains significantly more a high population of TC cells. Thus, depending on the goal of treatment, infusion into an individual of a population of T cells containing mainly TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells is isolated, it may be preferable to increase the number of this subset to a greater extent.

Moreover, in addition to the CD4 and CD8 markers, other phenotypic markers vary greatly, but for the most part, are reproducible during the cell expansion process. Thus, this reproducibility allows the activated T cell product to be tailored to specific targets.

Once a CAR has been constructed as described herein, various assays can be used to evaluate the activity of the molecule, such as, but not limited to, T cell expansion capacity following antigen challenge, maintenance of T cell expansion in the absence of restimulation, and anti-cancer activity in various in vitro and animal models. Assays for evaluating the effects of CARs of the present invention are described in more detail below.

Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, T cells (a 1:1 mixture of CD4 ⁺ and CD8 ⁺ T cells) expressing CAR are increased in vitro for more than 10 days, followed by lysis and SDS-PAGE under reducing conditions. CARs containing the full-length TCR-ζ cytoplasmic domain and the endogenous TCR-ζ chain are detected by Western blotting using an antibody to the TCR-ζ chain. The same subgroups of T cells are used for SDS-PAGE analysis under non-reducing conditions to be able to evaluate covalent dimer formation.

Expansion of CAR ⁺ T cells in vitro after antigen challenge can be measured by flow cytometry. For example, a mixture of CD4 ⁺ and CD8 ⁺ T cells are stimulated with APC αCD3/αCD28 followed by transduction with lentiviral vectors expressing GFP under the control of promoters to be analyzed. Illustrative promoters include the CMV IE, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) gene promoters. GFP fluorescence was assessed on day 6 of culture in CD4 ⁺ and/or CD8 ⁺ T cell subgroups using flow cytometry. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Alternatively, a mixture of CD4 ⁺ and CD8 ⁺ T cells are stimulated with αCD3/αCD28 coated magnetic beads on day 0 and transduced with CAR on day 1 using a bicistronic lentiviral vector expressing CAR together with eGFP using the ribosomal skip sequence 2A. After washing, cultures are restimulated with either cancer-associated antigen positive K562 cells (K562 expressing cancer-associated antigen as described herein) or wild-type K562 cells (wild-type K562) or hCD32-expressing K562 cells. and 4-1BBL, in the presence of an anti-CD3 antibody and an anti-CD28 antibody (K562-BBL-3/28). Exogenous IL-2 is added to the cultures every two days in an amount of 100 IU/ml. GFP ⁺ T cells are counted by flow cytometry using bead-based counting. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009).

It is also possible to measure the long-term expansion of CAR ⁺ T cells in the absence of restimulation. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, mean T cell volume (fl) is measured at day 8 of culture using a Coulter Multisizer III, Nexcelom Cellometer Vision, or Millipore Scepter particle counter, after stimulation with αCD3/αCD28 coated magnetic beads on day 0 and transduction with the indicated CAR at day 1. day.

Animal models can also be used to measure CART activity. For example, a xentorgraft model using CAR ⁺ T cells specific for the human cancer-associated antigen described herein can be used to treat primary human pre-B ALL in immunodeficient mice. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, once ALL is established, mice are randomly assigned to treatment groups. Different amounts of engineered CART cells specific for a cancer associated antigen were co-injected in a 1:1 ratio into B-ALL-bearing NOD-SCID-γ ^-/- mice. The number of copies of the vector with CAR specific for cancer associated antigen in spleen DNA from mice was assessed at various time points after T-cell injection. Animals are evaluated for leukemia at weekly intervals. B-ALL blast cell counts positive for the cancer-associated antigen described herein in peripheral blood are measured in mice injected with CAR ⁺ T cells against the cancer-associated antigen described herein and transduced with a mimic construct T cells. Group survival curves are compared using a log-rank test. In addition, it is also possible to analyze the absolute number of CD4 ⁺ and CD8 ⁺ T cells in peripheral blood 4 weeks after injection of T cells into NOD-SCID-γ ^-/- mice. Mice are injected with leukemia cells and 3 weeks later are injected with T cells engineered to express CAR by a bicistronic lentiviral vector that encodes an eGFP-linked CAR. T cells are normalized to 45-50% GFP ⁺ T cells inlet by mixing with transduced dummy construct cells prior to injection and confirmed by flow cytometry. Animals are evaluated for leukemia at 1 week intervals. Survival curves for groups of CAR ⁺ T cells are compared using a logarithmic rank test.

You can evaluate the dose-dependent response to the introduction of CAR. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after the onset of leukemia in mice that were injected on day 21 with CAR T cells, an equivalent number of T cells transduced with a mock construct, or no T cells were injected. Mice from each group were randomly bled to determine the numbers of peripheral blood ALL blasts positive for the cancer-associated antigen described herein, and then sacrificed on days 35 and 49. The rest of the animals were evaluated on days 57 and 70.

The evaluation of cell proliferation and cytokine production has been described previously, for example, in Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, the assessment of CAR-mediated proliferation is performed on microtiter plates by mixing washed T cells with K562 cells expressing the cancer-associated antigen described herein (K19) or CD32 and CD137 (KT32-BBL) in the final ratio T cells: K562 2:1. K562 cells are irradiated with gamma radiation prior to use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for the stimulation of T cell proliferation, since these signals support the long-term expansion of CD8 ⁺ T cells ex vivo . T cells are counted in cultures using CountBright ^™ fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry as described by the manufacturer. CAR ⁺ T cells are identified by GFP expression using T cells that are modified with eGFP-2A-linked CAR-expressing lentiviral vectors. For CAR+ T cells not expressing GFP, CAR+ T cells are detected using a biotinylated recombinant cancer-associated antigen protein as described herein and an avidin-PE secondary conjugate. Also simultaneously detect the expression of CD4+ and CD8 ⁺ on T-cells using specific monoclonal antibodies (BD Biosciences). Cytokine measurement was performed on supernatants collected 24 hours after restimulation using the TH1/TH2 cytometric bead array kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. Fluorescence is assessed using a FACScalibur flow cytometer and the data is analyzed according to the manufacturer's instructions.

Cytotoxicity can be assessed by the methods described in the present description, for example in the examples, or using the standard release of 51Cr. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, target cells (eg, BHK or CHO cells expressing mesothelin) are loaded with 51Cr (such as NaCrO4, New England Nuclear, Boston, MA) at 37°C for 2 hours with frequent shaking, washed twice in complete RPMI and plated on microtiter plates. Effector T cells are mixed with target cells in complete RPMI wells at various effector:target (E:T) ratios. Also prepare additional wells containing only medium (spontaneous release, SR) or 1% solution of triton-X 100 detergent (total release, TR). After incubation for 4 hours at 37°C, the supernatant of each well is collected. The release of ⁵¹ Cr is then measured using a gamma counter (Packard Instrument Co., Waltham, MA). Each condition is run in at least triplicate and percent lysis is calculated using the formula: % lysis=(ER-SR)/(TR-SR) where ER is the average amount of 51Cr released for each experimental condition. Alternative cytotoxicity assays, such as flow cytotoxicity assays, may also be used.

Imaging technologies can be used to evaluate the specific transport and proliferation of CARs in tumor-bearing animal models. Such assays are described, for example, in Barrett et al ., Human Gene Therapy 22:1575-1586 (2011). Briefly, NOD/SCID/γc ^−/− (NSG) mice are intravenously injected with Nalm-6 cells and then 7 days later with T cells 4 hours after injection of the CAR constructs by electroporation. T cells are stably transfected with a lentiviral construct to express firefly luciferase and mice are imaged for bioluminescence. Alternatively, the therapeutic efficacy of a single injection of CAR ⁺ T cells in the Nalm-6 xenograft model can be measured as follows: NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed by a single tail vein injection of T cells 7 days later. into which the CAR of the present invention has been introduced by electroporation. Animals are visualized at various time points after injection. For example, heat maps of the photon density of firefly luciferase-positive leukemia cells can be obtained from representative mice on day 5 (2 days before treatment) and day 8 (24 hours after CAR ⁺ PBL).

Other assays can also be used to evaluate the CAR constructs described herein, including those described in the Examples section of this document, as well as assays known in the art.

Therapeutic use

In one aspect, the invention provides methods for treating a disease associated with the expression of a cancer-associated antigen described herein.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express XCAR, where X is a tumor antigen, as described in the present description, and where malignant cells express the specified tumor antigen X.

In one aspect, the present invention relates to methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express the XCAR described herein, wherein the cancer cells express X. In one embodiment, X is expressed on both normal cells and malignant cells, but is expressed at lower levels on normal cells. In one embodiment, the method further comprises selecting a CAR that binds X with an affinity that allows the XCAR to bind and kill X-expressing cancer cells, but kill less than 30%, 25%, 20%, 15%, 10%, 5%, or less normal cells expressing X, for example, as determined using the assay described herein. For example, the assay described in FIGS. 13A and 13B or a kill assay such as flow cytometry based on Cr51 CTL can be used. In one embodiment, the selected CAR has an antigen binding domain that has a KD binding affinity of 10 ^-4 M to 10 ^-8 M, e.g., 10 ^-5 M to 10 ^-7 M, e.g., 10 ^-6 M or 10 ^-7 M , in relation to the target antigen. In one embodiment, the selected antigen-binding domain has a binding affinity that is at least five-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or 1000-fold less than a reference antibody, e.g. , the antibody described in the present description.

In one embodiment, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD19 CARs, wherein the cancer cells express CD19. In one embodiment, the cancer being treated is ALL (acute lymphoblastic leukemia), CLL (chronic lymphocytic leukemia), DLBCL (diffuse large B cell lymphoma), MCL (mantle cell lymphoma, or MM (multiple myeloma).

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express EGFRvIIICAR, wherein the cancer cells express EGFRvIII. In one embodiment, the cancer being treated is a glioblastoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-mesothelin CAR, wherein the cancer cells express mesothelin. In one embodiment, the cancer being treated is mesothelioma, pancreatic cancer, or ovarian cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD123CAR, wherein the cancer cells express CD123. In one embodiment, the cancer being treated is AML.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD22CAR, wherein the cancer cells express CD22. In one embodiment, the cancer being treated is B-cell cancers.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CS-1CAR, wherein the cancer cells express CS-1 . In one embodiment, the cancer being treated is multiple myeloma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CLL-1CAR, wherein the cancer cells express CLL-1 . In one embodiment, the cancer being treated is AML.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD33CAR, wherein the cancer cells express CD33. In one embodiment, the cancer being treated is AML.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express GD2CAR, wherein the cancer cells express GD2. In one embodiment, the cancer being treated is a neuroblastoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express BCMACAR, wherein the cancer cells express BCMA. In one embodiment, the cancer being treated is multiple myeloma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express TnCAR, wherein the cancer cells express a Tn antigen. In one embodiment, the cancer being treated is ovarian cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express PSMACAR, wherein the cancer cells express PSMA. In one embodiment, the cancer being treated is prostate cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express ROR1CAR, wherein the cancer cells express ROR1. In one embodiment, the cancer being treated is B-cell cancers.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express FLT3 CARs, wherein the cancer cells express FLT3. In one embodiment, the cancer being treated is AML.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express TAG72CAR, wherein the cancer cells express TAG72. In one embodiment, the cancer being treated is gastrointestinal cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express CD38CAR, wherein the cancer cells express CD38. In one embodiment, the cancer being treated is multiple myeloma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD44v6CAR, wherein the cancer cells express CD44v6. In one embodiment, the cancer being treated is cervical cancer, AML, or MM.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express CEACAR, wherein the cancer cells express CEA. In one embodiment, the cancer being treated is gastrointestinal cancer or pancreatic cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express EPCAMCAR, wherein the cancer cells express EPCAM. In one embodiment, the cancer being treated is gastrointestinal cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express B7H3CAR, wherein the cancer cells express B7H3.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express KITCAR, wherein the cancer cells express KIT. In one embodiment, the cancer being treated is gastrointestinal cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express IL-13Ra2CAR, wherein the cancer cells express IL-13Ra2 . In one embodiment, the cancer being treated is a glioblastoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express PRSS21CAR, wherein the cancer cells express PRSS21. In one embodiment, the cancer being treated is selected from ovarian cancer, pancreatic cancer, lung cancer, and breast cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD30CAR, wherein the cancer cells express CD30. In one embodiment, the cancer being treated is lymphomas or leukemias.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express GD3CAR, wherein the cancer cells express GD3. In one embodiment, the cancer being treated is melanoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD171CAR, wherein the cancer cells express CD171. In one embodiment, the cancer being treated is neuroblastoma, ovarian cancer, melanoma, breast cancer, pancreatic cancer, colon cancer, or NSCLC (non-small cell lung cancer).

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express IL-11RaCAR, wherein the cancer cells express IL-11Ra . In one embodiment, the cancer being treated is osteosarcoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express PSCACAR, wherein the cancer cells express PSCA. In one embodiment, the cancer being treated is prostate cancer.

In one aspect, the present invention relates to methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express VEGFR2CAR, wherein the cancer cells express VEGFR2. In one embodiment, the cancer being treated is a solid tumor.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express LewisYCAR, wherein the cancer cells express LewisY. In one embodiment, the cancer being treated is ovarian cancer or AML.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD24CAR, wherein the cancer cells express CD24. In one embodiment, the cancer being treated is pancreatic cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-PDGFR-beta CAR, wherein the cancer cells express PDGFR. -beta. In one embodiment, the cancer being treated is breast cancer, prostate cancer, GIST (gastrointestinal stromal tumor), CML, DFSP (dermatofibrosarcoma elevated), or glioma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express SSEA-4CAR, wherein the cancer cells express SSEA-4 . In one embodiment, the cancer being treated is glioblastoma, breast cancer, lung cancer, or stem cell cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD20CAR, wherein the cancer cells express CD20. In one embodiment, the cancer being treated is B-cell cancers.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-alpha folate receptor CAR, wherein the cancer cells express the receptor folate alpha. In one embodiment, the cancer being treated is ovarian cancer, NSCLC, endometrial cancer, kidney cancer, or other solid tumors.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express ERBB2CAR, wherein the cancer cells express ERBB2 (Her2/neu ). In one embodiment, the cancer being treated is breast cancer, stomach cancer, colon cancer, lung cancer, or other solid tumors.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express MUC1CAR, wherein the cancer cells express MUC1. In one embodiment, the cancer being treated is breast cancer, lung cancer, or other solid tumors.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express EGFRCAR, wherein the cancer cells express EGFR. In one embodiment, the cancer being treated is glioblastoma, SCLC (small cell lung cancer), SCCHN (head and neck squamous cell carcinoma), NSCLC, or other solid tumors.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express NCAMCAR, wherein the cancer cells express NCAM. In one embodiment, the cancer being treated is neuroblastoma or other solid tumors.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express CAIXCAR, wherein the cancer cells express CAIX. In one embodiment, the cancer being treated is kidney cancer, CRC, cervical cancer, or other solid tumors.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express EphA2CAR, wherein the cancer cells express EphA2. In one embodiment, the cancer being treated is GBM.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express GD3CAR, wherein the cancer cells express GD3. In one embodiment, the cancer being treated is melanoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-fucosyl-GM1 CAR, wherein the cancer cells express fucosyl -GM.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express sLeCAR, wherein the cancer cells express sLe. In one embodiment, the cancer being treated is NSCLC or AML.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express GM3CAR, wherein the cancer cells express GM3.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express TGS5CAR, wherein the cancer cells express TGS5.

In one aspect, the present invention relates to methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express HMWMAACAR, wherein the cancer cells express HMWMAA. In one embodiment, the cancer being treated is melanoma, glioblastoma, or breast cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express o-acetyl-GD2CAR, wherein the cancer cells express o -acetyl-GD2. In one embodiment, the cancer being treated is a neuroblastoma or melanoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express CD19CAR, wherein the cancer cells express CD19. In one embodiment, the cancer being treated is AML, myeloma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express TEM1/CD248CAR, wherein the cancer cells express TEM1/CD248 . In one embodiment, the cancer being treated is a solid tumor.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express TEM7RCAR, wherein the cancer cells express TEM7R. In one embodiment, the cancer being treated is a solid tumor.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CLDN6CAR, wherein the cancer cells express CLDN6. In one embodiment, the cancer being treated is ovarian cancer, lung cancer, or breast cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express TSHRCAR, wherein the cancer cells express TSHR. In one embodiment, the cancer being treated is thyroid cancer or multiple myeloma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express GPRC5DCAR, wherein the cancer cells express GPRC5D. In one embodiment, the cancer being treated is multiple myeloma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CXORF61CAR, wherein the cancer cells express CXORF61.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express CD97CAR, wherein the cancer cells express CD97. In one embodiment, the cancer being treated is B-cell cancers, gastric cancer, pancreatic cancer, esophageal cancer, glioblastoma, breast cancer, or colorectal cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD179aCAR, wherein the cancer cells express CD179a. In one embodiment, the cancer being treated is B-cell cancers.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express CAR ALK, wherein the cancer cells express ALK. In one embodiment, the cancer being treated is NSCLC, ALCL (Anaplastic Large Cell Lymphoma), IMT (Inflammatory Myofibroblastic Tumor), or Neuroblastoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express a polysialic acid CAR, wherein the cancer cells express polysialic acid. In one embodiment, the cancer being treated is small cell lung cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express PLAC1CAR, wherein the cancer cells express PLAC1. In one embodiment, the cancer being treated is HCC (hepatocellular carcinoma).

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express GloboHCAR, wherein the cancer cells express GloboH. In one embodiment, the cancer being treated is ovarian cancer, stomach cancer, prostate cancer, lung cancer, breast cancer, or pancreatic cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express NY-BR-1CAR, wherein the cancer cells express NY -BR-1. In one embodiment, the cancer being treated is breast cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express UPK2CAR, wherein the cancer cells express UPK2. In one embodiment, the cancer being treated is bladder cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express HAVCR1CAR, wherein the cancer cells express HAVCR1. In one embodiment, the cancer being treated is kidney cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express ADRB3CAR, wherein the cancer cells express ADRB3. In one embodiment, the cancer being treated is Ewing's sarcoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express PANX3CAR, wherein the cancer cells express PANX3. In one embodiment, the cancer being treated is osteosarcoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express GPR20CAR, wherein the cancer cells express GPR20. In one embodiment, the cancer being treated is a GIST.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express LY6KCAR, wherein the cancer cells express LY6K. In one embodiment, the cancer being treated is breast cancer, lung cancer, ovarian cancer, or cervical cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express OR51E2CAR, wherein the cancer cells express OR51E2. In one embodiment, the cancer being treated is prostate cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express TARPCAR, wherein the cancer cells express TARP. In one embodiment, the cancer being treated is prostate cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express WT1CAR, wherein the cancer cells express WT1.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express NY-ESO-1CAR, wherein the cancer cells express NY -ESO-1.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express LAGE-1a CAR, wherein the cancer cells express LAGE- 1a.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express MAGE-A1CAR, wherein the cancer cells express MAGE-A1 . In one embodiment, the cancer being treated is melanoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express MAGE A1CAR, wherein the cancer cells express MAGE A1.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express ETV6-AML CARs, wherein the cancer cells express ETV6- AML.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express the 17 CAR sperm protein, wherein the cancer cells express the sperm protein 17. In one embodiment, the cancer being treated is ovarian cancer, HCC or NSCLC.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express XAGE1CAR, wherein the cancer cells express XAGE1. In one embodiment, the cancer being treated is Ewing's sarcoma or rhabdomyosarcoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express Tie 2 CAR, wherein the cancer cells express Tie 2. In one embodiment, the cancer being treated is a solid tumor.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express MAD-CT-1CAR, wherein the cancer cells express MAD -CT-1. In one embodiment, the cancer being treated is prostate cancer or melanoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express MAD-CT-2CAR, wherein the cancer cells express MAD -CT-2. In one embodiment, the cancer being treated is prostate cancer, melanoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express anti-Fos-related antigen 1 CAR, wherein the cancer cells express Fos-related antigen 1. In one embodiment, the cancer being treated is glioma, squamous cell carcinoma, or pancreatic cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express p53CAR, wherein the cancer cells express p53.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express prosteine CAR, wherein the cancer cells express protein.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express anti-survivin and telomerase CARs, wherein the cancer cells express survivin. and telomerase.

In one aspect, the present invention relates to methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express anti-PCTA-1/galectin 8 CAR, wherein the cancer cells express PCTA-1/galectin 8.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express MelanA/MART1CAR, wherein the cancer cells express MelanA/MART1 .

In one aspect, the present invention relates to methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-mutant Ras CAR, wherein the cancer cells express the mutant Ras. .

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express a mutated p53 CAR, wherein the cancer cells express the mutated p53 .

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express hTERT CARs, wherein the cancer cells express hTERT.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CARs against breakpoints in sarcoma translocation, where the cancer cells express breakpoints in sarcoma translocation. In one embodiment, the cancer being treated is a sarcoma.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof, immune effector cells (eg, T cells, NK cells) engineered to express ML-IAP CAR, wherein the cancer cells express ML- IAP. In one embodiment, the cancer being treated is melanoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express ERGCAR, wherein the cancer cells express ERG (TMPRSS2 fusion gene). ETS).

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express NA17CAR, wherein the cancer cells express NA17. In one embodiment, the cancer being treated is melanoma.

In one aspect, the present invention relates to methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express PAX3CAR, wherein the cancer cells express PAX3. In one embodiment, the cancer being treated is alveolar rhabdomyosarcoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express androgen receptor CAR, wherein the cancer cells express the androgen receptor. In one embodiment, the cancer being treated is metastatic prostate cancer.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-cyclin B1 CAR, wherein the cancer cells express cyclin B1 .

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express MYCNCAR, wherein the cancer cells express MYCN.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express Rh°C CARs, wherein the cancer cells express RhoC.

In one aspect, the present invention relates to methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express TRP-2CAR, wherein the cancer cells express TRP-2 . In one embodiment, the cancer being treated is melanoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express CYP1B1CAR, wherein the cancer cells express CYP1B1. In one embodiment, the cancer being treated is breast cancer, colon cancer, lung cancer, esophageal cancer, skin cancer, lymph node cancer, brain cancer, or testicular cancer.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express BORIS CAR, wherein the cancer cells express BORIS. In one embodiment, the cancer being treated is lung cancer.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express SART3CAR, wherein the cancer cells express SART3.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express PAX5CAR, wherein the cancer cells express PAX5.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express OY-TES1CAR, wherein the cancer cells express OY-TES1 .

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express LCK CAR, wherein the cancer cells express LCK.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express AKAP-4CAR, wherein the cancer cells express AKAP-4 .

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express SSX2CAR, wherein the cancer cells express SSX2.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express RAGE-1CAR, wherein the cancer cells express RAGE-1 . In one embodiment, the cancer being treated is RCC (renal cell carcinoma) or other solid tumors.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof, immune effector cells (e.g., T cells, NK cells) engineered to express CAR against human telomerase reverse transcriptase, wherein the cancer cells express human telomerase reverse transcriptase. In one embodiment, the cancer being treated is a solid tumor.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express RU1CAR, wherein the cancer cells express RU1.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express RU2CAR, wherein the cancer cells express RU2.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-intestinal carboxylesterase CAR, wherein the cancer cells express intestinal carboxylesterase. . In one embodiment, the cancer being treated is thyroid cancer, RCC, CRC (colon and rectal cancer), breast cancer, or other solid tumors.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-prostate CAR, wherein the cancer cells express prostasis.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express PAPCAR, wherein the cancer cells express PAP.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-IGF-I receptor CAR, wherein the cancer cells express IGF-I receptor.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express gp100 CARs, wherein the cancer cells express gp100.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express bcr-abl CAR, wherein the cancer cells express bcr- abl.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-tyrosinase CAR, wherein the cancer cells express tyrosinase.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express an anti-fucosyl-GM1 CAR, wherein the cancer cells express fucosyl -GM1.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express mut hsp70-2CAR, wherein the cancer cells express mut hsp70 -2. In one embodiment, the cancer being treated is melanoma.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD79a CARs, wherein the cancer cells express CD79a.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD79b CARs, wherein the cancer cells express CD79b.

In one aspect, the present invention relates to methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD72 CAR, wherein the cancer cells express CD72.

In one aspect, the present invention relates to methods for treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express LAIR1 CAR, wherein the cancer cells express LAIR1.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (eg, T cells, NK cells) engineered to express FCAR CAR, wherein the cancer cells express FCAR.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express LILRA2 CAR, wherein the cancer cells express LILRA2.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CD300LF CAR, wherein the cancer cells express CD300LF.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express CLEC12A CARs, wherein the cancer cells express CLEC12A.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express BST2 CARs, wherein the cancer cells express BST2.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express EMR2 CARs, wherein the cancer cells express EMR2.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express LY75 CAR, wherein the cancer cells express LY75.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express GPC3 CAR, wherein the cancer cells express GPC3.

In one aspect, the present invention relates to methods of treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express FCRL5 CARs, wherein the cancer cells express FCRL5.

In one aspect, the present invention provides methods for treating cancer by providing an individual in need thereof with immune effector cells (e.g., T cells, NK cells) engineered to express IGLL1 CAR, wherein the cancer cells express IGLL1.

In one aspect, the present invention relates to the treatment of an individual in vivo using CAR against PD1, so that the growth of malignant tumors is inhibited. CAR against PD1 can be used alone to inhibit the growth of malignant tumors. Alternatively, the anti-PD1 CAR can be used in conjunction with other CARs, immunogenic agents, standard therapies, or other antibodies. In one embodiment, the individual is treated with CAR against PD1 and XCAR as described herein. In one embodiment, an anti-PD1 CAR is used in conjunction with another CAR, eg, the CAR described herein, and a kinase inhibitor, eg, the kinase inhibitor described herein.

In another aspect, a method is provided for treating, eg, reducing or alleviating, a hyperproliferative condition or disorder (eg, cancer), eg, a solid tumor, soft tissue tumor, or metastatic lesion, in an individual. As used herein, the term "cancer" includes all types of malignant growth or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasiveness. Examples of solid tumors include malignant tumors such as sarcomas, adenocarcinomas and carcinomas of various organ systems such as malignant tumors affecting the liver, lung, breast, lymphoid tissue, gastrointestinal tract (eg colon), urogenital tract (eg , kidneys, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung carcinoma, small intestine cancer, and esophageal cancer. In one embodiment, the cancer is a melanoma, such as an extended melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention. Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, endometrial cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, cancer testis, uterine corpus cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter , carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary lymphoma of the central nervous system C, tumor angiogenesis, spinal tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, environmentally induced malignant tumors, including asbestosis-induced tumors, and combinations of these malignant tumors. Treatment of metastatic cancers, eg, metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 17:133-144), can be performed using the antibody molecules described herein.

Exemplary cancers whose growth may be inhibited include cancers that typically respond to immunotherapy. Non-limiting examples of cancers to be treated include melanoma (eg, metastatic malignant melanoma), kidney cancer (eg, clear cell carcinoma), prostate cancer (eg, hormone-refractory adenocarcinoma of the prostate), breast cancer, colon cancer, and cancer lung (eg, non-small cell lung cancer). In addition, refractory or recurrent cancers can be treated using the molecules described herein.

In one aspect, the invention relates to a vector containing a CAR operably linked to a promoter for expression in mammalian immune effector cells (eg, T cells, NK cells). In one aspect, the invention relates to a recombinant immune effector cell expressing the CAR of the present invention for use in the treatment of cancer, expressing a cancer associated antigen as described herein. In one aspect, the CAR expressing cells of the invention are capable of contacting a tumor cell on whose surface at least one cancer associated antigen is expressed such that the CAR expressing cell targets the cancer cell and growth of the cancer cell is inhibited.

In one aspect, the invention relates to a method for inhibiting the growth of a cancer comprising bringing the cancer cell into contact with a CAR expressing cell of the present invention such that CART is activated in response to an antigen and targeted to the cancer, inhibiting tumor growth.

In one aspect, the invention relates to a method for treating cancer in an individual. The method includes administering to an individual a CAR expressing cell of the present invention so that the cancer in the individual is treated. In one aspect, the cancer associated with the expression of a cancer-associated antigen as described herein is a hematologic cancer. In one aspect, the hematological malignancy is a leukemia or lymphoma. In one aspect, a cancer associated with the expression of a cancer-associated antigen as described herein includes cancers and neoplasms, including, but not limited to, for example, one or more of acute leukemias, including, but not limited to, for example, B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL); one or more of chronic leukemias, including but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphoid leukemia (CLL). Additional cancers and hematologic conditions associated with expression of a cancer-associated antigen as described herein include, but are not limited to, e.g., B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B -cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, neoplasm from plasmacytoid dendritic cells, Waldenström's macroglobulinemia, and "preleukemia", which is a diverse set of hematological conditions characterized by ineffective production (or dysplasia) of myeloid blood cells, etc. The following diseases associated with the expression of a cancer-associated antigen as described herein include, but are not limited to, for example, atypical and/or non-classical cancers, neoplasms, pre-cancerous conditions, or proliferative diseases associated with expression of a cancer-associated antigen. antigen as described herein.

In some embodiments, the cancer that can be treated with a CAR expressing cell of the present invention is multiple myeloma. Multiple myeloma is a malignant blood tumor characterized by the accumulation of a clone of plasma cells in the bone marrow. Current therapies for multiple myeloma include, but are not limited to, treatment with lenalidomide, which is an analogue of thalidomide. Lenalidomide has activities that include antitumor activity, angiogenesis inhibition and immunomodulation. Typically, myeloid cells are considered negative for the expression of a cancer-associated antigen as described herein when determined using flow cytometry. Thus, in some embodiments, an anti-CD19 CAR, for example as described herein, can be used to target myeloma cells. In some embodiments, the CAR therapy of the present invention may be used in combination with one or more additional therapies, such as treatment with lenalidomide.

The invention relates to a type of cell therapy where immune effector cells (e.g. T cells, NK cells) are genetically modified to express a chimeric antigen receptor (CAR) and a CAR expressing T cell or NK cell is administered by infusion to a recipient in need of this. The cell introduced by infusion is capable of destroying the tumor cells of the recipient. In contrast to antibody therapy, CAR-modified immune effector cells (eg, T cells, NK cells) are able to replicate in vivo , leading to long-term persistence, which can lead to long-term tumor control. In various aspects, immune effector cells (e.g., T cells, NK cells) administered to a patient, or progeny thereof, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months. , ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty one months, twenty two months, twenty three months, two years, three years, four years, or five years after the introduction of a T cell or NK cell to a patient.

The invention also relates to a type of cellular therapy where immune effector cells (e.g. T cells, NK cells) are modified, for example with in vitro transcribed RNA, to transiently express a chimeric antigen receptor (CAR), and a CAR expressing T cell or NK The β-cell is administered by infusion to a recipient in need thereof. The cell introduced by infusion is capable of destroying the tumor cells of the recipient. Thus, in various aspects, immune effector cells (eg, T cells, NK cells) administered to a patient are maintained for less than one month, eg, three weeks, two weeks, one week after administration of the T cells to the patient.

Without wishing to be bound by any particular theory, the immune response against tumor cells induced by CAR-modified immune effector cells (e.g., T cells, NK cells) may be an active or passive immune response, or alternatively may result from direct versus indirect immune response. In one aspect, CAR-transduced immune effector cells (e.g., T cells, NK cells) exhibit specific secretion of pro-inflammatory cytokines and potent cytolytic activity in response to human cancer cells expressing the cancer-associated antigen described herein, withstand inhibition by soluble associated with a malignant tumor antigen described in the present description, and mediate non-specific cytolysis and mediate the regression of an expanded human tumor. For example, tumor cells lacking an antigen in a heterogeneous region of a tumor expressing the cancer-associated antigen described herein may be susceptible to indirect destruction of immune effector cells retargeted to the cancer-associated antigen described herein (e.g., T- cells, NK cells) that have previously reacted with neighboring antigen-positive malignant cells.

In one aspect, the fully human CAR-modified immune effector cells (eg, T cells, NK cells) of the invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.

With respect to ex vivo immunization, at least one of the following events occurs in vitro prior to cell introduction into a mammal: i) cell expansion, ii) introduction of a CAR-encoding nucleic acid into cells, or iii) cell cryopreservation.

Ex vivo techniques are well known in the art and are discussed in more detail below. Briefly, mammalian (eg, human) cells are isolated and genetically modified (ie, transduced or transfected in vitro ) with a CAR expressing vector as described herein. The CAR-modified cell may be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient may be a human, and the CAR-modified cell may be autologous to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or xenogeneic in relation to the recipient. In addition to the use of a cellular vaccine for ex vivo immunization, compositions and methods for in vivo immunization to induce an immune response directed against an antigen in a patient are also included in the present invention.

For the cells of the present invention, the ex vivo hematological stem and progenitor cell expansion technique described in US Pat. No. 5,199,942, incorporated herein by reference, can be used. Other suitable methods are known in the art, so the present invention is not limited to any particular method for expanding cells ex vivo . Briefly, culture and expansion of immune effector cells (eg, T cells, NK cells) ex vivo includes: (1) harvesting CD34+ hematological stem cells and progenitor cells from collected peripheral blood or bone marrow explants; and (2) ex vivo expansion of such cells. In addition to the cell growth factors described in US Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand can be used to culture and expand cells.

In addition to the use of a cellular vaccine for ex vivo immunization, the present invention also relates to in vivo immunization compositions and methods for inducing an immune response directed against an antigen in a patient.

Generally, cells activated and expanded as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In particular, the CAR-modified immune effector cells (eg, T cells, NK cells) of the invention are used to treat diseases, disorders, and conditions associated with the expression of a cancer-associated antigen described herein. In certain aspects, the cells of the invention are used to treat patients at risk of developing diseases, disorders, and conditions associated with expression of a cancer-associated antigen described herein. Thus, the invention provides methods for treating or preventing diseases, disorders, and conditions associated with the expression of a cancer-associated antigen described herein, comprising administering to an individual in need thereof a therapeutically effective amount of CAR-modified immune effector cells (e.g., T -cells, NK-cells) according to the invention.

In one aspect, the CAR expressing cells of the invention can be used to treat a proliferative disease such as cancer or neoplasm, or a premalignant condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia. In addition, a disease associated with the expression of a cancer-associated antigen as described herein includes, but is not limited to, for example, atypical and/or non-classical cancers, neoplasms, premalignant conditions, or proliferative diseases expressing a cancer-associated antigen. antigen as described herein. Non-cancer-related diseases associated with expression of a cancer-associated antigen as described herein include, but are not limited to, for example, autoimmune disease (eg, lupus), inflammatory disorders (allergy and asthma), and transplantation.

The CAR-modified immune effector cells (e.g., T cells, NK cells) of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations.

Hematological malignant tumor

Hematologic malignancies are types of malignancies such as leukemia, lymphoma, and malignant lympholiferative conditions that affect the blood, bone marrow, and lymphatic system.

Leukemia can be divided into acute leukemia and chronic leukemia. Acute leukemia can be further subdivided into acute myelogenous leukemia (AML) and acute lymphoid leukemia (ALL). Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, formerly known as "preleukemia"), which are a diverse collection of hematological conditions associated with inefficient production (or dysplasia) of myeloid blood cells and the risk of transformation into AML.

Lymphoma is a group of blood cell tumors that develop from lymphocytes. Illustrative lymphomas include non-Hodgkin's lymphoma and Hodgkin's lymphoma.

The present invention relates to compositions and methods for the treatment of cancer. In one aspect, the cancer is a hematological cancer, including, but not limited to, leukemia or lymphoma. In one aspect, the CAR-expressing cells of the invention can be used to treat cancers and neoplasms such as, but not limited to, for example, acute leukemias, including, but not limited to, for example, B-cell acute lymphoid leukemia ("BALL") , T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL); one or more of chronic leukemias, including but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic malignancies or hematologic conditions, including, but not limited to, for example, B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large follicular cell lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström's macroglobulinemia, and "preleukemia" which presents a variety of hematological conditions characterized by inefficient production (or dysplasia) of myeloid blood cells, and the like. The following diseases associated with the expression of a cancer-associated antigen as described herein include, but are not limited to, for example, atypical and/or non-classical cancers, neoplasms, pre-malignant conditions, or proliferative diseases associated with the expression of a cancer-associated antigen. antigen as described herein.

The present invention also relates to methods for inhibiting the proliferation or reducing a population of cells expressing a cancer associated antigen as described herein, the methods comprising contacting a cell population comprising a cell expressing a cancer associated antigen as described herein. , with a CAR-expressing T cell or NK cell of the invention that binds to a cell expressing a cancer-associated antigen as described herein. In a specific aspect, the present invention relates to methods for inhibiting the proliferation or reducing a population of cancer cells expressing a cancer associated antigen as described herein, the methods comprising contacting a population of cancer cells expressing a cancer associated antigen as described herein. described, with a CAR-expressing T cell or NK cell of the invention that binds to a cell expressing a cancer-associated antigen as described herein. In one aspect, the present invention relates to methods for inhibiting the proliferation or reducing the population of tumor cells expressing a cancer associated antigen as described herein, the methods comprising contacting a population of cancer cells expressing a cancer associated antigen as described herein. , with a CAR-expressing T cell or NK cell of the invention that binds to a cell expressing a cancer-associated antigen as described herein. In some aspects, the CAR-expressing T cell or NK cell of the invention reduces the number, number, size or percentage of cells and/or malignant cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in an individual with myeloid leukemia or other cancer associated with cells expressing a cancer-associated antigen as described herein or in an animal model for these diseases, relative to a negative control. In one aspect, the individual is a human.

The present invention also provides methods for preventing, treating and/or managing a disease associated with cells expressing a cancer-associated antigen as described herein (e.g., a hematologic cancer or an atypical cancer expressing a cancer-associated antigen, as described herein), the methods comprising administering to an individual in need thereof a CAR expressing T cell or NK cell of the invention that binds to a cell expressing a cancer associated antigen as described herein. In one aspect, the individual is a human. Non-limiting examples of disorders associated with cells expressing a cancer-associated antigen as described herein include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma), and cancers (such as hematologic malignancies or atypical cancers expressing a cancer-associated antigen as described herein).

The present invention also relates to methods for preventing, treating and/or managing a disease associated with cells expressing a cancer associated antigen as described herein, the methods comprising administering to an individual in need thereof a CAR-expressing T cell or NK a cell of the invention that binds to a cell expressing a cancer-associated antigen as described herein. In one aspect, the individual is a human.

The present invention relates to methods for preventing recurrence of a cancer associated with cells expressing a cancer associated antigen as described herein, the methods comprising administering to an individual in need thereof a CAR expressing T cell or NK cell of the invention which binds to a cell expressing a cancer-associated antigen as described herein. In one aspect, the methods comprise administering to an individual in need thereof an effective amount of a CAR-expressing T cell or NK cell described herein that binds to a cell expressing a cancer-associated antigen as described herein, in combination with an effective amount of another therapeutic agent.

Combined therapies

The CAR-expressing cell described herein can be used in combination with other known therapies and therapies. Administration "in combination" as used herein means that two (or more) different treatments are administered to an individual while the individual has a disorder, e.g., two or more treatments are administered after the patient has been diagnosed with a disorder and until cure or elimination of the disorder, or until treatment is stopped for other reasons. In some embodiments, the implementation of the introduction of one drug is still carried out when the introduction of the second begins, so that there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "joint" delivery. In other embodiments, delivery of one drug ends before delivery of another drug begins. In some embodiments, in any case, the treatment is more effective due to the combined administration. For example, the second drug is more effective, e.g., an equivalent effect is observed using less of the second drug, or the second drug reduces symptoms more than would be observed if the second drug was administered in the absence of the first drug, or the like. the situation is observed for the first drug. In some embodiments, delivery is such that the reduction in a symptom or other parameter associated with a disorder is greater than would be observed with delivery of one drug in the absence of the other. The effect of the two treatments may be partially additive, fully additive, or more than additive. The delivery may be such that the effect of the first drug delivered is still detectable when the second drug is delivered.

Expressing CAR cell described in the present description, and at least one additional drug can be administered simultaneously, in one or in separate compositions, or sequentially. For sequential administration, the CAR expressing cell described herein may be administered first, and then the additional agent may be administered, or the order of administration may be reversed.

CAR therapy and/or administration of other drugs, procedures and treatments may be carried out during periods of active impairment or during periods of remission or less active disease. CAR therapy may be given prior to another treatment, concurrently with treatment, after treatment, or during remission of the disorder.

When administered in combination, CAR therapy and administration of an additional agent (e.g., a second or third agent), or all of them, can be administered in an amount or dose that is greater than, lower, or the same as the amount or dose of each means separately, for example, as monotherapy. In certain embodiments, the administered amount or dose of CAR therapy, the additional agent (e.g., second or third agent), or all of them, is lower (e.g., at least 20%, at least 30%, at least 40% or at least 50%) than the amount or dose of each agent used alone, for example, as monotherapy. In other embodiments, the amount or dose of the CAR therapy, the additional agent (e.g., second or third agent), or all of them that results in the desired effect (e.g., cancer treatment) is lower (e.g., at least 20% , at least 30%, at least 40%, or at least 50% lower) than the amount or dose of each agent used alone, for example, as monotherapy, required to achieve the same therapeutic effect.

In the following aspects, the CAR expressing cell described herein can be used in a therapeutic regimen in combination with surgery, chemotherapy, radiation therapy, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunodestructive agents such as such as CAMPATH, anti-CD3 antibodies, or other antibody based therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiotherapy, peptide vaccine such as the peptide vaccine described in Izumoto et al . 2008 J Neurosurg 108:963-971.

In one embodiment, the CAR expressing cell described herein can be used in combination with another chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (eg, doxorubicin (eg, liposomal doxorubicin)), vinca alkaloid (eg, vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (eg, cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), antibody against immune cells (eg, alemtuzumab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), antimetabolite (including, for example, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (eg, fludarabine)), mTOR inhibitor, agonist of glucocorticoid-induced TNFR- related protein (GITR), a proteasome inhibitor (eg aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (eg lenalidomide).

The main chemicals contemplated for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injectable (Busulfex®), capecitabine (Xeloda®) , N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar® ), Cytarabine, Cytosine Arabinoside (Cytosar-U®), Injectable Liposomal Cytarabine (DepoCyt®), Dacarbazine (DTIC-Dome®), Dactinomycin (Actinomycin D, Cosmegan), Daunorubicin Hydrochloride (Cerubidine®), Injectable Liposomal Daunorubicin Citrate (DaunoXome ®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®) , tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), and darubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (yttrium-90/MX-DTPA), pentostatin, polyfeprosan 20 implant with carmustine (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6 -thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, and triazenes: uracil mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost ®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune ^TM ), ifosfamide (Mitoxana®), melphalan (Alkeran®) , chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®) , carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (also known as Actinomycin-D, Cosmegen®); melphalan (also known as L-PAM, L-sarcolysin and phenylalanine mustard, Alkeran®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (also known as CCNU, CeeNU®); cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); chlorambucil (Leukeran®); cyclophosphamide (Cytoxan® and Neosar®); dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); ifosfamide (Ifex®); prenumustine; procarbazine (Matulane®); mechlorethamine (also known as nitrogen mustard, mustine and mechlorethamine hydrochloride, Mustargen®); streptozocin (Zanosar®); thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®) and bendamustine HCl (Treanda®).

In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with fludarabine, cyclophosphamide, and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with fludarabine, cyclophosphamide, and rituximab (FCR). In embodiments, the individual has a CLL. For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have del(17p). In embodiments, the individual has a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene. In other embodiments, the individual does not have a leukemic cell containing a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene. In embodiments, fludarabine is administered at a dosage ^of about 10-50 mg/m mg / m ² ), for example, intravenously. In embodiments, cyclophosphamide is administered at a dosage of about 200-300 mg/m ² (eg, about 200-225, 225-250, 250-275, or 275-300 mg/m ² ), eg, intravenously. In embodiments, rituximab is administered at a dosage of about 400-600 mg/m ² (eg, 400-450, 450-500, 500-550, or 550-600 mg/m ² ), eg, intravenously.

In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with bendamustine and rituximab. In embodiments, the individual has a CLL. For example, the individual has a deletion in the short arm of chromosome 17 (del(17p), e.g., in a leukemia cell. In other examples, the individual does not have del(17p). In embodiments, the individual has a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region gene ( IgV _H ).In other embodiments, the individual does not have a leukemic cell containing a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene.In embodiments, bendamustine is administered at a dosage ^of about 70-110 mg/m , 80-90, 90-100, or 100-110 mg/m ² ), for example, intravenously.In embodiments, rituximab is administered at a dosage of about 400-600 mg/m ² (for example, 400-450, 450-500, 500- 550 or 550-600 mg/m ² ), for example, intravenously.

In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and/or a corticosteroid (eg, prednisone). In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). In embodiments, the individual has diffuse large B-cell lymphoma (DLBCL). In embodiments, the individual has a non-bulky stage limited DLBCL (eg, has a tumor having a size/diameter of less than 7 cm). In embodiments, the individual is treated with radiation therapy in combination with R-CHOP. For example, the subject is given R-CHOP therapy (eg, 1-6 courses, eg, 1, 2, 3, 4, 5, or 6 courses of R-CHOP) followed by radiation therapy. In some instances, an individual is given R-CHOP therapy (eg, 1-6 courses, eg, 1, 2, 3, 4, 5, or 6 courses of R-CHOP) after radiation therapy.

In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R). In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with an adjusted dose of EPOCH-R (DA-EPOCH-R). In embodiments, the individual has B-cell lymphoma, eg, aggressive B-cell lymphoma with rearranged Myc.

In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab and/or lenalidomide. Lenalidomide (( RS )-3-(4-amino-1-oxo-1,3-dihydro-2H- isoindol -2-yl)piperidine-2,6-dione) is an immunomodulator. In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab and lenalidomide. In some embodiments, the individual has follicular lymphoma (FL) or mantle cell lymphoma (MCL). In some embodiments, the individual has FL and has not been previously treated with cancer therapy. In some embodiments, the implementation of lenalidomide is administered at a dose of approximately 10-20 mg (eg, 10-15 or 15-20 mg), for example, every day. In some embodiments, rituximab is administered at a dosage of about 350-550 mg/m ² (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m ² ), e.g., intravenously.

Exemplary mTOR inhibitors include, for example, temsirolimus; ridaforolimus (formerly known as deferolimus, (1 R ,2 R ,4 S )-4-[(2 R )-2 [(1 R ,9 S ,12 S ,15 R ,16 E ,18 R ,19 R , 21 R , 23 S ,24 E ,26 E ,28 Z ,30 S ,32 S ,35 R )-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl -2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 ^4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2 -methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669 and described in PCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-bis[( 3S )-3-methylmorpholin-4-yl]pyrido[2,3- d ]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methylpyrido[2,3- d ]pyrimidin-7( 8H )-one (PF04691502, CAS 1013101-36-4) and N ² -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl- 4H -1-benzopyran-2-yl)morpholinium-4 -yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartyl-L-serine-, internal salt (SF1126, CAS 936487-67-1) (SEQ ID NO: 1262) and XL765.

Exemplary immunomodulators include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimide (CC4047) and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2 and interferon γ, CAS 951209-71-5, available from IRX Therapeutics).

Illustrative anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin and rubidomycin hydrochloride, Cerubidine®); liposomal daunorubicin (liposomal daunorubicin citrate, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence ^™ ); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and deacetylgavidomycin.

Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®) and vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleukoblastin and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

Exemplary proteasome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, ( S )-4-methyl- N -(( S )-1-((( S )-4-methyl-1-(( R )-2-methyloxiran-2-yl) -1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-(( S )-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O -methyl- N -[(2-methyl-5-thiazolyl)carbonyl]-L-seryl- O -methyl- N -[( 1S )-2-[( 2R )-2-methyl-2- oxiranil]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with brentuximab. Brentuximab is an antibody-drug conjugate between an anti-CD30 antibody and monomethylauristatin E. In embodiments, the individual has Hodgkin's lymphoma (HL), eg, relapsed or refractory HL. In embodiments, the individual has CD30+ HL. In embodiments, the subject has been subjected to an autologous stem cell transplant (ASCT). In embodiments, the subject was not subjected to ASCT. In embodiments, brentuximab is administered at a dosage of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), such as intravenously, e.g. every 3 weeks.

In embodiments, a CAR expressing cell described herein is administered to an individual in combination with brentuximab and dacarbazine, or in combination with brentuximab and bendamustine. Dacarbazine is an alkylating agent with the chemical name 5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide. Bendamustine is an alkylating agent with the chemical name 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid. In embodiments, the individual has Hodgkin's lymphoma (HL). In embodiments, the subject has not previously been treated with cancer therapy. In embodiments, the individual is at least 60 years of age, such as 60, 65, 70, 75, 80, 85, or older. In embodiments, dacarbazine is administered at a dosage of about 300-450 mg/m ² (e.g., about 300-325, 325-350, 350-375, 375-400, 400-425, or 425-450 mg/m ² ), e.g., intravenously. In embodiments, bendamustine is administered at a dosage of about 75-125 mg/m ² (eg, 75-100 or 100-125 mg/m ² , eg, about 90 mg/m ² ), eg, intravenously. In embodiments, brentuximab is administered at a dosage of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), such as intravenously, e.g. every 3 weeks.

In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a CD20 inhibitor, such as an anti-CD20 antibody (eg, a monospecific or bispecific anti-CD20 antibody), or a fragment thereof. Exemplary CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Pro131921 (Genentech). See, for example, Lim et al. haematologica. 95.1(2010):135-43.

In some embodiments, the anti-CD20 antibody comprises rituximab. Rituximab is a chimeric mouse/human IgG1 kappa monoclonal antibody that binds to CD20 and induces cytolysis of a CD20 expressing cell, for example, as described in www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl.pdf. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab. In embodiments, the individual has a CLL or SLL.

In some embodiments, the implementation of rituximab is administered intravenously, for example, as an intravenous infusion. For example, each infusion provides approximately 500-2000 mg (e.g., approximately 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900 or 1900-2000 mg) rituximab. In some embodiments, rituximab is administered at a dose of 150 mg/m ² to 750 mg/m ² , e.g., about 150-175 mg/m ² , 175-200 mg/m ² , 200-225 mg/m ² , 225- 250 mg/ ^m2 , 250-300 mg/ ^m2 , 300-325 mg/ ^m2 , 325-350 mg/ ^m2 , 350-375 mg/ ^m2 , 375-400 mg/ ^m2 , 400-425 mg / m ² , 425-450 mg / m ² , 450-475 mg / m ² , 475-500 mg / m ² , 500-525 mg / m ² , 525-550 mg / m ² , 550-575 mg / m ² , 575-600 mg/m ² , 600-625 mg/m ² , 625-650 mg/m ² , 650-675 mg/m ² or 675-700 mg/m ² where m ² indicates body surface area individual. In some embodiments, rituximab is administered at a dosing interval of at least 4 days, such as 4, 7, 14, 21, 28, 35 days or more. For example, rituximab is administered at a dosing interval of at least 0.5 weeks, such as 0.5, 1, 2, 3, 4, 5, 6, 7, 8 weeks, or more. In some embodiments, rituximab is administered at the dose and dosing interval described herein over a period of time, such as at least 2 weeks, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more. For example, rituximab is administered at the dose and dosing interval described herein for a total of at least 4 doses per course of treatment (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16 or more doses per course of treatment).

In some embodiments, the CD20 antibody comprises ofatumumab. Ofatumumab is an anti-CD20 human IgG1κ monoclonal antibody having a molecular weight of approximately 149 kDa. For example, ofatumumab is produced using transgenic mice and hybridoma technology, and is expressed and purified from a recombinant mouse (NS0) cell line. See, for example, www.accessdata.fda.gov/drugsatfda_docs/label/2009/125326lbl.pdf; and clinical trials under the identification number NCT01363128, NCT01515176, NCT01626352 and NCT01397591. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with ofatumumab. In embodiments, the individual has a CLL or SLL.

In some embodiments, ofatumumab is administered as an intravenous infusion. For example, each infusion provides approximately 150-3000 mg (eg, approximately 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 600-650 650-700 700-750 750-800 800-850 850-900 900-950 950-1000 1000-1200 1200-1400 1400-1600 1600-1800 1800- 2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800 or 2800-3000 mg) ofatumumab. In embodiments, ofatumumab is administered at an initial dosage of about 300 mg followed by 2000 mg, eg, for about 11 doses, eg, over 24 weeks. In some embodiments, ofatumumab is administered at a dosing interval of at least 4 days, such as 4, 7, 14, 21, 28, 35 days or more. For example, ofatumumab is administered at a dosing interval of at least 1 week, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28, 20, 22 , 24, 26, 28, 30 weeks or more. In some embodiments, ofatumumab is administered at the dose and dosing interval described herein over a period of time, such as at least 1 week, such as 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50, 60 weeks or more, or 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or 1, 2, 3, 4, 5 years or more. For example, ofatumumab is administered at the dose and dosing interval described herein for a total of at least 2 doses per course of treatment (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 18, 20 or more doses per course of treatment).

In some cases, the CD20 antibody includes ocrelizumab. Ocrelizumab is a humanized anti-CD20 monoclonal antibody, for example, as described in clinical trials with identification numbers NCT00077870, NCT01412333, NCT00779220, NCT00673920, NCT01194570, and Kappos et al. Lancet. 19,378(2011):1779-87.

In some cases, the anti-CD20 antibody includes veltuzumab. Veltuzumab is a humanized anti-CD20 monoclonal antibody. See, for example, clinical trials with identification numbers NCT00547066, NCT00546793, NCT01101581, and Goldenberg et al. Leuk Lymphoma. 51(5)(2010):747-55.

In some cases, the anti-CD20 antibody includes GA101. GA101 (also called obinutuzumab or RO5072759) is a humanized glyco-modified anti-CD20 monoclonal antibody. See, for example, Robak. Curr. Opin. Investig. drugs. 10.6(2009):588-96; clinical trials under identification numbers: NCT01995669, NCT01889797, NCT02229422 and NCT01414205; and www.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s000lbl.pdf.

In some instances, the CD20 antibody includes AME-133v. AME-133v (also called LY2469298 or ocaratuzumab) is a humanized anti-CD20 IgG1 monoclonal antibody with increased affinity for the FcγRIIIa receptor and enhanced antibody-dependent cellular cytotoxicity (ADCC) activity compared to rituximab. See, for example, Robak et al. BioDrugs 25.1(2011):13-25; and Forero-Torres et al. Clinic Cancer Res. 18.5(2012):1395-403.

In some instances, the CD20 antibody includes PRO131921. PRO131921 is a humanized anti-CD20 monoclonal antibody engineered to have improved FcγRIIIa binding and enhanced ADCC compared to rituximab. See, for example, Robak et al. BioDrugs 25.1(2011):13-25; and Casulo et al. Clinic Immunol. 154.1(2014):37-46; and clinical trial identification number NCT00452127.

In some cases, the CD20 antibody includes TRU-015. TRU-015 is a CD20 fusion protein formed by anti-CD20 antibody domains. TRU-015 is smaller than monoclonal antibodies but retains Fc-mediated effector functions. See, for example, Robak et al. BioDrugs 25.1(2011):13-25. TRU-015 contains an anti-CD20 single chain variable fragment (scFv) associated with the human IgG1 hinge, CH2 and CH3 domains, but lacks the CH1 and CL domains.

In some embodiments, an anti-CD20 antibody described herein is conjugated or otherwise linked to a drug, e.g., a chemotherapeutic agent (e.g., cytoxan, fludarabine, histone deacetylase inhibitor, demethylating agent, peptide vaccine, anticancer antibiotic, tyrosine kinase inhibitor, alkylating agent). agent, antitubulin or antimitotic), anti-allergy, anti-nausea (or anti-emetic), pain-relieving, or cytoprotective agent as described herein.

In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a B-cell lymphoma 2 (BCL-2) inhibitor (eg, venetoclax, also referred to as ABT-199 or GDC-0199) and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with venetoclax and rituximab. Venetoclax is a small molecule compound that inhibits the anti-apoptotic protein BCL-2. Structure of Ventoclax -[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-( 1H - pyrrolo [2,3- b ]pyridin-5-yloxy)benzamide) is shown below.

In embodiments, the individual has a CLL. In embodiments, the individual has recurrent CLL, eg, the individual has previously received cancer therapy. In embodiments, venetoclax is administered at a dosage of about 15-600 mg (e.g., 15-20, 20-50, 50-75, 75-100, 100-200, 200-300, 300-400, 400-500, or 500-600 mg), for example, every day. In embodiments, rituximab is administered at a dosage of about 350-550 mg/m ² (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m ² ), e.g., intravenously, for example, every month.

In one embodiment, cells expressing the CAR described herein are administered to an individual in combination with a molecule that reduces the Treg cell population. Methods that reduce (eg, deplete) Treg cells are known in the art and include, for example, CD25 depletion, administration of cyclophosphamide, modulation of GITR function. Without wishing to be bound by theory, it is believed that reducing the number of Treg cells in an individual prior to apheresis or prior to administration of a CAR expressing cell described herein reduces the number of unwanted immune cells (e.g., Treg) in the tumor microenvironment and reduces the individual's risk of recurrence. In one embodiment, cells expressing the CAR described herein are administered to an individual in combination with a GITR-targeting and/or GITR-modulating molecule, such as a GITR agonist and/or an anti-GITR antibody that depletes regulatory T cells (Treg ). In embodiments, cells expressing the CAR described herein are administered to an individual in combination with cyclophosphamide. In one embodiment, GITR binding molecules and/or molecules that modulate GITR functions (eg, a GITR agonist and/or Treg-depleting anti-GITR antibodies) are administered prior to administration of the CAR expressing cell. For example, in one embodiment, the GITR agonist may be administered prior to cell apheresis. In embodiments, cyclophosphamide is administered to the individual prior to administration (eg, infusion or reinfusion) of the CAR-expressing cell or prior to apheresis of the cells. In embodiments, the cyclophosphamide and the anti-GITR antibody are administered to the individual prior to administration (eg, infusion or reinfusion) of the CAR-expressing cell or prior to apheresis of the cells. In one embodiment, the individual has a cancer (eg, a solid cancer or a hematological cancer such as ALL or CLL). In one embodiment, the individual has a CLL. In embodiments, the individual has ALL. In embodiments, the subject has a solid cancer, such as the solid cancer described herein. Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as the GITR fusion protein described in US Pat. WO 2010/003118 and 2011/090754, or an anti-GITR antibody as described, for example, in US Patent No. 7025962, EP No. 1947183B1, US Patent No. 7812135, US Patent No. 8388967, US Patent No. 8591886, EP Patent No. EP 1866339, PCT Publication No. WO 2011/028683, PCT Publication No. WO 2013/039954, PCT Publication No. WO2005/007190, PCT Publication No. WO 2007/133822, PCT Publication No. WO2005/055808, PCT Publication No. WO 99/40196, PCT Publication No. WO 2001/03720, PCT Publication No. WO99/20758, PCT Publication No. WO2006/083289, PCT Publication No. WO 2005/115451, US Patent No. 7618632, and PCT Publication No. WO 2011/051726.

In one embodiment, a CAR expressing cell described herein is administered to an individual in combination with an mTOR inhibitor, eg an mTOR inhibitor described herein, eg rapalogue such as everolimus. In one embodiment, the mTOR inhibitor is administered prior to the introduction of the CAR expressing cell. For example, in one embodiment, the mTOR inhibitor can be administered prior to cell apheresis. In one embodiment, the individual has a CLL.

In one embodiment, a combination of a CAR expressing cell described herein and a kinase inhibitor described herein is administered to an individual in combination with a GITR agonist, eg, a GITR agonist described herein. In one embodiment, the GITR agonist is administered prior to the introduction of the CAR expressing cell. For example, in one embodiment, the GITR agonist may be administered prior to cell apheresis. In one embodiment, the individual has a CLL.

In one embodiment, the CAR expressing cell described herein can be used in combination with a kinase inhibitor. In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitor as described herein, e.g., a CD4/6 inhibitor, such as, for example, 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazine -1-ylpyridin-2-ylamino) -8H -pyrido[2,3- d ]pyrimidin-7-one hydrochloride (also called palbociclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, such as a BTK inhibitor as described herein, such as ibrutinib, for example. In one embodiment, the kinase inhibitor is an mTOR inhibitor, eg, an mTOR inhibitor as described herein, such as eg rapamycin, rapamycin analogs, OSI-027. An mTOR inhibitor may be, for example, an mTORC1 inhibitor and/or an mTORC2 inhibitor, such as an mTORC1 inhibitor and/or an mTORC2 inhibitor as described herein. In one embodiment, the kinase inhibitor is an MNK inhibitor, such as an MNK inhibitor as described herein, such as 4-amino-5-(4-fluoroanilino)pyrazolo[3,4- d ]pyrimidine, for example. The MNK inhibitor may be, for example, an inhibitor of MNK1a, MNK1b, MNK2a and/or MNK2b. In one embodiment, the kinase inhibitor is a dual PI3K/mTOR inhibitor as described herein, eg, PF-04695102.

In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from aloisin A; flavopyridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066); 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2R.3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one hydrochloride (P276-00); 1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazole-2-amine (RAF265); indisulama (E7070); roscovitine (CYC202); palbociclib (PD0332991);dinacyclib (SCH727965); N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide (BMS 387032); 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]benzoic acid (MLN8054); 5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519); 4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438) and XL281 (BMS908662).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g., palbociclib (PD0332991) and palbociclib is administered at a dose of approximately 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg . every day for 7-12 days of the course of 21 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of palbociclib are administered.

In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a cyclin-dependent kinase (CDK) 4 or 6 inhibitor, such as a CDK4 inhibitor or a CDK6 inhibitor described herein. In some embodiments, a CAR expressing cell described herein is administered to an individual in combination with a CDK4/6 inhibitor (e.g., an inhibitor that targets both CDK4 and CDK6), e.g., a CDK4/6 inhibitor described herein. . In one embodiment, the individual has an MCL. MCL is an aggressive malignant tumor that does not respond well to currently available therapies, i. essentially incurable. In many cases of MCL, cyclin D1 (CDK4/6 regulator) is expressed in MCL cells (eg, due to a chromosomal translocation involving the genes for immunoglobulins and cyclin D1). Thus, without being bound by theory, it is believed that MCL cells are highly sensitive to CDK4/6 inhibition with high specificity (ie, minimal effect on normal immune cells). CDK4/6 inhibitors alone have some efficacy in treating MCL, but achieve only partial remission with a high relapse rate. An exemplary CDK4/6 inhibitor is LEE011 (also referred to as ribociclib), whose structure is shown below.

Without wishing to be bound by theory, it is believed that administration of a CAR expressing cell as described herein with a CDK4/6 inhibitor (e.g., LEE011 or another CDK4/6 inhibitor as described herein) can achieve higher responsiveness, e.g., with higher remission rates and/or lower relapse rates, for example, compared to a CDK4/6 inhibitor alone.

In one embodiment, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774 and LFM-A13. In one embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2 inducible kinase (ITK) and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774 and LFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, such as ibrutinib (PCI-32765). In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a BTK inhibitor (eg, ibrutinib). In some embodiments, a CAR expressing cell described herein is administered to an individual in combination with ibrutinib (also referred to as PCI-32765). Structure of ibrutinib (1-[( 3R )-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop -2-en-1-one) is shown below.

In some embodiments, the individual has CLL, mantle cell lymphoma (MCL), or small lymphocytic lymphoma (SLL). For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have del(17p). In some embodiments, the individual has recurrent CLL or SLL, eg, the individual has had prior cancer therapy (eg, one, two, three, or four previous cancer therapies). In some embodiments, the individual has refractory CLL or SLL. In other embodiments, the individual has follicular lymphoma, such as relapsed or refractory follicular lymphoma. In some embodiments, ibrutinib is administered at a dosage of about 300-600 mg/day (e.g., about 300-350, 350-400, 400-450, 450-500, 500-550, or 550-600 mg/day, e.g., about 420 mg/day or approximately 560 mg/day), for example, orally. In some embodiments, ibrutinib is administered at a dose of approximately 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (eg 250 mg, 420 mg or 560 mg) every day for a period of time, for example every day for a course of 21 days or every day for a course of 28 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of ibrutinib are administered. Without being bound by theory, it is believed that the addition of ibrutinib enhances the T cell proliferative response and may shift T cells from the T helper-2 (Th2) phenotype to the T helper-1 (Th1) phenotype. Th1 and Th2 are helper T cell phenotypes, with Th1 relative to Th2 providing different immune response cascades. The Th1 phenotype is associated with pro-inflammatory responses, for example to kill cells such as intracellular pathogens/viruses or malignant cells, or indefinite autoimmune responses. The Th2 phenotype is associated with eosinophil accumulation and anti-inflammatory responses.

In one embodiment, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (1 R ,2 R ,4 S )-4-[(2 R )-2 [(1 R ,9 S ,12 S ,15 R ,16 E ,18 R ,19 R ,21 R , 23 S , 24 E ,26 E ,28 Z ,30 S ,32 S ,35 R )-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23, 29,35-hexamethyl-2,3,10 ,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 ^4.9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate , also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); simapimoda; (5-{2,4-bis[( 3S )-3-methylmorpholin-4-yl]pyrido[2,3- d ]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3- d ]pyrimidine-7(8 H ) -one (PF04691502); and N ² -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl- 4H -1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L- arginylglycyl-L-α-aspartyl-L-serine-, internal salt (SF1126) (SEQ ID NO: 1262) and XL765.

In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) every day for a period of time, for example, every day for a course of 21 days or every day for a course of 28 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of rapamycin are administered. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as everolimus, and everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (eg 10 mg) every day for a period of time, eg every day for a course of 28 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of everolimus are administered.

In one embodiment, the kinase inhibitor is an MNK inhibitor selected from CGP052088; 4-amino-3-(p-fluorophenylamino)pyrazolo[3,4- d ]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2 and 4-amino-5-(4-fluoroanilino)pyrazolo[3,4- d ]pyrimidine.

In some embodiments, a CAR expressing cell described herein is administered to an individual in combination with a phosphoinositide 3-kinase (PI3K) inhibitor (e.g., a PI3K inhibitor as described herein, e.g., idelicib or duvelisib) and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with idelisib and rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with duvelisib and rituximab. Idelalisib (also called GS-1101 or CAL-101; Gilead) is a small molecule compound that blocks the delta isoform of PI3K. The structure of Idelalisib (5-fluoro-3-phenyl-2-[(1 S )-1-(7 H -purin-6-ylamino) propyl]-4(3 H )-quinazolinone) is shown below.

Duvelysib (also called IPI-145; Infinity Pharmaceuticals and Abbvie) is a small molecule compound that blocks PI3K-δ,γ. The structure of duvelysib (8-chloro-2-phenyl-3-[(1S)-1-(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone) is shown below.

In embodiments, the individual has a CLL. In embodiments, the subject has recurrent CLL, eg, the subject has previously received cancer therapy (eg, previously received an anti-CD20 antibody or previously received ibrutinib). For example, an individual has a deletion on the short arm of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have del(17p). In embodiments, the individual has a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene. In other embodiments, the individual does not contain leukemic cells. Containing a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene. In embodiments, the individual has a deletion in the long arm of chromosome 11 (del(11q)). In other embodiments, the individual does not have del(11q). In embodiments, idelicib is administered at a dosage of about 100-400 mg (e.g., 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-300, 325-350 , 350-375, or 375-400 mg), eg BID. In embodiments, duvelisib is administered at a dosage of about 15-100 mg (eg, about 15-25, 25-50, 50-75, or 75-100 mg), eg, twice daily. In embodiments, rituximab is administered at a dosage of about 350-550 mg/m ² (eg, 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m ² ), such as intravenously .

In one embodiment, the kinase inhibitor is a dual phosphatidylinositol 3-kinase (PI3K) inhibitor and an mTOR inhibitor selected from 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy- 3-pyridinyl)-4-methylpyrido[2,3- d ]pyrimidin-7( 8H )-one (PF-04691502); N- [4-[[4-(dimethylamino)-1-piperidinyl]carbonyl]phenyl]- N '-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl )phenyl]urea (PF-05212384, PKI-587); 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1 H -imidazo[4,5-c]quinolin-1-yl ]phenyl}propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinoline-2 (3H)-one-maleic acid (NVP-BGT226); 3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9-isopropyl-8- methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-dimethoxyphenyl)amino]quinoxalin-3-yl]- 4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765).

In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK kinase inhibitors include, but are not limited to, crizotinib (Pfizer), ceritinib (Novartis), alectinib (Chugai), brigatinib (also referred to as AP26113; Ariad), entrectinib (Ignyta), PF-06463922 (Pfizer), TSR-011 (Tesaro) (see, for example, clinical trial ID no. NCT02048488), CEP-37440 (Teva), and X-396 (Xcovery). In some embodiments, the individual has a solid cancer, such as a solid cancer described herein, such as lung cancer.

The chemical name of crizotinib is 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine. The chemical name of ceritinib is 5-chloro- N ² -[2-isopropoxy-5-methyl-4-(4-piperidinyl)phenyl] -N ⁴ -[2-(isopropylsulfonyl)phenyl]-2,4-pyrimidinediamine. The chemical name of alectinib is 9-ethyl-6,6-dimethyl-8-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile. The chemical name of brigatinib is 5-chloro-N ² -{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N ⁴ -[2-(dimethylphosphoryl)phenyl]-2,4-pyrimidinediamine. The chemical name of entrectinib is N-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-4 -yl)amino)benzamide. The chemical name of PF-06463922 is (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno) pyrazolo[4,3-h][2,5,11]-benzoxodiazacyclotetradecine-3-carbonitrile. The chemical structure of CEP-37440 is (S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-1-methoxy-6,7,8, 9-tetrahydro-5H-benzo[7]annulen-2-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide. The chemical name of X-396 is (R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methylpiperazine-1-carbonyl)phenyl) pyridazine-3-carboxamide.

Drugs that either inhibit calcium-dependent calcineurin phosphatase (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signaling (rapamycin), can also be used. (Liu et al ., Cell 66:807-815, 1991; Henderson et al ., Immun. 73:316-321, 1991; Bierer et al ., Curr. Opin. Immun. 5:763-773, 1993). In a further aspect, the cell compositions of the present invention may be administered to a patient in conjunction with (e.g., before, concurrently, or after) bone marrow transplantation, T-cell depleting therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide and/or antibodies such as OKT3 or CAMPATH. In one aspect, the cell compositions of the present invention are administered following a B-cell depleting therapy, such as agents that react with CD20, such as Rituxan. For example, in one embodiment, individuals may be treated with standard high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, individuals are infused with increased immune cells of the present invention. In a further embodiment, the expanded cells are administered before or after surgery.

In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with an indolamine-2,3-dioxygenase (IDO) inhibitor. IDO is an enzyme that catalyzes the degradation of the amino acid L-tryptophan to kynurenine. Many cancers overexpress IDO, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer, and lung cancer. pDC, macrophages and dendritic cells (DC) can express IDO. Without being bound by theory, it is believed that a decrease in L-tryptophan (eg, catalyzed by IDO) leads to an immunosuppressive environment by inducing anergy and T cell apoptosis. Thus, without being bound by theory, it is believed that an IDO inhibitor can increase the efficiency of the CAR-expressing cell described herein, for example, by reducing the suppression or death of CAR-expressing immune cells. In embodiments, the individual has a solid tumor, e.g., a solid tumor as described herein, e.g., prostate cancer, colon cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer, and lung cancer. Exemplary IDO inhibitors include, but are not limited to, 1-methyltryptophan, indoxymod (NewLink Genetics) (see, e.g., clinical trials id no. NCT01191216; NCT01792050) and INCB024360 (Incyte Corp.) (see, e.g., clinical trials under identification numbers NCT01604889; NCT01685255).

In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a myeloid-derived suppressor cell (MDSC) modulator. MDSCs accumulate in the periphery and in the tumor region of many solid tumors. These cells suppress T-cell responses, thereby interfering with the efficiency of CAR-expressing cells. Without wishing to be bound by theory, it is believed that administration of the MDSC modulator enhances the efficiency of the CAR-expressing cells described herein. In one embodiment, the subject has a solid tumor, such as a solid tumor as described herein, such as a glioblastoma. Exemplary MDSC modulators include, but are not limited to, MCS110 and BLZ945. MCS110 is a monoclonal antibody (mAb) against macrophage colony stimulating factor (M-CSF). See, for example, clinical trials with reference number NCT00757757. BLZ945 is a small molecule inhibitor of colony stimulating factor receptor 1 (CSF1R). See, for example, Pyonteck et al. Nat. Med. 19(2013):1264-72. The structure of BLZ945 is shown below.

In some embodiments, a CAR expressing cell described herein is administered to an individual in combination with a CD19 CART cell (eg, CTL019, eg, as described in WO2012/079000, incorporated herein by reference). In some embodiments, the individual has a CD19+ lymphoma, such as CD19+ non-Hodgkin's lymphoma (NHL), CD19+ FL, or CD19+ DLBCL. In some embodiments, the individual has relapsed or refractory CD19+ lymphoma. In some embodiments, the individual is given lymphocyte-depleting chemotherapy before, simultaneously with, or after administration (eg, infusion) of CD19 CART cells. In one example, lymphocyte-depleting chemotherapy is administered to the subject prior to administration of CD19 CART cells. For example, lymphocyte-depleting chemotherapy ends 1-4 days (eg, 1, 2, 3, or 4 days) prior to infusion of CD19 CART cells. In some embodiments, multiple doses of CD19 CART cells are administered, for example, as described herein. For example, a single dose contains approximately 5×10 ⁸ CD19 CART cells. In some embodiments, lymphocyte-depleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (eg, infusion) of a CAR-expressing cell described herein, eg, a non-anti-CD19 CAR-expressing cell. In some embodiments, CD19 CART is administered to an individual prior to, concurrently with, or after administration (eg, infusion) of a non-anti-CD19 CAR-expressing cell, eg, a non-anti-CD19 CAR-expressing cell.

In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or with a combination of both an IL-15 polypeptide, and an IL-15Ra polypeptide, such as hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimeric non-covalent complex of IL-15 and IL-15Ra. hetIL-15 is described, for example, in U.S. 8124084, U.S. 2012/0177598, U.S. 2009/0082299, U.S. 2012/0141413 and U.S. 2011/0081311 included in the present description by reference. In embodiments, het-IL-15 is administered subcutaneously. In embodiments, the subject has a cancer, such as a solid cancer, such as melanoma or colon cancer. In embodiments, the subject has metastatic cancer.

In one embodiment, an agent can be administered to an individual that reduces or alleviates a side effect associated with administration of a CAR expressing cell. Side effects associated with administration of a CAR expressing cell include, but are not limited to, CRS and hemophagocytic lymphohistiocytosis (HLH), also referred to as macrophage activation syndrome (MAS). Symptoms of CRS include high fever, nausea, temporary hypotension, hypoxia, and the like. CRS may include clinical systemic signs and symptoms such as fever, fatigue, anorexia, myalgias, arthralgias, nausea, vomiting, and headache. CRS may include clinical skin signs and symptoms such as a rash. CRS may include clinical gastrointestinal signs and symptoms such as nausea, vomiting, and diarrhea. CRS may include clinical respiratory signs and symptoms such as tachypnea and hypoxemia. CRS may include clinical cardiovascular signs and symptoms such as tachycardia, increased pulse pressure, hypotension, increased cardiac output (early stage) and potentially reduced cardiac output (late stage). CRS may include clinical signs and symptoms of coagulation such as elevated d-dimer levels, hypofibrinogenemia with or without bleeding. CRS may include clinical renal signs and symptoms such as azotemia. CRS may include clinical hepatic signs and symptoms such as transaminitis and hyperbilirubinemia. CRS may include clinical neurological signs and symptoms such as headache, mental status changes, confusion, delirium, word difficulty or Frank's aphasia, hallucinations, tremors, dysmetria, gait changes, and seizures.

Thus, the methods described herein may include administering a CAR expressing cell as described herein to an individual and further administering one or more agents to control elevated levels of soluble factor due to treatment with the CAR expressing cell. In one embodiment, the soluble factor elevated in the individual is one or more of IFN-γ, TNFα, IL-2, and IL-6. In one embodiment, the factor elevated in the individual is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5, and fractalkine. Thus, the agent administered to treat this side effect may be an agent that neutralizes one or more of these soluble factors. In one embodiment, the agent that neutralizes one or more of these soluble forms is an antibody or an antigen-binding fragment thereof. Examples of such agents include, but are not limited to, a steroid (eg, a corticosteroid), a TNFα inhibitor, and an IL-6 inhibitor. An example of a TNFα inhibitor is an anti-TNFα antibody molecule such as infliximab, adalimumab, certolizumab pegol and golimumab. Another example of a TNFα inhibitor is a fusion protein such as etanercept. Small molecule TNFα inhibitors include, but are not limited to, xanthine derivatives (eg pentoxifylline) and bupropion. An example of an IL-6 inhibitor is an anti-IL-6 antibody molecule or an anti-IL-6 receptor antibody molecule such as tocilizumab (toc), tocilizumab (toc), sarilumab, elsilimoab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI -2364, CDP6038, VX30, ARGX-109, FE301 and FM101. In one embodiment, the anti-IL-6 receptor antibody molecule is tocilizumab. An example of an IL-1R inhibitor is anakinra.

In one embodiment, an agent can be administered to an individual that increases the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, such as Programmed Death 1 (PD-1), in some embodiments, may reduce the ability of a CAR-expressing cell to induce an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 and TGF-beta. Inhibition of an inhibitory molecule, for example by inhibiting the level of DNA, RNA, or protein, can optimize the efficiency of a CAR-expressing cell. In embodiments, an inhibitory nucleic acid, such as an inhibitory nucleic acid, such as dsRNA, such as siRNA or shRNA, can be used to inhibit the expression of an inhibitory molecule in a CAR-expressing cell; regularly clustered short palindromic repeats (CRISPR), transcription activator-like nuclease (TALEN) or zinc finger endonuclease (ZFN), for example, as described herein. In one embodiment, the inhibitor is shRNA. In one embodiment, the inhibitory molecule is inhibited in a CAR expressing cell. In these embodiments, the dsRNA molecule that inhibits the expression of the inhibitory molecule is linked to a nucleic acid that encodes a component, eg all of the components, of CAR. In one embodiment, an inhibitor of an inhibitory signal may be, for example, an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent may be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2, or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketed as Yervoy®; Bristol- Myers Squibb; tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675.206)) In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is an antibody or an antibody fragment that binds to CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5) In one embodiment, the agent is an antibody or antibody fragment that binds to LAG3.

PD-1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD-1, PD-L1 and PD-L2, have been shown to suppress T cell activation when bound to PD-1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8 Carter et al 2002 Eur J Immunol 32:634-43). PD-L1 is prevalent in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094) . Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1. Antibodies, antibody fragments and other inhibitors of PD-1, PD-L1 and PD-L2 are known in the art and can be used in combination with the CARs of the present invention described herein. For example, nivolumab (also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are described in US 8008449 and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are described in WO2009/101611. Pembrolizumab (formerly known as lambrolizumab and also referred to as MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are described in US 8354509 and WO2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PDL1 and inhibits ligand interaction with PD1. MDPL3280A (Genentech/Roche) is an Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are described in US Pat. No. 7,943,743 and US Publication No. 20120039906. Other PD-L1 binding agents include YW243.55.S70 (heavy and light chain variable regions are shown in SEQ ID NOs: 20 and 21 in WO2010/077634) and MDX-1 105 (also referred to as BMS-936559, and for example PD-L1 binding agents described in WO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g. described in WO2010/027827 and WO2011/066342) is a fused soluble PD-L2-Fc receptor that blocks the interaction between PD-1 and B7-H1. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), among others, for example the anti-PD-1 antibodies described in US 8609089, US 2010028330 and/or US 20120114649.

TIM-3 (T cell immunoglobulin 3) also suppresses T cell function, particularly in IFN-γ secreting CD4+ T helper 1 and CD8+ cytotoxic T cells 1, and it plays an important role in T cell elimination. Inhibition of the interaction between TIM3 and its ligands, such as galectin-9 (Gal9), phosphatidylserine (PS) and HMGB1, can increase the immune response. Antibodies, antibody fragments and other inhibitors of TIM3 and its ligands are available in the art and can be used in combination with the anti-CD19 CAR described herein. For example, antibodies, antibody fragments, small molecule compounds, or peptidic inhibitors that target TIM3 bind to the IgV domain of TIM3, inhibiting interactions with its ligands. Antibodies and peptides that inhibit TIM3 are described in WO2013/006490 and US20100247521. Other anti-TIM3 antibodies include humanized versions of RMT3-23 (described in Ngiow et al., 2011, Cancer Res, 71:3540-3551) and clone 8B.2C12 (described in Monney et al., 2002, Nature, 415:536- 541). Bispecific antibodies that inhibit TIM3 and PD-1 are described in US20130156774.

In other embodiments, the agent that increases the activity of a CAR-expressing cell is a CEACAM inhibitor (eg, an inhibitor of CEACAM-1, CEACAM-3, and/or CEACAM-5). In one embodiment, the CEACAM inhibitor is an anti-CEACAM antibody molecule. Illustrative antibodies against CEACAM-1 are described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO 2014/022332, for example monoclonal antibody 34B1, 26H7 and 5F4; or their recombinant forms, as described, for example, in US 2004/0047858, US 7132255 and WO 99/052552. In other embodiments, the anti-CEACAM antibody binds to CEACAM-5 as described, for example, in Zheng et al.PLOS One. 2010 Sep 2;5(9). pii: e12529 (DOI:10:1371/journal.pone.0021146) or cross-reacts with CEACAM-1 and CEACAM-5 as described for example in WO 2013/054331 and US 2014/0271618.

Without wishing to be bound by theory, it is believed that carcinoembryonic antigenic cell adhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, mediate, at least in part, inhibition of the antitumor immune response (see, for example, Markel et al . J Immunol . 2002 Mar 15;168(6):2803-10 Markel et al J Immunol 2006 Nov 1;177(9):6062-71 Markel et al Immunology 2009 Feb 126(2):186-200 Markel et al Cancer Immunol Immunother 2010 Feb;59(2):215-30 Ortenberg et al Mol Cancer Ther 2012 Jun;11(6):1300-10 Stern et al J Immunol 2005 Jun 1;174(11):6692-701 Zheng et al PLoS One 2010 Sep 2;5(9) pii: e12529). For example, CEACAM-1 has been described as a heterophilic ligand for TIM-3 with a role in TIM-3 mediated tolerance and T cell elimination (see e.g. WO 2014/022332; Huang, et al . (2014) Nature doi:10.1038 /nature13848). In embodiments, co-blockade of CEACAM-1 and TIM-3 has been shown to enhance the antitumor immune response in colon cancer xenograft models (see, for example, WO 2014/022332; Huang, et al . (2014), supra). In other embodiments, the implementation of the joint blockade of CEACAM-1 and PD-1 reduces the tolerance of T cells, as described, for example, in WO 2014/059251. Thus, CEACAM inhibitors can be used with other immunomodulators described herein (e.g., PD-1 inhibitors and/or TIM-3 inhibitors) to enhance the immune response against cancer, e.g., melanoma, lung cancer (e.g., NSCLC), bladder cancer, colon cancer, ovarian cancer and other malignancies as described herein.

LAG-3 (lymphocyte activation gene 3 or CD223) is a cell surface molecule expressed on activated T cells and B cells that has been shown to play a role in the elimination of CD8+ T cells. Antibodies, antibody fragments and other inhibitors of LAG-3 and its ligands are available in the art and can be used in combination with the anti-CD19 CAR described herein. For example, BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targets LAG-3. IMP701 (Immutep) is a LAG-3 antagonist antibody and IMP731 (Immutep and GlaxoSmithKline) is an anti-LAG-3 depleting antibody. Other LAG-3 inhibitors include IMP321 (Immutep), which is a recombinant fusion protein of the soluble portion of LAG-3 and Ig that binds to MHC class II molecules and activates antigen presenting cells (APCs). Other antibodies are described, for example, in WO2010/019570.

In some embodiments, an agent that increases the activity of a CAR-expressing cell may be, for example, a fusion protein containing a first domain and a second domain, where the first domain is an inhibitory molecule or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, for example, a polypeptide containing an intracellular signaling domain, as described in the present description. In some embodiments, a polypeptide that is associated with a positive signal may include a co-stimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27, and/or ICOS, and/or a primary signaling domain, e.g., from CD3-zeta, e.g. described in the present description. In one embodiment, the fusion protein is expressed by the same cell that expresses the CAR. In another embodiment, the fusion protein is expressed by a cell, such as a T cell, that does not express the CAR of the present invention.

In one embodiment, an agent that increases the activity of a CAR-expressing cell described herein is miR-17-92.

In one embodiment, the agent that enhances the activity of a CAR described herein is a cytokine. Cytokines have important functions related to T cell expansion, differentiation, survival, and homeostasis. Cytokines that can be administered to an individual receiving a CAR-expressing cell described herein include: IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, and IL-21 or their combination. In preferred embodiments, the cytokine administered is IL-7, IL-15, or IL-21, or a combination thereof. The cytokine can be administered once a day or more than once a day, for example twice a day, three times a day, or four times a day. The cytokine can be administered over more than one day, for example, the cytokine is administered over 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, a cytokine once a day for 7 days.

In embodiments, the cytokine is administered in combination with CAR-expressing T cells. The cytokine can be administered simultaneously or co-administered with CAR expressing T cells, eg, administered on the same day. The cytokine can be formulated into the same pharmaceutical composition as the CAR expressing T cells, or it can be formulated into a separate pharmaceutical composition. Alternatively, the cytokine can be administered shortly after the administration of the CAR expressing T cells, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the administration of the CAR expressing T cells. In embodiments in which the cytokine is administered on a dosing regimen lasting more than one day, the first day of the cytokine dosing regimen may be the same as the day of administration of CAR-expressing T cells, or the first day of the cytokine dosing regimen may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of CAR-expressing T cells. In one embodiment, on the first day, CAR expressing T cells are administered to the individual, and on the second day, the cytokine is administered once a day for a further 7 days. In a preferred embodiment, the cytokine to be administered in combination with the CAR expressing T cells is IL-7, IL-15 or IL-21.

In other embodiments, the cytokine is administered after a period of time following the administration of CAR expressing cells, e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months , 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of CAR-expressing cells. In one embodiment, the cytokine is administered after evaluating the individual's response to CAR-expressing cells. For example, an individual is administered CAR expressing cells in accordance with the dosage and regimens described herein. The individual's response to CAR-expressing cell therapy is assessed at 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months. , 11 months or 1 year or more after administration of CAR expressing cells using any of the methods described herein, including tumor growth inhibition, reduction in circulating tumor cells, or tumor regression. Individuals who do not show a sufficient response to therapy expressing CAR cells, you can enter the cytokine. Administration of a cytokine to an individual who has a suboptimal response to therapy with CAR-expressing cells increases the efficacy of CAR-expressing cells or antitumor activity. In a preferred embodiment, the cytokine administered following the administration of the CAR expressing cells is IL-7.

Combination with a low dose mTOR inhibitor

In one embodiment, cells expressing a CAR molecule, such as the CAR molecule described herein, are administered in combination with a low immune-boosting dose of an mTOR inhibitor.

In one embodiment, the dose of mTOR inhibitor is associated with or provides mTOR inhibition of at least 5% but not more than 90%, at least 10% but not more than 90%, at least 15% but not more than 90%, at least at least 20 but not more than 90%, at least 30 but not more than 90%, at least 40 but not more than 90%, at least 50 but not more than 90%, at least 60 but not more 90%, or at least 70 but not more than 90%.

In one embodiment, the dose of mTOR inhibitor is associated with or provides mTOR inhibition of at least 5% but not more than 80%, at least 10% but not more than 80%, at least 15% but not more than 80%, at least at least 20 but not more than 80%, at least 30 but not more than 80%, at least 40 but not more than 80%, at least 50 but not more than 80%, or at least 60 but not over 80%.

In one embodiment, the dose of mTOR inhibitor is associated with or provides mTOR inhibition of at least 5% but not more than 70%, at least 10% but not more than 70%, at least 15% but not more than 70%, at least at least 20 but not more than 70%, at least 30 but not more than 70%, at least 40 but not more than 70%, or at least 50 but not more than 70%.

In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5% but not more than 60%, at least 10% but not more than 60%, at least 15% but not more than 60%, at least at least 20 but not more than 60%, at least 30 but not more than 60%, or at least 40 but not more than 60%.

In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5% but not more than 50%, at least 10% but not more than 50%, at least 15% but not more than 50%, at least at least 20 but not more than 50%, at least 30 but not more than 50%, or at least 40 but not more than 50%.

In one embodiment, the dose of mTOR inhibitor is associated with or provides mTOR inhibition of at least 5% but not more than 40%, at least 10% but not more than 40%, at least 15% but not more than 40%, at least at least 20 but not more than 40%, at least 30 but not more than 40%, or at least 35 but not more than 40%.

In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5% but not more than 30%, at least 10% but not more than 30%, at least 15% but not more than 30%, at least at least 20 but not more than 30%, or at least 25 but not more than 30%.

In one embodiment, the dose of mTOR inhibitor is associated with or provides mTOR inhibition of at least 1%, 2%, 3%, 4%, or 5%, but not more than 20%, at least 1%, 2%, 3%, 4%, or 5%, but not more than 30%, at least 1, 2, 3, 4 or 5, but not more than 35, at least 1, 2, 3, 4 or 5, but not more than 40%, or at least 1, 2, 3, 4 or 5, but not more than 45%.

In one embodiment, the dose of mTOR inhibitor is associated with or provides mTOR inhibition of at least 1, 2, 3, 4, or 5, but not more than 90%.

[0658] As described herein, the degree of mTOR inhibition can be expressed as the degree of inhibition of P70 S6 kinase, for example, the degree of mTOR inhibition can be determined by the level of decrease in P70 S6 kinase activity, for example, the decrease in P70 S6 kinase substrate phosphorylation. The level of mTOR inhibition can be assessed by the method described herein, for example, using the Boulay assay, or measuring levels of phosphorylated S6 using Western blotting.

EXAMPLE mTOR INHIBITORS

As used herein, the term "mTOR inhibitor" refers to a compound or ligand, or a pharmaceutically acceptable salt thereof, that inhibits mTOR kinase in a cell. In one embodiment, the mTOR inhibitor is an allosteric inhibitor. In one embodiment, the mTOR inhibitor is a catalytic inhibitor.

Allosteric mTOR inhibitors include the neutral tricyclic compound rapamycin (sirolimus), rapamycin-related compounds, which are compounds that have structural and functional similarities to rapamycin, including, for example, rapamycin derivatives, rapamycin analogs (also called rapalogues), and other macrolide compounds that inhibit mTOR activity.

[0661] Rapamycin is a known macrolide antibiotic produced by Streptomyces hygroscopicus having the structure shown in Formula A.

See, for example, McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S. L., et al., J. Am. Chem. soc. (1991) 113: 7433; US patent No. 3929992. There are various numbering schemes proposed for rapamycin. To avoid confusion, when specific analogs of rapamycin are referred to herein, the names are relative to rapamycin using the formula A numbering scheme.

Rapamycin analogs useful in the context of the invention are, for example, O-substituted analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin is replaced by OR ₁ , where R ₁ is hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl or aminoalkyl; for example RAD001, also known as everolimus, as described in US 5665772 and WO94/09010, the contents of which are incorporated by reference. Other suitable analogs of rapamycin include analogs of rapamycin substituted at position 26 or 28. The analog of rapamycin may be an epimer of the analog mentioned above, in particular the epimer of the analog substituted at position 40, 28 or 26, and it may optionally be further hydrogenated, for example , as described in US 6015815, WO95/14023 and WO99/15530, the contents of which are incorporated herein by reference , e.g. which is incorporated by reference, eg AP23573, also known as ridaphorolimus.

Examples of rapamycin analogues suitable for use in the present invention from US 5665772 include, but are not limited to, 40-O-benzylrapamycin, 40-O-(4'-hydroxymethyl)benzylrapamycin, 40-O-[4'-(1 ,2-dihydroxyethyl)]benzylrapamycin, 40-O-allylrapamycin, 40-O-[3'-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2'-en- 1'-yl]-rapamycin, (2'E,4'S)-40-O-(4',5'-dihydroxypent-2'-en-1'-yl)rapamycin, 40-O-(2-hydroxy) ethoxycarbonylmethylrapamycin, 40-O-(2-hydroxy)ethylrapamycin, 40-O-(3-hydroxy)propylrapamycin, 40-O-(6-hydroxy)hexylrapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethylrapamycin , 40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methylrapamycin, 40-O-[(2S)-2,3-dihydroxyprop-1-yl]rapamycin, 40-O-(2- acetoxy)ethylrapamycin, 40-O-(2-nicotinoyloxy)ethylrapamycin, 40-O-[2-(N-morpholino)acetoxy]ethylrapamycin, 40-O-(2-N-imidazolylacetoxy)ethylrapamycin, 40-O-[2 -(N-methyl-N'-piperazinyl)acetoxy]ethylrapamycin, 39-O-desmethyl-39,40-O,O-ethylenerapamycin, (26R)-26-dihydro-40-O-(2-hydroxy)ethylrapamycin, 40-O-( 2-aminoethyl)rapamycin, 40-O-(2-acetaminoethyl)rapamycin, 40-O-(2-nicotinamidoethyl)rapamycin, 40-O-(2-(N-methylimidazo-2'-ylcarbethoxamido)ethyl)rapamycin, 40 -O-(2-ethoxycarbonylaminoethyl)rapamycin, 40-O-(2-tolylsulfonamidoethyl)rapamycin and 40-O-[2-(4',5'-dicarboethoxy-1',2',3'-triazole-1' -yl)ethyl]rapamycin.

Other rapamycin analogs useful in the present invention are analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin and/or the hydroxyl group at position 28 is replaced by a hydroxyester group, such as the rapamycin analogs described in US RE44768, such as temsirolimus.

Other analogs of rapamycin useful in the present invention include analogs in which the methoxy group at position 16 is replaced by another substituent, preferably (optionally hydroxy-substituted) alkynyloxy, benzyl, orthomethoxybenzyl or chlorobenzyl and/or where the methoxy group at position 39 is removed along with carbon 39 , so that the cyclohexyl ring of rapamycin becomes a cyclopentyl ring lacking the methoxy group at position 39; for example, as described in WO95/16691 and WO96/41807, the contents of which are incorporated by reference. The analogs can be further modified such that the hydroxy at the 40 position of rapamycin is alkylated and/or the 32-carbonyl is reduced.

Rapamycin analogs from WO95/16691 include, but are not limited to, 16-demethoxy-16-(pent-2-ynyl)oxyrapamycin, 16-demethoxy-16-(but-2-ynyl)oxyrapamycin, 16-demethoxy-16-( propargyl)oxyrapamycin, 16-demethoxy-16-(4-hydroxybut-2-ynyl)oxyrapamycin, 16-demethoxy-16-benzyloxy-40-O-(2-hydroxyethyl)rapamycin, 16-demethoxy-16-benzyloxyrapamycin, 16- demethoxy-16-orthomethoxybenzylrapamycin, 16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxyrapamycin, 39-demethoxy-40-deoxy-39-formyl-42-nor-rapamycin, 39- demethoxy-40-deoxy-39-hydroxymethyl-42-norrapamycin, 39-demethoxy-40-deoxy-39-carboxy-42-norrapamycin, 39-demethoxy-40-deoxy-39-(4-methylpiperazin-1-yl)carbonyl -42-norrapamycin, 39-demethoxy-40-deoxy-39-(morpholin-4-yl)carbonyl-42-norrapamycin, 39-demethoxy-40-deoxy-39-[N-methyl,N-(2-pyridine- 2-ylethyl)]carbamoyl-42-norrapamycin; and 39-demethoxy-40-deoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-norrapamycin.

Rapamycin analogs from WO96/41807 include, but are not limited to, 32-deoxorapamycin, 16-O-pent-2-ynyl-32-deoxorapamycin, 16-O-pent-2-ynyl-32-deoxo-40-O-( 2-hydroxyethyl)rapamycin, 16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)rapamycin, 32(S)-dihydro-40-O-(2-methoxy )ethylrapamycin; and 32(S)-dihydro-40-O-(2-hydroxyethyl)rapamycin.

Another suitable analog of rapamycin is mirolimus, as described in US2005/0101624, the content of which is incorporated by reference.

RAD001, otherwise known as everolimus (Afinitor®), has the chemical name (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18- dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21, 23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30,3,1,04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20- pentaon

The following examples of allosteric mTOR inhibitors include sirolimus (rapamycin, AY-22989), 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin (also called temsirolimus or CCI-779), and ridaforolimus (AP-23573/MK -8669). Other examples of allosteric mTor inhibitors include zotarolimus (ABT578) and umirolimus.

Alternatively or additionally, catalytic ATP-competing mTOR inhibitors have been found to target the mTOR kinase domain directly and target both mTORC1 and mTORC2. They are also more potent mTORC1 inhibitors than allosteric mTOR inhibitors such as rapamycin because they modulate rapamycin-resistant mTORC1 activity such as 4EBP1-T37/46 phosphorylation and cap-dependent translation.

Catalytic inhibitors include: BEZ235 or 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinoline-1- yl)phenyl]propionitrile, or its monotosylate form. The synthesis of BEZ235 is described in WO2006/122806; CCG168 (otherwise known as AZD-8055, Chresta, C.M., et al., Cancer Res, 2010, 70(1), 288-298), which has the chemical name {5-[2,4-bis-((S) -3-methylmorpholin-4-yl)pyrido[2,3d]pyrimidin-7-yl]-2-methoxyphenyl}methanol; 3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide (WO09104019); 3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidine-4-amine (WO10051043 and WO2013023184); N-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxalin-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide (WO07044729 and WO12006552); PKI-587 (Venkatesan, A.M., J. Med.Chem., 2010, 53, 2636-2645), which has the chemical name 1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3- [4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl]urea; GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43), which has the chemical name 2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6 -quinolinyl]-3-pyridinyl}benzenesulfonamide; 5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (WO10114484); (E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl -1H-imidazo[4,5-c]quinoline-2(3H)-ylidene)cyanamide (WO12007926).

Further examples of catalytic mTOR inhibitors include 8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethylphenyl)-1,3-dihydroimidazo[4,5- c]quinolin-2-one (WO2006/122806) and Ku-0063794 (Garcia-Martinez JM, et al., Biochem J., 2009, 421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)). WYE-354 is another example of a catalytic mTor inhibitor (Yu K, et al. (2009). Biochemical, Cellular, and In vivo Activity of Novel ATP-Competitive and Selective Inhibitors of the Mammalian Target of Rapamycin. Cancer Res. 69(15) : 6232-6240).

mTOR inhibitors useful in accordance with the present invention also include prodrugs, derivatives, pharmaceutically acceptable salts or analogues of any of the above agents.

mTOR inhibitors such as RAD001 can be formulated for delivery based on methods well known in the art based on the specific dosages described herein. In particular, US Pat. No. 6,004,973 (incorporated herein by reference) provides examples of formulations that can be used with the mTOR inhibitors described herein.

ASSESSMENT OF mTOR INHIBITION

mTOR phosphorylates the P70 S6 kinase, thereby activating the P70 S6 kinase and allowing it to phosphorylate the substrate. The degree of mTOR inhibition can be expressed as the degree of inhibition of P70 S6 kinase, for example, the degree of mTOR inhibition can be determined by the level of decrease in P70 S6 kinase activity, for example, by the decrease in P70 S6 kinase substrate phosphorylation. You can determine the level of mTOR inhibition by measuring the activity of P70 S6 kinase (the ability of P70 S6 kinase to phosphorylate the substrate), in the absence of an inhibitor, for example, before the administration of the inhibitor, and in the presence of the inhibitor, or after the administration of the inhibitor. The level of P70 S6 kinase inhibition provides the level of mTOR inhibition. Thus, if P70 S6 kinase is inhibited by 40%, mTOR activity, as measured by P70 S6 kinase activity, is inhibited by 40%. The degree or level of inhibition referred to herein is the average level of inhibition over a dosing period. By way of example, if the inhibitor is administered once a week, the level of inhibition is given as the average level of inhibition over that interval, ie a week.

Boulay et al., Cancer Res, 2004, 64:252-61, incorporated herein by reference, describes an assay that can be used to assess the level of mTOR inhibition (referred to herein as the Boulay assay). In one embodiment, the assay is based on measuring P70 S6 kinase activity from biological samples before and after administration of an mTOR inhibitor, such as RAD001. Sampling can be performed at predetermined time points after administration of the mTOR inhibitor, for example, 24, 48 and 72 hours after administration. You can use biological samples, for example, from the skin or peripheral blood mononuclear cells (PBMC). Extracts of total protein are obtained from the samples. The P70 S6 kinase is isolated from the protein extracts by immunoprecipitation using an antibody that specifically recognizes the P70 S6 kinase. The activity of the isolated P70 S6 kinase can be measured in an in vitro kinase assay. The isolated kinase can be incubated with 40S ribosomal subunits (which are the endogenous substrate of P70 S6 kinase) and ^32P gamma under conditions that allow the substrate to be phosphorylated. The reaction mixture can then be separated on an SDS-PAGE gel and the ³² P signal can be analyzed using a FluoroImager. The ³² P signal, corresponding to the size of the 40S ribosomal subunit, indicates a phosphorylated substrate and P70 S6 kinase activity. The increase and decrease in kinase activity can be calculated by quantifying the area and intensity of the ³² P signal at the phosphorylated substrate (e.g., using ImageQuant, Molecular Dynamics), assigning arbitrary unit values to the quantified signal, and comparing post-administration values with pre-administration values or with a reference value. . For example, percent inhibition of kinase activity can be calculated using the following formula: 1-(value obtained after administration/value obtained before administration)×100. As described above, the degree or level of inhibition referred to herein is the average level of inhibition over a dosing interval.

Methods for assessing kinase activity, for example P70 S6 kinase activity, are also provided in US 7,727,950, incorporated herein by reference.

The level of mTOR inhibition can also be assessed by the change in the ratio of PD1 negative to PD1 positive T cells. T cells from peripheral blood can be identified as PD1 positive or negative by methods known in the art.

Low dose mTOR inhibitors

The methods described herein use low immune-boosting doses of mTOR inhibitors, eg allosteric mTOR inhibitors, including rapalogues such as RAD001. In contrast, inhibitor levels that completely or substantially completely inhibit the mTOR cascade are immunosuppressive, and are used, for example, to prevent organ transplant rejection. In addition, high doses of rapalogues, which completely inhibit mTOR, also inhibit the growth of tumor cells and are used to treat various malignant tumors (see, for example, Antineoplastic effects of mammalian target of rapamycine inhibitors. Salvadori M. World J Transplant. 2012 Oct 24; Finn RS Liver Cancer 2012 Nov;1(3-4):247-256 Emerging Signaling Pathways in Hepatocellular Carcinoma Moeini A, Cornellà H, Villanueva A. Liver Cancer, 2012 Sep;1(2):83-93; 2013 Sep 20. Role of natural and adaptive immunity in renal cell carcinoma response to VEGFR-TKIs and mTOR inhibitor Santoni M, Berardi R, Amantini C, Burattini L, Santini D, Santoni G, Cascinu S. Int J Cancer. 2013 Oct 2).

The present invention is based, at least in part, on the surprising discovery that doses of mTOR inhibitors that are significantly lower than doses used in current clinical settings have an improved effect in terms of enhancing the immune response in humans and increasing the ratio of negative for PD-1 T cells/PD-1 positive T cells. It was surprising that low doses of mTOR inhibitors, causing only partial inhibition of mTOR activity, are able to effectively increase the human immune response and increase the ratio of PD-1 negative T cells/PD-1 positive T cells.

Alternatively or additionally, without wishing to be bound by any theory, it is believed that a low immune-enhancing dose of an mTOR inhibitor can increase the numbers of naive T cells, eg, at least temporarily, eg, compared to an untreated individual. Alternatively or additionally, again without wishing to be bound by theory, it is believed that such treatment with an mTOR inhibitor, after a sufficient period of time or sufficient dosing, results in one or more of the following:

increased expression of one or more of the following markers: CD62L ^high , CD127 ^high , CD27 ⁺ and BCL2, for example, on T-memory cells, for example, progenitors of T-memory cells;

downregulation of KLRG1, eg on memory T cells, eg memory T cell precursors; and

an increase in memory T cell precursors, eg, cells with one or a combination of the following: increased CD62L ^high , increased CD127 ^high , increased CD27 ⁺ , decreased KLRG1, and increased BCL2;

and where any of the changes described above occurs, for example, at least temporarily, for example, compared with an individual without treatment (Araki, K et al. (2009) Nature 460:108-112). Memory T cell progenitors are memory T cells that are at an early stage in the differentiation program. For example, memory T cells have one or more of the following: increased CD62L ^high , increased CD127 ^high , increased CD27 ⁺ , decreased KLRG1, and/or increased BCL2.

In one embodiment, the invention relates to a composition or dosage form of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g. , associated with: the level of mTOR inhibition, which is not associated with complete or significant immune suppression, but is associated with increased immune response.

An mTOR inhibitor such as an allosteric mTOR inhibitor such as rapalogue, rapamycin or RAD001 or a catalytic mTOR inhibitor may be provided in a sustained release formulation. Any of the compositions or unit dosage forms described herein may be provided in a sustained release formulation. In some embodiments, a sustained release formulation has lower bioavailability than an immediate release formulation. For example, in embodiments to achieve a similar therapeutic effect to an immediate release formulation, the sustained release formulation will have about 2 to about 5, about 2.5 to about 3.5, or about 3 times the amount of inhibitor than provided in the immediate release formulation.

In one embodiment, immediate release formulations are provided, such as RAD001, typically used for one administration per week, having 0.1 to 20, 0.5 to 10, 2.5 to 7.5, 3 to 6 or about 5 mg per unit dosage form. For once weekly administration, these immediate release formulations correspond to sustained release formulations having 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9 to 18, respectively, or approximately 15 mg of an mTOR inhibitor such as an allosteric mTOR inhibitor such as rapamycin or RAD001. In embodiments, both forms are administered once per week.

In one embodiment, immediate release formulations are provided, such as RAD001 formulations typically used for one administration per day, having 0.005 to 1.5, 0.01 to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to 1.5, 0.5 to 1.5, 0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to 0.6, or about 0.5 mg per unit dosage form. For once daily administration, these immediate release forms correspond to sustained release forms having, respectively, 0.015 to 4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 up to 4.5, from 0.9 to 4.5, from 1.2 to 4.5, from 1.5 to 4.5, from 1.8 to 4.5, from 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5, 0.9 to 1.8, or about 1.5 mg of an mTOR inhibitor such as an allosteric mTOR inhibitor such as rapamycin or RAD001. For once weekly administration, these immediate release forms correspond to sustained release forms having, respectively, 0.1 to 30, 0.2 to 30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30, 14 to 30, 16 to 30, 20 to 30, 6 to 12, or about 10 mg of an mTOR inhibitor, e.g. an allosteric mTOR inhibitor, e.g. rapamycin or RAD001.

In one embodiment, immediate release forms are contemplated, such as RAD001 forms typically used for once daily administration, having 0.01 to 1.0 mg per unit dosage form. For once daily administration, these immediate release forms correspond to sustained release forms having, respectively, 0.03 to 3 mg of an mTOR inhibitor, eg an allosteric mTOR inhibitor, eg rapamycin or RAD001. For once weekly administration, these immediate release forms correspond to sustained release forms having, respectively, 0.2 to 20 mg of an mTOR inhibitor, eg an allosteric mTOR inhibitor, eg rapamycin or RAD001.

In one embodiment, immediate release forms are provided, eg RAD001 forms, typically used for one administration per week, having 0.5 to 5.0 mg per unit dosage form. For once weekly administration, these immediate release forms correspond to sustained release forms having, respectively, 1.5 to 15 mg of an mTOR inhibitor, eg an allosteric mTOR inhibitor, eg rapamycin or RAD001.

As described above, one target of the mTOR cascade is the P70 S6 kinase. Thus, doses of mTOR inhibitors that are useful in the methods and compositions described herein are doses that are sufficient to achieve no more than 80% inhibition of P70S6 kinase activity relative to P70S6 kinase activity in the absence of an mTOR inhibitor, e.g. , when measured using the assay described herein, eg the Boulay assay. In a further aspect, the invention relates to an amount of mTOR inhibitor sufficient to achieve no more than 38% inhibition of P70 S6 kinase activity relative to P70 S6 kinase activity in the absence of the mTOR inhibitor.

In one aspect, the dose of an mTOR inhibitor useful in the methods and compositions of the invention is sufficient to achieve, for example, when administered to a human, 90 +/- 5% (i.e., 85-95%), 89 +/- 5% , 88 +/- 5%, 87 +/- 5%, 86 +/- 5%, 85 +/- 5%, 84 +/- 5%, 83 +/- 5%, 82 +/- 5%, 81 +/- 5%, 80 +/- 5%, 79 +/- 5%, 78 +/- 5%, 77 +/- 5%, 76 +/- 5%, 75 +/- 5%, 74 +/- 5%, 73 +/- 5%, 72 +/- 5%, 71 +/- 5%, 70 +/- 5%, 69 +/- 5%, 68 +/- 5%, 67 + /- 5%, 66 +/- 5%, 65 +/- 5%, 64 +/- 5%, 63 +/- 5%, 62 +/- 5%, 61 +/- 5%, 60 +/ - 5%, 59 +/- 5%, 58 +/- 5%, 57 +/- 5%, 56 +/- 5%, 55 +/- 5%, 54 +/- 5%, 54 +/- 5%, 53 +/- 5%, 52 +/- 5%, 51 +/- 5%, 50 +/- 5%, 49 +/- 5%, 48 +/- 5%, 47 +/- 5 %, 46 +/- 5%, 45 +/- 5%, 44 +/- 5%, 43 +/- 5%, 42 +/- 5%, 41 +/- 5%, 40 +/- 5% , 39 +/- 5%, 38 +/- 5%, 37 +/- 5%, 36 +/- 5%, 35 +/- 5%, 34 +/- 5%, 33 +/- 5%, 32 +/- 5%, 31 +/- 5%, 30 +/- 5%, 29 +/- 5%, 28 +/- 5%, 27 +/- 5%, 26 +/- 5%, 25 +/- 5%, 24 +/- 5%, 23 +/- 5%, 22 +/- 5%, 21 +/- 5%, 20 +/- 5%, 19 +/- 5%, 18 + /- 5%, 17 +/- 5%, 16 +/- 5%, 15 +/- 5%, 14 +/- 5%, 13 +/- 5%, 12 +/- 5%, 11 +/- 5% or 10 +/- 5%, inhibition P70 S6 kinase activity, for example, when measured using the assay described herein, for example, the Boulay assay.

P70 S6 kinase activity in an individual can be measured using methods known in the art, for example, according to the methods described in US Pat. activity in vitro .

As used herein, the term "about" in relation to the dose of an mTOR inhibitor refers to up to +/-10% variability in the amount of mTOR inhibitor, but may include no variability in said dose.

In some embodiments, the invention relates to methods comprising administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage within a predetermined residual level. In some embodiments, the residual level is significantly lower than the residual levels associated with dosing regimens used in organ transplant and cancer patients. In one embodiment, an mTOR inhibitor, such as RAD001 or rapamycin, is administered to achieve a residual level that is less than 1/2, 1/4, 1/10, or 1/20 of the residual level that results in an immunosuppressive or anti-cancer effect. In one embodiment, an mTOR inhibitor, such as RAD001 or rapamycin, is administered to achieve a residual level that is less than 1/2, 1/4, 1/10, or 1/20 of the residual level indicated on the FDA-approved package insert for use for immunosuppression or anticancer indications.

In one embodiment, the method described herein comprises administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a desired residual level of 0.1 to 10 ng/mL, 0.1 to 5 ng/mL ml, 0.1 to 3 ng/ml, 0.1 to 2 ng/ml, or 0.1 to 1 ng/ml.

In one embodiment, the method described herein comprises administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a desired residual level of 0.2 to 10 ng/mL, 0.2 to 5 ng/mL ml, 0.2 to 3 ng/ml, 0.2 to 2 ng/ml, or 0.2 to 1 ng/ml.

In one embodiment, the method described herein comprises administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a desired residual level of 0.3 to 10 ng/mL, 0.3 to 5 ng/mL ml, 0.3 to 3 ng/ml, 0.3 to 2 ng/ml, or 0.3 to 1 ng/ml.

In one embodiment, the method described herein comprises administering to an individual an mTOR inhibitor, such as an allosteric inhibitor such as RAD001, at a dosage that provides a desired residual level of 0.4 to 10 ng/mL, 0.4 to 5 ng/mL ml, 0.4 to 3 ng/ml, 0.4 to 2 ng/ml, or 0.4 to 1 ng/ml.

In one embodiment, the method described herein comprises administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a desired residual level of 0.5 to 10 ng/mL, 0.5 to 5 ng/mL ml, 0.5 to 3 ng/ml, 0.5 to 2 ng/ml, or 0.5 to 1 ng/ml.

In one embodiment, the method described herein comprises administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a desired residual level of 1 to 10 ng/mL, 1 to 5 ng/mL, 1 up to 3 ng/ml or from 1 to 2 ng/ml.

As used in the context of the invention, the term "residual level" refers to the plasma drug concentration immediately prior to the next dose or the trough drug concentration between two doses.

In some embodiments, the desired residual level of RAD001 is in the range of approximately 0.1 to 4.9 ng/mL. In one embodiment, the desired residual level is less than 3 ng/ml, for example less than 0.3 or less than 3 ng/ml. In one embodiment, the desired residual level is less than 3 ng/ml, for example, is from about 0.3 or less to 1 ng/ml.

In a further aspect, an mTOR inhibitor other than RAD001 may be used within the scope of the invention in an amount that is associated with a target residual level that is biologically equivalent to the specified target residual level for RAD001. In one embodiment, the target residual level for an mTOR inhibitor other than RAD001 is a level that provides the same level of mTOR inhibition (e.g., as measured by the method described herein, e.g., P70 S6 kinase inhibition) as the residual level RAD001 described in the present description.

Pharmaceutical compositions: mTOR inhibitors

In one aspect, the present invention relates to pharmaceutical compositions containing an mTOR inhibitor, such as an mTOR inhibitor as described herein, formulated for use in combination with the CAR cells described herein.

In some embodiments, the mTOR inhibitor is formulated to be administered in combination with an additional agent, for example, as described herein.

In particular, the compounds of the invention may be administered in therapeutically effective amounts as described above by any of the usual and acceptable routes known in the art, either alone or in combination with one or more drugs.

Pharmaceutical formulations can be prepared using conventional dissolution and mixing techniques. For example, the bulk drug (eg, mTOR inhibitor or stabilized form of the compound (eg, complexed with a cyclodextrin derivative or other known complexing agent)) is dissolved in a suitable solvent in the presence of one or more of the excipients described herein. The mTOR inhibitor is typically formulated into pharmaceutical dosage forms to allow for easy control of drug dosing and to provide the patient with a convenient and easy to use product.

The compounds of the invention can be administered as pharmaceutical compositions in the usual way, in particular enterally, for example orally, for example in the form of tablets or capsules, or parenterally, for example in the form of injectable solutions or suspensions, topically, for example in the form of lotions, gels, ointments or creams, or in nasal or suppository form. When an mTOR inhibitor is administered in combination with (either simultaneously or separately) another agent as described herein, in one aspect, both components can be administered by the same route (eg, parenterally). Alternatively, the other agent may be administered in a different route than the route of administration of the mTOR inhibitor. For example, an mTOR inhibitor may be administered orally and another agent may be administered parenterally.

DELAYED RELEASE

mTOR inhibitors, such as allosteric mTOR inhibitors or catalytic mTOR inhibitors described herein, can be provided as pharmaceutical formulations in the form of oral solid dosage forms containing an mTOR inhibitor described herein, such as rapamycin or RAD001, which satisfy stability requirements. product and/or have favorable pharmacokinetic properties relative to immediate release (IR) tablets, such as reduced mean peak plasma concentrations, reduced inter-patient and intra-patient variability in drug absorption and maximum plasma concentration, reduced C _max ratio /C _min and/or reduced nutritional effects. The provided pharmaceutical formulations may allow for more precise dose titration and/or a reduction in the incidence of adverse events, thus allowing safer treatment of patients with an mTOR inhibitor as described herein, such as rapamycin or RAD001.

In some embodiments, the present invention relates to stable sustained release formulations of an mTOR inhibitor described herein, such as rapamycin or RAD001, which are multiparticulate systems and may have functional layers and coatings.

The term "sustained release multiparticulate formulation" as used herein refers to a formulation that releases an mTOR inhibitor described herein, such as rapamycin or RAD001, over an extended period of time, such as for at least 1, 2, 3, 4, 5 or 6 hours. A sustained release formulation may contain matrices and coatings of specialty excipients, for example as described herein, which are formulated so that the active ingredient is available for an extended period of time after ingestion.

The term "sustained release" may be used interchangeably with the terms "sustained release" (SR) or "extended release". The term "sustained release" refers to a pharmaceutical composition that does not release the active substance immediately after oral administration, but releases it over an extended period of time, as defined in Ph. Eur. (7th Edition) on Tablets and Capsules and USP <1151> General Chapter on Pharmaceutical Dosage Forms. The term "immediate release" (IR), as used herein, refers to a pharmaceutical formulation that releases 85% of the active drug in less than 60 minutes, as defined by "Guidance for Industry:" Dissolution Testing of Immediate Release Solid Oral Dosage Forms" (FDA CDER, 1997). In some embodiments, the term "immediate release" means the release of everolimus from tablets within 30 minutes, for example, as measured in a dissolution assay described herein.

Stable sustained release formulations of the mTOR inhibitor described herein, such as rapamycin or RAD001, can be characterized by in vitro release profile using assays known in the art such as dissolution assay as described herein: dissolution vessel filled 900 ml of phosphate buffer, pH 6.8, containing sodium dodecyl sulfate 0.2% at 37°C, and dissolution is carried out using the paddle method at 75 rpm in accordance with USP according to USP article on testing 711, and article Ph.Eur. about testing 2.9.3, respectively.

In some embodiments, sustained release formulations of an mTOR inhibitor described herein, such as rapamycin or RAD001, release the mTOR inhibitor in an in vitro release assay according to the following release characteristics:

0.5 h: <45%, or <40, e.g. <30%

1 hour: 20-80% e.g. 30-60%

2 hours: >50%, or >70%, e.g. >75%

3 hours: >60%, or >65%, eg >85%, eg >90%.

In some embodiments, stable sustained release formulations of the mTOR inhibitor described herein, e.g., rapamycin or RAD001, release 50% of the mTOR inhibitor for at least 45, 60, 75, 90, 105 min, or 120 min in a dissolution assay in vitro .

Delivery methods via biopolymers

In some embodiments, one or more CAR-expressing cells, as described herein, can be administered or delivered to an individual via a biopolymer scaffold, such as a biopolymer implant. Biopolymer scaffolds can promote or enhance the delivery, expansion and/or distribution of the CAR-expressing cells described herein. The biopolymer scaffold contains a biocompatible (eg, does not substantially induce an inflammatory or immune response) and/or biodegradable polymer, which may be naturally occurring or synthetic.

Examples of suitable biopolymers include, but are not limited to, agar, agarose, alginate, alginate/calcium phosphate cement (CPC), beta-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose ), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid, collagen, hydroxyapatite, 3-hydroxybutyrate-3-hydroxyhexanoate copolymer) (PHBHHx), polylactide, polycaprolactone (PCL), lactide-glycolide copolymer (PLG), polyethylene oxide (PEO), lactic acid copolymer (PLGA), polypropylene oxide (PPO), polyvinyl alcohol (PVA), silk, soy protein and soy protein isolate, alone or in combination with any other polymer composition, in any concentration and in any ratio . The biopolymer can be enhanced or modified with adhesion or migration enhancing molecules, such as collagen mimetic peptides that bind to the collagen receptor on lymphocytes, and/or stimulatory molecules to increase delivery, expansion, or function, such as antitumor activity, of the cells to be delivered. . The biopolymer scaffold may be injectable, for example in the form of a gel or semi-solid, or it may be a solid composition.

In some embodiments, the CAR-expressing cells described herein are seeded onto a biopolymer scaffold prior to delivery to an individual. In embodiments, the biopolymer scaffold further comprises one or more additional drugs described herein (e.g., another CAR-expressing cell, antibody, or small molecule), or agents that enhance the activity of the CAR-expressing cell, e.g., incorporated or conjugated with scaffold biopolymers. In embodiments, the biopolymer scaffold is injected, for example, into the tumor, or surgically implanted into the tumor or close enough to the tumor to mediate an antitumor effect. Additional examples of biopolymer compositions and methods for their delivery are described in Stephan et al.,Nature Biotechnology, 2015, 33:97-101; and WO2014/110591.

Pharmaceutical compositions and methods of treatment

Pharmaceutical compositions of the present invention may contain a CAR expressing cell, for example a plurality of CAR expressing cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide); and preservatives. In one aspect, the compositions of the present invention are formulated for intravenous administration.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease being treated (or prevented). The number and frequency of administrations will be determined by such factors as the condition of the patient and the type and severity of the patient's disease, although suitable dosages may be determined in clinical trials.

In one embodiment, the pharmaceutical composition is substantially free of impurities, e.g., no detectable levels of impurities, e.g., selected from the group consisting of endotoxin, mycoplasma, replication-competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag , anti-CD3/anti-CD28 coated residual beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cells or plasmid components, bacteria and fungi. In one embodiment, the bacterium is at least one bacterium selected from the group consisting of Alcaligenes faecalis , Candida albicans , Escherichia coli , Haemophilus influenza , Neisseria meningitides , Pseudomonas aeruginosa , Staphylococcus aureus , Streptococcus pneumoniae , and Group A Streptococcus pyogenes .

When an "immunologically effective amount", "tumor-effective amount", "tumor-inhibiting effective amount", or "therapeutic amount" is indicated, the exact amount of the compositions of the present invention to be administered may be determined by the clinician, taking into account individual differences in age, body weight, tumor size , the degree of infection or metastasis, and the condition of the patient (individual). In general, it can be argued that a pharmaceutical composition containing immune effector cells (eg, T cells, NK cells) described herein can be administered at a dosage of 10 ⁴ to 10 ⁹ cells/kg body weight, in some cases 10 ⁵ to 10 ⁶ cells/kg body weight, including all integers in these ranges. T cell compositions can also be administered multiple times at these dosages. Cells can be administered using infusion techniques that are well known in immunotherapy (see, for example, Rosenberg et al ., New Eng. J. of Med. 319:1676, 1988).

In some aspects, it may be desirable to administer activated immune effector cells (e.g., T cells, NK cells) to an individual, and then subsequently redraw blood (or perform apheresis), activate immune effector cells (e.g., T cells, NK cells ) from it in accordance with the present invention, and re-infusion of activated and increased in the number of immune effector cells (eg, T cells, NK cells) to the patient. This process can be repeated multiple times every few weeks. In some aspects, immune effector cells (eg, T cells, NK cells) can be activated from 10 cm ³ to 400 cm ³ of drawn blood. In some aspects, immune effector cells (eg, T cells, NK cells) are activated from 20 cm ³ , 30 cm ³ , 40 cm ³ , 50 cm ³ , 60 cm ³ , 70 cm ³ , 80 cm ³ drawn blood. , 90 cm ³ or 100 cm ³ .

Administration of the subject compositions may be by any convenient means, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intralymph nodeally, intramuscularly, intramuscularly, by intravenous (IV) injection, or intraperitoneally. In one aspect, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present invention are administered by intravenous injection. Immune effector cell compositions (eg, T cells, NK cells) can be injected directly into a tumor, lymph node, or site of infection.

In a specific exemplary aspect, individuals may be subjected to leukoapheresis, where leukocytes are harvested, expanded, or depleted ex vivo to select and/or isolate cells of interest, eg, T cells. These isolated T cells can be expanded by methods known in the art and processed so that one or more CAR constructs can be introduced, thereby creating a CAR T cell of the invention. Individuals in need thereof may subsequently be treated with standard high dose chemotherapy followed by peripheral blood stem cell transplantation. In some aspects, after or simultaneously with transplantation, individuals are infused with increased CAR T cells of the present invention. In a further aspect, the expanded cells are administered before or after surgery.

The dosage of the drugs described above to be administered to a patient varies depending on the exact nature of the condition being treated and the recipient of the treatment. Dosage scaling up for human administration can be done in accordance with accepted practice in the art. The dose of CAMPATH, for example, is typically in the range of 1 to about 100 mg for an adult patient, typically administered over a period of 1 to 30 days. The preferred daily dose is from 1 to 10 mg per day, although in some cases you can use higher doses up to 40 mg per day (as described in US patent No. 6120766).

In one embodiment, CARs are introduced into immune effector cells (e.g., T cells, NK cells), e.g., using in vitro transcription, and the subject (e.g., human) is initially administered immune effector cells with CAR (e.g., T- cells, NK cells) of the invention, and one or more subsequent administrations of immune effector cells with CARs (e.g. T cells, NK cells) of the invention, where one or more subsequent administrations are carried out for less than 15 days, for example 14 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days after the previous administration. In one embodiment, an individual (e.g., human) receives more than one administration of CAR immune effector cells (e.g., T cells, NK cells) of the invention per week, e.g., 2, 3, or 4 administrations of CAR immune effector cells (e.g., , T-cells, NK-cells) according to the invention per week. In one embodiment, the subject (e.g., human) receives more than one administration of CAR immune effector cells (e.g., T cells, NK cells) per week (e.g., 2, 3, or 4 administrations per week) (also referred to herein as course) followed by a week without administration of CAR immune effector cells (eg, T cells, NK cells) and then one or more additional administrations of CAR immune effector cells (eg, T cells, NK cells). ) (eg, more than one administration of CAR immune effector cells (eg, T cells, NK cells) per week). In another embodiment, an individual (e.g., a human) is given more than one course of administration of CAR immune effector cells (e.g., T cells, NK cells) and the time between courses in each case is less than 10, 9, 8, 7, 6, 5, 4 or 3 days. In one embodiment, CAR immune effector cells (eg, T cells, NK cells) are administered every two days for 3 administrations per week. In one embodiment, the CAR immune effector cells (eg, T cells, NK cells) of the invention are administered for at least two, three, four, five, six, seven, eight, or more weeks.

In one aspect, the CAR expressing cells of the present invention are obtained using viral vectors such as lentivirus. Cells such as CART thus obtained will have stable CAR expression.

In one aspect, CAR expressing cells, eg, CART, are generated using a viral vector, such as a gamma retroviral vector, eg, the gamma retroviral vector described herein. CARTs generated using these vectors can have stable CAR expression.

In one aspect, CART transiently express CAR vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of a CAR can be achieved by delivering a vector RNA with a CAR. In one aspect, the CAR RNA is transduced into a T cell by electroporation.

A potential problem that may arise in patients treated with transiently expressed CAR immune effector cells (eg, T cells, NK cells) (particularly mouse scFv-containing CARTs) is anaphylaxis after multiple doses.

Without being bound by theory, it is believed that such an anaphylactic response may be due to the development of a humoral anti-CAR response in the patient, ie. anti-CAR antibodies having an anti-IgE isotype. A patient's antibody-producing cells are believed to undergo a class switch from the IgG isotype (which does not cause anaphylaxis) to the IgE isotype when there is a period of ten to fourteen days of antigen exposure hiatus.

If a patient is at high risk of inducing an anti-CAR antibody response during temporary CAR therapy (such as RNA transduction therapy), the intervals between CART infusions should not exceed ten to fourteen days.

EXAMPLES

Further, the invention is described in detail with the help of the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed as covering any and all variations that will become apparent from the teachings provided herein.

Treatment of a patient with CLL with CART19

Patient UPCC04409-10 was treated with autologous CART19 T cells from CLL. Treatment resulted in complete remission of CLL.

Analysis of the CART cell population

As shown in Figure 1, monitoring of CART cells in patient UPCC04409-10 over time was performed by taking blood samples. The expression level of BBZ in cells was determined (red). The number of copies of the TCR family sequence Vbeta5.1 (blue) was determined. Both measurements were performed on samples obtained on the indicated day after the second infusion of CART cells. As shown in Figure 2, the T-cell receptor repertoire of the UPCC04409-10 patient was determined in a sample obtained 28 days (Figure 2A) or 51 days (Figure 2B) post-CART infusion. This demonstrates a large number of T-cell receptors of the TCRBV05-01 family at day 51, indicating clonal expansion over time. As shown in Figure 3, isolated T cells from patient UPCC04409-10 were analyzed for co-expression of CAR19 and 2 different TCR family genes over time (day 50 and day 51) and compared to the original dosed material (product): in the top panel TCR Vb13.1 family introduced; the bottom panel shows the TCR Vb5.1 family. The data demonstrate that CAR19 positive cells contain one gene of the TCR family (Vb5.1), which increases rapidly between days 50 and 51. As shown in Figure 4, the T cell receptor repertoire for CD8 positive cells from patient UPCC04409-10 was determined in a sample obtained 51 days after CART infusion. This demonstrates a large number of T cell receptors of the TCRBV05-01 family at day 51, indicating a clonal expansion of CD8 positive cells over time.

CART clone persistence analysis

As shown in FIG. 6, sonicated DNA was obtained from T cells of patient #. This material was used to amplify genomic sequences adjacent to the CAR19 insertion region. These genes adjacent to CAR19 in the genome were identified as having increased expression relative to the infused product (D0). At different time points after said CART infusion (d=day; m=month), different relative increases in neighboring gene expression were observed, with the increase in Tet2 expression peaking in both peripheral blood (PBMC) and CAR+CD8+ T cell samples at 51 days.

As shown in Figure 7, the CAR19 gene insertion site was mapped to the Tet2 gene. More specifically, insertion occurred between exons 9 and 10 of the Tet2 gene. The catalytic domain for Tet2 is located in exon 11. Insertion at this position may result in expression of aberrant mRNA transcripts or reduced expression of functional Tet2 (wild type).

As shown in FIG. 8, Tet2 gene transcripts from the isolated UPCC04409-10 patient mRNA were evaluated by RT-PCR using primers spanning the indicated Tet2 or CAR19 regions, or both, as indicated in the figure on the right. Rxn 3 contains primers designed to amplify the region of the Tet2 transcript spanning exons 9 and 10. Rxn, 6, 7, 8, 9 and 10 are primers designed to amplify the indicated portions of the CAR19 lentivirus. Rxn 12-16 are primer pairs that contain the sequence of exon 9 of the Tet2 transcript as well as the sequence from the CAR19 lentiviral construct. These data show that the transcripts are derived from the Tet2 locus, which contains both the Tet2 sequence and the CAR19 sequence.

Analysis of the Tet2 function

As shown in Fig. 10, it schematically represents the enzymatic activity of Tet2 (Fig. 10A). The Tet family protein converts 5-methylcytosine (5-mc) to 5-hydroxymethylcytosine (5-hmc) and then to 5-formylcytosine (5-fmc), resulting in demethylated cytosine. Methylated DNA is an epigenetic state known to affect the transcription profile. The methylation status of T cells from patient UPCC04409-10 was assessed (FIG. 10B). Patient T cells were stained for TCRVb5.1 (which contains the CAR19 insertion in Tet2) and 5-hmc and 5-fmc were evaluated in TCRVb5.1 positive (red) and TCRVb5.1 negative (blue) populations by flow cytometry. These data indicate that cells containing the CAR19 insertion in the Tet2 gene have a demethylation defect.

Treatment of T cells with shRNA-based Tet2 inhibitors

Materials and methods

Production of lentivirus and infection of Jurkat cells

Lentiviruses were obtained from 293T cells on 15 cm dishes. Briefly, 10 million 293T cells were seeded onto collagen-coated 15 cm dishes on day -1. On day 0, 15 µg of shRNA vector (ie, a vector containing a sequence encoding TET2 or control shRNA), 15 µg of Gag/pol vector, and 5 µg of VSV-G vector were transfected using Lipofectamin 2000 (Invitrogen). After 24 hours (1 day) the medium was replaced. After changing the medium, supernatants with viruses were collected on days 2 and 3. Viruses were concentrated using a Lenti-X Concentrator (3:1 v/v ratio, Clonetech, cat #: 631231). 100 μl of viruses were added to 0.5 million jurkat cells in the presence of 6 μg/ml polybrene. Cells were infected by rotating 2000 rpm for 90 min at 32°C. After incubation for 1 hour in an incubator at 37 degrees, fresh RPMI 1640 medium was added and transferred to a 24-well plate. On day 6, cells were transferred to a 6-well plate in the presence of puromycin at a final concentration of 2 μg/ml for 6 days.

Antibodies

The antibodies used for Western blotting were as follows: β-actin (clone #: 8H10D10, Cell Signaling); TET2 (clone #: hT2H21F11, Millipore); Mouse IgG (H+L) (HRP-conjugated, catalog #: 115-035-166, Jackson ImmunoResearch); Rabbit IgG (H+L) (HRP conjugated, catalog #: 111-035-114, Jackson ImmunoResearch).

Western blotting

To study the effectiveness of shRNA knockdown against TET2 at the protein level in jurkat cells, cell lysates were prepared in RIPA buffer containing a cocktail of protease inhibitors (Sigma). Protein concentration was measured using BCA protein assay kits (Pierce, Item#: 3603904). 20 μg of the protein was subjected to SDS-PAGE and then the protein was transferred to a nitrocellulose membrane using an iBot transfer system (Invitrogen, 20 V, 11 min 30 s). The membrane was blocked with 5% BSA containing TBST for 30 minutes at room temperature. Antibodies were incubated overnight with membranes at a dilution of 1:000 at 4°C. After incubation with HRP conjugated secondary antibodies, signal detection was performed using a chemiluminescence detection device (Chemidoc; Bio-Rad).

Quantitative RT-PCR

To study the effectiveness of shRNA knockdown against TET2 at the DNA level in jurkat cells, a quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed. The RNeasy Micro Kit (Qiagen) was used for RNA extraction. mRNA was reverse transcribed into single stranded complementary DNA (cDNA) using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time PCR was performed using a C1000 Touch Thermal Cycler (Biorad). SYBR-based protocol (SsoAdvanced Universal SYBR Green Supermix, Biorad) was used to detect gene expression. PCR reactions were run in 96-well plates and the manufacturer's recommended cycle parameters were used in triplicate (95°C for 3 minutes followed by 40 cycles at 95°C for 15 seconds and 60°C for 30 seconds). Cycle threshold (Ct) values for genes of interest were normalized to Ct for β-actin. The primers used for qRT-PCR were as follows: β-actin #1 (forward primer: CAT GTA CGT TGC TAT CCA GGC (SEQ ID NO: 1263), reverse primer: CTC CTT AAT GTC ACG CAC GAT (SEQ ID NO: 1264); product size 250 bp); β-actin #2 (forward primer: CTC ACC ATG GAT GAT GAT ATC GC (SEQ ID NO: 1265), reverse primer: CCA CAT AGG AAT CCT TCT GAC CC (SEQ ID NO: 1266); product size 169 bp .); TET1 (forward primer: CAG AAC CTA AAC CAC CCG TG (SEQ ID NO: 1267), reverse primer: TGC TTC GTA GCG CCA TTG TAA (SEQ ID NO: 1268); product size 141 bp); TET2 (forward primer: ATA CCC TGT ATG AAG GGA AGC C (SEQ ID NO: 1269), reverse primer: CTT ACC CCG AAG TTA CGT CTT TC (SEQ ID NO: 1270); product size 197 bp); TET3 (forward primer: CAC CCG GCT CTA TGA AAC CTT (SEQ ID NO: 1271), reverse primer: CCA GCC ACT CGA GGT AGT CA (SEQ ID NO: 1272); product size 209 bp); RPLP1 (catalog #: PPH17813G-200, Qiagen).

flow cytometry

Cells were obtained from FACS Fortessa (BD). Data processing for presentation was performed using the program FlowJo (Treestar Inc.).

results

Validation of Tet2 knockdown efficiency using shRNA

As shown in FIG. 27, it is a schematic representation of the validation of TET2 knockdown efficiency by shRNA. shRNA against TET2 and shRNA shRNA control constructs with shuffle expressing red fluorescent protein (RPF) and puromycin resistance gene were introduced into jurkat cells to validate the efficiency of TET2 knockdown by shRNA using qRT-PCR and Western blot.

As shown in FIG. 28, shRNA-infected jurkat cells express RFP. RFP expression was determined by FACS on day 6 after puromycin treatment. Based on RFP expression, over 99% of shRNA-introduced jurkat cells were selected by puromycin treatment. Of note, jurkat cells infected with shRNA against TET2 #3 and #4 did not grow in the presence of puromycin. Thus, jurkat cells infected with shRNA against TET2 #3 and #4 were not further used. These data indicate that puromycin is effective in selecting shRNA-infected jurkat cells.

As shown in FIG. 29, the efficiency of tet2 knockdown depends on shRNA. To determine the level of expression of mRNA tet1 and tet2 and tet3 in jurkat cells infected with shRNA against TET2, an experiment was performed using qRT-PCR. Compared to the shRNA with shuffle, shRNA against TET2 #1, #2, #8 and #9 knocked down the tet2 gene at 35.6%, 22.7%, 21.6% and 76.7%, respectively. Surprisingly, while anti-TET2 #9 shRNA effectively knocks down tet2 , it also downregulates tet1 and tet3 expression at 43.4% and 67.3%, respectively. β-actin serves as an internal control to quantify relative gene expression among the samples tested. To increase the reliability of qRT-PCR, two primers for β-actin and one primer for RPLP1 were used in this experiment. These data indicate that several TET2-targeted shRNAs are able to reduce TET2 mRNA levels, with shRNA #9 showing the most pronounced tet2 knockdown effect, also affecting TET1 and TET2 levels.

As shown in Figure 30, knockdown of the TET2 protein in response to shRNA correlates with knockdown of TET2 mRNA levels. Western blotting was performed to determine the expression level of the TET2 protein in jurkat cells infected with anti-TET2 shRNA. Similar to the qRT-PCR data shown in Fig. 29, anti-TET2 shRNA #1, #2, #8, and #9 reduced the level of TET2 protein compared to shRNA with shuffling, while β-actin was constitutively expressed in all samples tested. . These data indicate that several TET2-targeted shRNAs are able to reduce TET2 protein levels in Jurkat cells, with shRNA #9 showing the most pronounced Tet2 knockdown effect.

Treatment of primary T cells with shRNA-based Tet2 inhibitors

As shown in Figure 11, anti-TET2 shRNAs reduce 5-hmc levels in normal human T cells. anti-TET2 shRNA and mCherry-expressing shRNA shRNA control constructs were introduced into normal human T cells. 5-hmc levels were determined by intracellular staining with FACS on day 6 post-expansion using anti-CD3/CD28 antibody beads. TET2 knockdown reduced overall 5-hmc levels.

As shown in FIG. 12, anti-TET2 shRNA is increased in Tscm T cells. anti-TET2 shRNA and mCherry-expressing shRNA shRNA control constructs were introduced into normal human T cells. CD45RA+CD62L+CCR7+CD27+CD95+ Tscm T cells were detected by FACS staining on day 11 after expansion using anti-CD3/CD28 antibody beads. Knockdown of TET2 stimulated the expansion of T cells with the Tscm phenotype.

Tet2 Inhibition in CAR T Cells Using the CRISPR/Cas Gene Editing System

In this example, TET2 inhibition was investigated in T cells expressing a chimeric antigen receptor (CAR).

Ways

Guide RNA molecules

For the experiments described in this sub-example, gRNA molecules containing the targeting sequences shown in Table 5 were used. Unless otherwise indicated, all gRNA molecules were tested as double gRNA molecules containing the tracr and cr-RNA sequences described in this sub-example.

Table 5

target area Anti-TET2 gRNA targeting sequence Guide designation Exon 9 CAGAGCACCAGAGUGCCGUC (SEQ ID NO: 1273) 9_1 (also referred to as Tet2_E9_1_Tet2) Exon 9 AGAGCACCAGAGUGCCGUCU (SEQ ID NO: 1274) 9_2 (also referred to as Tet2_E9_2_Tet2) Exon 9 UUCAGACCCAGACGGCACUC (SEQ ID NO: 1275) 9_3 (also referred to as Tet2_E9_3_Tet2) Exon 9 AUGGCAGCACAUUGGUAAGU (SEQ ID NO: 1276) 9_4 (also referred to as Tet2_E9_4_Tet2) Exon 9 CACAUUGGUAAGUUGGGCUG (SEQ ID NO: 1277) 9_5 (also referred to as Tet2_E9_5_Tet2) Exon 9 GACUUGCACAACAUGCAGAA (SEQ ID NO: 1278) 9_6 (also referred to as Tet2_E9_5_Tet2, Ex9-3 or crEx9-3) exon 7 UCAUGGAGCAUGUACUACAAA (SEQ ID NO: 1279) 7_1 exon 7 AACUUGCGCCUGUCAGGGGC (SEQ ID NO: 1280) 7_2 exon 7 CCAAGGAAGUUUAAGCUGCU (SEQ ID NO: 1281) 7_3 exon 7 CCAAGCAGCUUAAACUUCCU (SEQ ID NO: 1282) 7_4 exon 8 UUGGUGCCAUAAGAGUGGAC (SEQ ID NO: 1283) 8_1 exon 8 GCAAAACCUGUCCACUCUUA (SEQ ID NO: 1284) 8_2 exon 8 AUAUGUUGGUGCCAUAAGAG (SEQ ID NO: 1285) 8_3 exon 10 AAAACGGAGUGGUGCCAUUC (SEQ ID NO: 1286) 10_1 exon 10 GUCUCUGACGUGGAUGAGUU (SEQ ID NO: 1287) 10_2 exon 10 UUUAUACAAAGUCUCUGACG (SEQ ID NO: 1288) 10_3 exon 10 AGAGAAGACAAUCGAGAAUU (SEQ ID NO: 1289) 10_4 exon 10 ACGUCAGAGACUUUGUAUAA (SEQ ID NO: 1290) 10_5 exon 3 GGAUAGAACCAACCAUGUUG (SEQ ID NO: 1291) 3_1 (also referred to as Tet2_E3_1_Tet2) exon 3 UUGUAGCCAGAGGUUCUGUC (SEQ ID NO: 1292) 3_2 (also referred to as Tet2_E3_2_Tet2) exon 3 UCUGUUGCCCUCAACAUGGU (SEQ ID NO: 1293) 3_3 (also referred to as Tet2_E3_3_Tet2, Ex3-3 or crEx3-3) exon 3 GAUGAAACCAACCAUGUUGA (SEQ ID NO: 1294) 3_4 (also referred to as Tet2_E3_4_Tet2) exon 3 UUCUGGAGCUUUGUAGCCAG (SEQ ID NO: 1295) 3_5 (also referred to as Tet2_E3_5_Tet2)

Obtaining CRISPR CAR T cells

Isolated and frozen total T cells were thawed and activated using anti-CD3/CD28 antibody beads (CD3/CD28 CTS Dynabeads® 43205D) on day 0. Activated T cells were transduced with a lentivirus encoding either BCMA CAR (BCMA-10 (139109) as described in WO2016/0046724; referred to herein as BCMA-10 CAR) or CD19 CAR (CD19 CAR having the amino acid sequence of SEQ ID NO : 12 WO2012/079000; referred to herein as CD19 CAR) for 1 day. On day 3, transduced CAR T cells were electroporated to introduce CRISPR/Cas systems in the form of pre-gRNA/Cas9 complex ribonucleoprotein ("RNP"). To obtain RNP, all RNA samples were heated at 95°C. S. pyogenes CAS9 protein (NLS CAS9 iPROT106154, 37 μM) was diluted in buffer before addition of tracr RNA (having the sequence: AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGUUUUUUU (SEQ ID NO: 1296); AXO Labs). After mixing the CAS9 protein with tracr RNA, CRISPR RNA was added (in each case, each cr RNA contained the sequence nnnnnnnnnnnnnnnnnnGUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 40), where the n residues correspond to 20 ribonucleic acid residues of the indicated targeting sequence). The precomplex RNPs were then added to a total of 1 million cells, the RNA concentration was 3.2 μM. Electroporation was performed with a neon electroporator using the Neon® Transfection System 100 μL Kit (MPK10096) at 1600 V, 10 ms, 3 pulses. The cells were kept in culture for 7 additional days. Cells were then separated, some used for flow cytometry: staining for CAR (PE), CD3 (PerCP-Cy5.5), CD4 (V450) and CD8 (APC). The remaining T cells were frozen and used for functional assays and next generation sequencing (NGS) samples.

Sample acquisition for next generation sequencing (NGS)

Cell-edited frozen pellets (described above) were thawed and processed using the DNeasy Blood & Tissue Kit (Qiagen, 69506) for genomic DNA isolation. The eluted DNA was used for PCR using the Titan Taq PCR kit (Clontech Laboratories, 639211) and TET2 primers (primers designed to flank the expected gRNA target site). The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen, 28104). The purified PCR product was then used for T7E1 analysis to detect base mismatches and confirm gene editing. PCR amplicons were subjected to standard conversion to the Nextera NGS library prep (Illumina) and sequenced using a paired-end read on an Illumina MiSeq sequencer. The sequenced sequences were aligned with the reference genome and variants were identified.

Analysis of cytokine production

Effector cells (CAR T cells) were thawed on the day of analysis and counted on Cellometer (Nexelcom). These cells were then co-cultured with different target cells in an effector:target cell ratio of 2:1. After 20 hours, 100 μl of the coculture supernatant was collected. These supernatants were then used to quantify released IL-2 and IFN-g cytokines using the Meso Scale Discovery System, Proinflammatory Panel 1, cat # N05049A-1, according to the manufacturer's protocol.

T cell proliferation assay

Proliferation of CAR T cells was assessed in response to BCMA or CD19 expressing target cells. The target cell lines were: BCMA positive multiple myeloma cell lines, NCI-H929-luc, KMS-11-luc and BCMA negative Nalm6luc (CD19 positive cell line). CAR T cells were thawed and incubated for 2 hours in T cell medium for recovery. Cells were counted on a Cellometer. Target cells were irradiated at 10,000 rad. After irradiation, target cells were washed twice in complete T cell medium and counted. Then 30,000 irradiated cells were co-cultured with CART cells in a ratio of 1:1. Medium alone was added to CAR T cells as a negative control.

The coculture was incubated for 4 days at 37°C. On day 4, coculture cells were stained for 20 min on ice with CD3-percp cy5.5(Ebioscience:45-0037), CD4-eflor450(Ebioscience:48-0047) and CD8-APC (Ebioscience 17-0087) and quantified flow cytometry against CountBright Absolute Counting Beads (Life Technologies) to determine cell numbers. CAR expression was quantified by incubation in two stages of 20 min each on ice: biotinylated protein L + streptavidin-PE (Jackson immuno research). Flow cytometry data were acquired using a BD 5 Fortessa laser and analyzed using FlowJo software.

results

On figa and 13B shows the expression of CAR in cells subjected to electroporation with the introduction of CRISPR against Tet2 or without it. 13A shows a gating strategy for detecting CAR+ T cells. Lymphocyte selection was performed using forward scatter (FSC-A) and side scatter (SSC-A) as indicated by circling. CD3 positive cells were then selected (middle panel). Then, CAR positive (PE positive cells using CAR detection with biotinylated protein and streptavidin-PE) barred cells were determined by gating against the peak of the negative control. On figv presents a quantitative determination of the percentage of positive for CAR cells. Cells were transduced with either BCMA-10 CAR or CD19 CAR as indicated. RNP containing the Cas9 protein, tracer RNA and the indicated guide RNA targeting Tet2 (Ex3-3 targeting exon 3 or Ex9-6 targeting exon 9) were introduced into cells by electroporation, or no electroporation was performed. CAR expression was determined 10 days after cell activation with granules. These data indicate that Tet2 editing does not affect CAR expression in T cells.

Figure 14 shows the quantification of CD4 and CD8 positive cells after CAR transduction and Tet2 editing. Cells were stained for CD3, CD4, CD8 and CAR expression at the end of bead expansion for 10 days. The left panel shows the percentage of CD4 and CD8 positive cells in the total CD3+ cell population. The right panel shows the percentage of CD4 and CD8 positive cells in the CD3+ cell population, which are also CAR+. Cells were transduced with either BCMA-10 CAR or CD19 CAR or left untransduced (UTD). RNP containing the Cas9 protein, tracer RNA and the indicated guide RNA targeting Tet2 (Ex3-3 targeting exon 3 or Ex9-6 targeting exon 9) were introduced into cells by electroporation, or no electroporation was performed. These data showed that editing Tet2 causes a small but steady decrease in the percentage of CD8 cells and an increase in the percentage of CD4 during the bead-based expansion period.

Figure 15 shows the yield and viability of cells after expansion using pellets for 10 days. Cells per ml (left panel) and cell viability (right panel) were quantified using a Cellometer after a 10 day pellet expansion process. Cells were transduced with either BCMA-10 CAR or CD19 CAR or left untransduced (UTD). RNP containing the Cas9 protein, tracer RNA and the indicated guide RNA targeting Tet2 (Ex3-3 targeting exon 3 or Ex9-6 targeting exon 9) were introduced into cells by electroporation, or no electroporation was performed. These data indicate that Tet2 editing results in an increase in cell number and viability relative to cells without CRISPR/Cas9, with the 9-targeted guide (ex9-6) showing the greatest effect in non-transduced as well as transduced CAR cells.

16 shows IL-2 production in response to cells that are either positive or negative for an antigen recognized by CAR. CART cells or non-transduced cells (UTD) were co-cultured with BCMA positive/CD19 negative cells (KMS11 and NCIH929) or BCMA negative/CD19 positive cells (NALM6) and cytokine secretion into the medium was quantified. IL-2 levels (pg/ml) are shown. Cells were obtained as described above. These data indicate that Tet2 editing causes an increase in T cell production of IL-2 in response to antigen, with the exon 9-targeted guide (Ex9-6) showing the greatest effect.

Figure 17 shows the production of interferon-gamma. CART cells or non-transduced cells (UTD) were co-cultured with BCMA positive/CD19 negative cells (KMS11 and NCIH929) or BCMA negative/CD19 positive cells (NALM6) and cytokine secretion into the medium was determined. Interferon-gamma (IFN-g) levels (pg/mL) are shown. Cells were obtained as described above. These data indicate that Tet2 editing results in increased production of interferon-gamma by T cells in response to antigen, with the exon 9-targeted guide (Ex9-6) showing the greatest effect.

18 shows antigen-driven proliferation of CART cells. CART cells or non-transduced cells (UTD) were co-cultured with BCMA positive/CD19 negative cells (KMS11 and NCIH929) or BCMA negative/CD19 positive cells (NALM6) or cultured without target cells (medium) and quantified CAR proliferation -positive T cells. Cells were obtained as described above. These data indicate that Tet2 editing leads to increased proliferation of CAR+ T cells in response to antigen, with the exon 9-targeted guide (CrEx9-6) showing the greatest effect.

Figure 19 shows antigen-driven proliferation of total T cells. CART cells or non-transduced cells (UTD) were co-cultured with BCMA positive/CD19 negative cells (KMS11 and NCIH929) or BCMA negative/CD19 positive cells (NALM6) or cultured without target cells (medium) and the proliferation of all CD3+ T cells. Cells were obtained as described above. These data indicate that editing Tet2 leads to increased proliferation of CD3+ T cells in response to antigen, with the exon 9-targeted guide (CrEx9-6) showing the greatest effect.

FIG. 20 shows antigen-driven proliferation of CAR+ T cells. CART cells or non-transduced cells (UTD) were co-cultured with BCMA positive/CD19 negative cells (KMS11 and NCIH929) or BCMA negative/CD19 positive cells (NALM6) or cultured without target cells (medium) and the proliferation of all CD4+ CD3+ T cells. Cells were obtained as described above. These data indicate that editing Tet2 results in increased proliferation of CD4+ T cells in response to antigen, with the exon 9-targeted guide (CrEx9-6) showing the greatest effect.

FIG. 21 shows antigen-driven proliferation of CAR+ CD4+ T cells. CART cells or non-transduced cells (UTD) were co-cultured with BCMA positive/CD19 negative cells (KMS11 and NCIH929) or BCMA negative/CD19 positive cells (NALM6) or cultured without target cells (medium) and the proliferation of all CAR+CD4+ CD3+ T cells. Cells were obtained as described above. These data indicate that Tet2 editing leads to increased proliferation of CAR+CD4+ T cells in response to antigen, with the exon 9-targeted guide (CrEx9-6) showing the greatest effect.

FIG. 22 shows antigen-driven proliferation of CD8+ T cells. CART cells or non-transduced cells (UTD) were co-cultured with BCMA positive/CD19 negative cells (KMS11 and NCIH929) or BCMA negative/CD19 positive cells (NALM6) or cultured without target cells (medium) and the proliferation of all CD8+ CD3+ T cells. Cells were obtained as described above. These data indicate that editing Tet2 results in increased proliferation of CD8+ T cells in response to antigen, with the exon 9-targeted guide (CrEx9-6) showing the greatest effect.

FIG. 23 shows antigen-driven proliferation of CAR+ CD8+ T cells. CART cells or non-transduced cells (UTD) were co-cultured with BCMA positive/CD19 negative cells (KMS11 and NCIH929) or BCMA negative/CD19 positive cells (NALM6) or cultured without target cells (medium) and the proliferation of all CAR+CD8+ CD3+ T cells. Cells were obtained as described above. These data indicate that Tet2 editing leads to increased proliferation of CAR+CD8+ T cells in response to antigen, with the exon 9-targeted guide (CrEx9-6) showing the greatest effect.

24 shows % edit and % frameshift edit by introducing Tet2-targeted CRISPR/Cas systems. The level of editing in primary T cells after electroporation of RNP containing the Cas9 protein, tracer RNA and the indicated guide RNAs targeting either exon 3 or exon 9 of Tet2 is shown. The percentage of nucleotide insertions or deletions observed relative to the reference genome is presented in the middle column (mean % insertions/deletions). The edit that results in an open reading frame shift is shown in the far right column (middle % with frame shift). These data represent the average values for three repetitions of the experiment. These data indicate highly efficient genome editing in primary T cells using these guide RNA sequences.

The profile of insertions and deletions at or near the target site for each gRNA was assessed using next generation sequencing. Briefly, RNP containing the indicated RNG guide was introduced into T cells by electroporation. After 48 hours, DNA was isolated and processed for sequencing. 25 shows the 5 most common insertions/deletions (insertions and/or deletions) seen in primary T cells for guide RNAs targeting exon 3 of Tet2. FIG. 26 shows the 5 most common insertions/deletions (insertions and/or deletions) seen in primary T cells for guide RNAs targeting exon 9 of Tet2. The percentages indicate the frequency with which a given editing profile was observed. Insertions are indicated by nucleotide designation in capital letters ("a", "g", "c" or "t"), while deletions are indicated by dashes ("-").

Experiments with ATAC-Seq

CD8+ T cells with or without Tet2 insertion were increased in number from the patient sample after infusion. Chromatin accessibility was assessed using high throughput sequencing transposase accessible chromatin assay (ATAC-seq). It is essentially a method for global chromatin mapping based on the transposition of barcoded DNA fragments. These DNA fragments are inserted into regions of open chromatin, allowing determination of which regions of chromatin are open and which are closed. Based on the position of open or closed regions, it is possible to analyze the cascades, assuming that "open" is equivalent to "expressed" and "closed" is equivalent to "repressed". FIG. 30A shows Venn plots of ATAC peaks in CAR+CD8+ T cells from a Tet2 impaired patient compared to CAR-CD8+ T cells from the same patient at the corresponding time point without Tet2 impaired. FIG. 28B shows the GO terms associated with ATAC peaks being the most occluded in a Tet2 deficient cell population compared to its counterpart. The significance of Figure 30B is, at least in part, that the chromatin pattern of Tet2-disrupted CD8+ T cells is possibly consistent with a less differentiated cell that may be more "early memory cell-like" and less "effector-like". ". They are a type of cells that are expected to persist for a long time, providing a stable and long-lasting antitumor activity.

shRNA studies

T cells from healthy donors were activated for 24 hours using CD3/CD28 coated beads and then transduced with either untargeted (control) shRNA or anti-Tet2 shRNA with lentivirus. As shown in FIG. 32A, knockdown efficiency was assessed by qPCR and shown to be 50% (may reflect what happened in the patient where Tet2 was destroyed by lentivirus insertion). The differentiation phenotype was examined at 14 by CCR7, CD45RO assay. Central memory cells are defined as CCR7+CD45RO+ while effector cells are CCR7-CD45RO-. For a specific study of the differentiation phenotype in cells with a 50% Tet2 knockdown, the GFP indicator was used in the shRNA construct. The results are shown in FIGS. 32B and 32C.

Without further description, it is believed that one skilled in the art can, using the foregoing description and the following illustrative examples, make and use the compounds of the present invention and practice the claimed methods. The following working examples specifically indicate various aspects of the present invention, and should not be considered limiting in any way the rest of the description.

EQUIVALENTS

The content of each patent, patent application and publication cited in the present description, is incorporated into the present description by reference in its entirety. Although the present invention has been described in relation to specific aspects, it is obvious that other aspects and variations of the present invention can be established by other experts without deviating from the true essence and scope of the invention. The appended claims are intended to be construed as including all such aspects and equivalent variations.

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SEQUENCE LIST

<110> NOVARTIS AG

THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA

<120> CAR T CELL THERAPIES WITH ENHANCED EFFICACY

<130> N2067-7098WO

<140>

<141>

<150> 62/220.196

<151> 2015-09-17

<160> 1363

<170>PatentIn version 3.5

<210> 1

<211> 1184

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 1

cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120

180

gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240

gtgccgtgtg tggttcccgc gggcctggcc tctttacgggg ttatggccct tgcgtgcctt 300

gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360

ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg 420

cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480

ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540

tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg 600

gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660

tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 720

tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 780

caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat 840

ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900

ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc 960

tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020

cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080

tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140

gtga 1184

<210> 2

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 3

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 3

atggccctgc ctgtgacagc cctgctgctg ccctctggctc tgctgctgca tgccgctaga 60

ccc 63

<210> 4

<211> 45

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 4

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 5

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 5

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 6

<211> 230

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 6

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Met

225 230

<210> 7

<211> 690

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 7

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac 180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc 240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag 360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgtccct gggcaagatg 690

<210> 8

<211> 282

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 8

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 9

<211> 847

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 9

aggtggcccg aaagtcccaa ggcccaggca tctagtgttc ctactgcaca gccccaggca 60

gaaggcagcc tagccaaagc tactactgca cctgcacta cgcgcaatac tggccgtggc 120

ggggaggaga agaaaaagga gaaagagaaa gaagaacagg aagagaggga gaccaagacc 180

cctgaatgtc catcccatac ccagccgctg ggcgtctatc tcttgactcc cgcagtacag 240

gacttgtggc ttagagataa ggccaccttt acatgtttcg tcgtgggctc tgacctgaag 300

gatgcccatt tgacttggga ggttgccgga aaggtaccca cagggggggt tgaggaaggg 360

ttgctggagc gccattccaa tggctctcag agccagcact caagactcac ccttccgaga 420

tccctgtgga acgccgggac ctctgtcaca tgtactctaa atcatcctag cctgccccca 480

cagcgtctga tggcccttag agagccagcc gcccaggcac cagttaagct tagcctgaat 540

ctgctcgcca gtagtgatcc cccagaggcc gccagctggc tcttatgcga agtgtccggc 600

tttagcccgc ccaacatctt gctcatgtgg ctggaggacc agcgagaagt gaacaccagc 660

ggcttcgctc cagcccggcc cccaccccag ccgggttcta ccacattctg ggcctggagt 720

gtcttaaggg tcccagcacc acctagcccc cagccagcca catacacctg tgttgtgtcc 780

catgaagata gcaggaccct gctaaatgct tctaggagtc tggaggtttc ctacgtgact 840

gaccatt 847

<210> 10

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 10

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 11

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 11

ggtggcggag gttctgggagg tggaggttcc 30

<210> 12

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 12

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 13

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 13

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 14

<211> 42

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 14

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 15

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 15

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 16

<211> 48

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 16

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 40 45

<210> 17

<211> 123

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 17

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggccccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 18

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 18

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 19

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 19

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 20

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 20

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 21

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 21

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 22

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 22

Gly Gly Gly Gly Ser

15

<210> 23

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 23

ggtggcggag gttctgggagg tggaggttcc 30

<210> 24

<211> 150

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 24

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 25

<211> 450

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 25

cccggatggt ttctggactc tccggatcgc ccgtggaatc ccccaacctt ctcaccggca 60

ctcttggttg tgactgaggg cgataatgcg accttcacgt gctcgttctc caacacctcc 120

gaatcattcg tgctgaactg gtaccgcatg agcccgtcaa accagaccga caagctcgcc 180

gcgtttccgg aagatcggtc gcaaccggga caggattgtc ggttccgcgt gactcaactg 240

ccgaatggca gagacttcca catgagcgtg gtccgcgcta ggcgaaacga ctccgggacc 300

tacctgtgcg gagccatctc gctggcgcct aaggcccaaa tcaaagagag cttgagggcc 360

gaactgagag tgaccgagcg cagagctgag gtgccaactg cacatccatc cccatcgcct 420

cggcctgcgg ggcagtttca gaccctggtc 450

<210> 26

<211> 394

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 26

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro

20 25 30

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

35 40 45

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

50 55 60

Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

65 70 75 80

Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe

85 90 95

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

100 105 110

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser

115 120 125

Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

130 135 140

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

145 150 155 160

Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala

165 170 175

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

180 185 190

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

195 200 205

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

210 215 220

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

225 230 235 240

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

245 250 255

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

260 265 270

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

275 280 285

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

290 295 300

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

305 310 315 320

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

325 330 335

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

340 345 350

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

355 360 365

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

370 375 380

Ala Leu His Met Gln Ala Leu Pro Pro Arg

385 390

<210> 27

<211> 1182

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 27

atggccctcc ctgtcactgc cctgcttctc cccctcgcac tcctgctcca cgccgctaga 60

ccacccggat ggtttctgga ctctccggat cgcccgtgga atcccccaac cttctcaccg 120

gcactcttgg ttgtgactga gggcgataat gcgaccttca cgtgctcgtt ctccaacacc 180

tccgaatcat tcgtgctgaa ctggtaccgc atgagcccgt caaaccagac cgacaagctc 240

gccgcgtttc cggaagatcg gtcgcaaccg ggacaggatt gtcggttccg cgtgactcaa 300

ctgccgaatg gcagagactt ccacatgagc gtggtccgcg ctaggcgaaa cgactccggg 360

acctacctgt gcggagccat ctcgctggcg cctaaggccc aaatcaaaga gagcttgagg 420

gccgaactga gagtgaccga gcgcagagct gaggtgccaa ctgcacatcc atccccatcg 480

cctcggcctg cggggcagtt tcagaccctg gtcacgacca ctccggcgcc gcgcccaccg 540

actccggccc caactatcgc gagccagccc ctgtcgctga ggccggaagc atgccgccct 600

gccgccggag gtgctgtgca tacccgggga ttggacttcg catgcgacat ctacatttgg 660

gctcctctcg ccggaacttg tggcgtgctc cttctgtccc tggtcatcac cctgtactgc 720

aagcggggtc ggaaaaagct tctgtacatt ttcaagcagc ccttcatgag gcccgtgcaa 780

accacccagg aggaggacgg ttgctcctgc cggttccccg aagaggaaga aggaggttgc 840

gagctgcgcg tgaagttctc ccggagcgcc gacgcccccg cctataagca gggccagaac 900

cagctgtaca acgaactgaa cctgggacgg cgggaagagt acgatgtgct ggacaagcgg 960

cgcggccggg accccgaaat gggcgggaag cctagaagaa agaaccctca ggaaggcctg 1020

tataacgagc tgcagaagga caagatggcc gaggcctact ccgaaattgg gatgaaggga 1080

gagcggcgga ggggaaaggg gcacgacggc ctgtaccaag gactgtccac cgccaccaag 1140

gacacatacg atgccctgca catgcaggcc cttccccctc gc 1182

<210> 28

<211> 40

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<220>

<221> OPTIONAL SIGN

<222> (1)..(40)

<223> /note="this sequence can span 1-10

repeating elements "Gly-Gly-Gly-Ser""

<400> 28

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser Gly Gly Gly Ser

35 40

<210> 29

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 29

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 30

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 30

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 31

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 31

Gly Gly Gly Ser

1

<210> 32

<211> 2000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<220>

<221> additional feature

<222> (1)..(2000)

<223> /note="this sequence can span 50-2000

nucleotides"

<220>

<221> source

<223> /note="See attached description for details on replacements and

preferred embodiments"

<400> 32

60

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1740

1800

1860

1920

1980

aaaaaaaaaaaaaaaaaaaa 2000

<210> 33

<211> 150

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 33

60

120

150

<210> 34

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<220>

<221> additional attribute

<222> (1)..(5000)

<223> /note="this sequence can span 50-5000

nucleotides"

<220>

<221> source

<223> /note="See attached description for details on replacements and

preferred embodiments"

<400> 34

60

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1740

1800

1860

1920

1980

2040

2100

2160

2220

2280

2340

2400

2460

2520

2580

2640

2700

2760

2820

2880

2940

3000

3060

3120

3180

3240

3300

3360

3420

3480

3540

3600

3660

3720

3780

3840

3900

3960

4020

4080

4140

4200

4260

4320

4380

4440

4500

4560

4620

4680

4740

4800

4860

4920

4980

aaaaaaaaaaaaaaaaaaaa 5000

<210> 35

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 35

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60

tttttttttt tttttttttt tttttttttt tttttttttt 100

<210> 36

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<220>

<221> additional attribute

<222> (1)..(5000)

<223> /note="this sequence can span 50-5000

nucleotides"

<220>

<221> source

<223> /note="See attached description for details on replacements and

preferred embodiments"

<400> 36

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1980

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2040

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2100

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2160

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2220

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2280

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2340

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2400

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2460

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2520

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2580

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2640

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2700

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2760

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2820

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2880

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 2940

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3000

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3060

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 3960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 4980

tttttttttt tttttttttt 5000

<210> 37

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<220>

<221> additional attribute

<222> (1)..(5000)

<223> /note="this sequence can span 100-5000

nucleotides"

<220>

<221> source

<223> /note="See attached description for details on replacements and

preferred embodiments"

<400> 37

60

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1740

1800

1860

1920

1980

2040

2100

2160

2220

2280

2340

2400

2460

2520

2580

2640

2700

2760

2820

2880

2940

3000

3060

3120

3180

3240

3300

3360

3420

3480

3540

3600

3660

3720

3780

3840

3900

3960

4020

4080

4140

4200

4260

4320

4380

4440

4500

4560

4620

4680

4740

4800

4860

4920

4980

aaaaaaaaaaaaaaaaaaaa 5000

<210> 38

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<220>

<221> additional attribute

<222> (1)..(400)

<223> /note="this sequence can span 100-400

nucleotides"

<220>

<221> source

<223> /note="See attached description for details on replacements and

preferred embodiments"

<400> 38

60

120

180

240

300

360

400

<210> 39

<211> 373

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 39

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

145 150 155 160

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

165 170 175

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

180 185 190

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

195 200 205

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

210 215 220

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

225 230 235 240

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

245 250 255

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

260 265 270

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

275 280 285

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

290 295 300

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

305 310 315 320

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

325 330 335

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

340 345 350

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

355 360 365

Ala Leu Pro Pro Arg

370

<210> 40

<211> 42

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<220>

<221> modified base

<222> (1)..(20)

<223> a, c, u, g, unknown or other

<400> 40

nnnnnnnnnn nnnnnnnnnn guuuuagagc uaugcuguuu ug 42

<210> 41

<211> 86

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<220>

<221> additional attribute

<222> (80)..(86)

<223> /note="This region can span 1-7 nucleotides"

<220>

<221> source

<223> /note="See attached description for details on replacements and

preferred embodiments"

<400> 41

aacuuaccaa ggaacagcau agcaaguuaa aauaaggcua guccguuauc aacuugaaaa 60

aguggcaccg agucggugcu uuuuuu 86

<210> 42

<211> 74

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<220>

<221> additional feature

<222> (68)..(74)

<223> /note="This region can span 1-7 nucleotides"

<220>

<221> source

<223> /note="See attached description for details on replacements and

preferred embodiments"

<400> 42

aacagcauag caaguuaaaa uaaggcuagu ccguuaucaa cuugaaaaag uggcaccgag 60

ucggugcuuuuuuu 74

<210> 43

<211> 103

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<220>

<221> modified base

<222> (1)..(20)

<223> a, c, u, g, unknown or other

<220>

<221> variation

<222> (97)..(103)

<223> /note="This region can span 1-7 nucleotides"

<220>

<221> source

<223> /note="See attached description for details on replacements and

preferred embodiments"

<400> 43

nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 44

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 44

ccuuggacac cuucuccucc 20

<210> 45

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 45

gaaccugug gaagaggagg 20

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 46

ggaacctgtg gaagaggagg 20

<210> 47

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 47

gaaggaagct gaggaacctg 20

<210> 48

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 48

atgacctcca aacaatacac 20

<210> 49

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 49

caagtgctgt ttcaacactg 20

<210> 50

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 50

gggagatgtg aactctggga 20

<210> 51

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 51

ggaggtgatg gtatcaggaa 20

<210> 52

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 52

ggttctgtct ggcaaatggg 20

<210> 53

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 53

ggatgagctc tctcaggcag 20

<210> 54

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 54

Asp Val Pro Asp Tyr Ala Ser Leu Gly Pro Ser Ser Pro Lys Lys

1 5 10 15

Lys Arg Lys Val Ser Arg Gly Val Gln Val Glu Thr Ile Ser Pro Gly

20 25 30

Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr

35 40 45

Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg

50 55 60

Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly

65 70 75 80

Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu

85 90 95

Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile

100 105 110

Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu

115 120 125

Glu Thr Ser Tyr

130

<210> 55

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 55

Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys

1 5 10 15

Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly

20 25 30

Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met

35 40 45

Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Glu Gly Val Ala Gln

50 55 60

Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala

65 70 75 80

Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu

85 90 95

Val Phe Asp Val Glu Leu Leu Lys Leu Glu Thr Ser

100 105

<210> 56

<211> 93

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 56

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys

85 90

<210> 57

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 57

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 58

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 58

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 59

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 59

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 60

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<220>

<221> MODIFIED REMAIN

<222> (12)..(12)

<223> Any amino acid

<220>

<221> MODIFIED REMAIN

<222> (78)..(78)

<223> Any amino acid

<400> 60

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Xaa Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Xaa Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 61

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 61

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 62

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 62

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 63

<211> 1132

<212> PROTEIN

<213> Homo sapiens

<400> 63

Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser

1 5 10 15

His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly

20 25 30

Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg

35 40 45

Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro

50 55 60

Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu

65 70 75 80

Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val

85 90 95

Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro

100 105 110

Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr

115 120 125

Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val

130 135 140

Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val

145 150 155 160

Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr

165 170 175

Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly

180 185 190

Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg

195 200 205

Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg

210 215 220

Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg

225 230 235 240

Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp

245 250 255

Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val

260 265 270

Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala

275 280 285

Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His

290 295 300

Ala Gly Pro Pro Ser Thr Ser Arg Pro Arg Pro Trp Asp Thr Pro

305 310 315 320

Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly

325 330 335

Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro

340 345 350

Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser

355 360 365

Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln

370 375 380

Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His

385 390 395 400

Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg

405 410 415

Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln

420 425 430

Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu

435 440 445

Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe

450 455 460

Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser

465 470 475 480

Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser

485 490 495

Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met

500 505 510

Ser Val Arg Gly Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys

515 520 525

Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe

530 535 540

Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe

545 550 555 560

Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr

565 570 575

Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His

580 585 590

Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln

595 600 605

His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

610 615 620

Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val

625 630 635 640

Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser

645 650 655

Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg

660 665 670

Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg

675 680 685

Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro

690 695 700

Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile

705 710 715 720

Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln

725 730 735

Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His

740 745 750

Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp

755 760 765

Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser

770 775 780

Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu

785 790 795 800

Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His

805 810 815

Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro

820 825 830

Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp

835 840 845

Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu

850 855 860

Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala

865 870 875 880

Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys

885 890 895

Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu

900 905 910

Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe

915 920 925

Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser

930 935 940

Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe

945 950 955 960

Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly

965 970 975

Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn

980 985 990

Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln

995 1000 1005

Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln

1010 1015 1020

Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp

1025 1030 1035

Thr Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly

1040 1045 1050

Met Ser Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu

1055 1060 1065

Ala Val Gln Trp Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr

1070 1075 1080

Arg His Arg Val Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr

1085 1090 1095

Ala Gln Thr Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr

1100 1105 1110

Ala Leu Glu Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys

1115 1120 1125

Thr Ile Leu Asp

1130

<210> 64

<211> 4027

<212> DNA

<213> Homo sapiens

<400> 64

caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc 60

cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc 120

tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg 180

gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg 240

cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg 300

cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg 360

cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc gtgcgcagct 420

acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc 480

gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg 540

tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca 600

ctcaggccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg 660

cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga 720

ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg 780

ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga 840

cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag 900

ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc 960

agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc 1020

ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc 1080

ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg 1140

agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc 1200

tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc 1260

agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag 1320

cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg 1380

acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt 1440

acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc 1500

acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca 1560

agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca 1620

ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg 1680

ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt 1740

1800

gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt 1860

cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc 1920

gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag 1980

ccagaacgtt ccgcagagaa aaggggccg agcgtctcac ctcgagggtg aaggcactgt 2040

tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg 2100

gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc 2160

cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc 2220

aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc 2280

gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc 2340

acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg 2400

agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca 2460

gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg 2520

gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct 2580

gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacggggc 2640

tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa 2700

ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga 2760

agacagtggt gaacttccct gtagaagacg aggccctgggg tggcacggct tttgttcaga 2820

tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg 2880

tgcagagcga ctactccagc tatgcccggga cctccatcag agccagtctc accttcaacc 2940

gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt 3000

gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc acccaacatct 3060

acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc 3120

atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc 3180

tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg 3240

ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc 3300

tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gccagacgc 3360

agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg 3420

cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg 3480

agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc 3540

ccacacccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct 3600

gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc 3660

tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc 3720

agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc 3780

cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc 3840

caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt 3900

gccctgtaca caggcgagga ccctgcacct ggatggggggt ccctgtggggt caaattgggg 3960

ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa 4020

aaaaaaa 4027

<210> 65

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 65

tgaaggagcc cagagagaga 20

<210> 66

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 66

gtaagccaag aaagaaatcc 20

<210> 67

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 67

Gly Gly Ser Gly Gly Ser

15

<210> 68

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(3)

<223> /replace=" "

<220>

<221> additional feature

<222> (1)..(21)

<223> /note="Variants of residues given in sequences are not

have a preference for remainders in annotations for positions

options"

<400> 68

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 69

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(3)

<223> /replace=" "

<220>

<221> additional feature

<222> (1)..(22)

<223> / note="Residue variants given in sequences are not

have a preference for remainders in annotations for positions

options"

<400> 69

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 70

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(3)

<223> /replace=" "

<220>

<221> additional attribute

<222> (1)..(23)

<223> / note="Residue variants given in sequences are not

have a preference for remainders in annotations for positions

options"

<400> 70

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 71

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(3)

<223> /replace=" "

<220>

<221> additional feature

<222> (1)..(25)

<223> / note="Residue variants given in sequences are not

have a preference for remainders in annotations for positions

options"

<400> 71

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210> 72

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<220>

<221> OPTIONAL SIGN

<222> (1)..(30)

<223> /note="this sequence can span 1-6 repeats

elements "Gly Gly Gly Gly Ser""

<400> 72

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 73

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 73

ugucgggucu uuaaaaauac 20

<210> 74

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 74

uacaggcccc uaaagcacua 20

<210> 75

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 75

acaggccccu aaagcacuaa 20

<210> 76

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 76

aagcacuaag ggcaugcccu 20

<210> 77

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 77

gggcaugccc ucggugaaac 20

<210> 78

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 78

ggcaugcccu cggugaaaca 20

<210> 79

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 79

gcaugccuc ggugaaacag 20

<210> 80

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 80

ugagauuaaa gcgacagaaa 20

<210> 81

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 81

gagauuaaag cgacagaaaa 20

<210> 82

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 82

uaaagcgaca gaaaagggaa 20

<210> 83

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 83

agaaaaggga aggagagcg 20

<210> 84

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 84

gaaaagggaa aggagagcgc 20

<210> 85

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 85

ggaaaggaga gcgcgggcaa 20

<210> 86

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 86

gaaaggagag cgcgggcaac 20

<210> 87

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 87

gcgcgggcaa cgggaucuaa 20

<210> 88

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 88

cgcgggcaac gggaucuaaa 20

<210> 89

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 89

ucuaaaggga gauagagacg 20

<210> 90

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 90

cuaaagggag auagagacgc 20

<210> 91

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 91

gauagagacg cgggccucug 20

<210> 92

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 92

auagagacgc gggccucuga 20

<210> 93

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 93

gacgcgggcc ucugagggua 20

<210> 94

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 94

gcgggccucu gaggguaagg 20

<210> 95

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 95

cgggccucug aggguaaggu 20

<210> 96

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 96

gaggguaagg ugggcgcaag 20

<210> 97

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 97

gcaugcccuu agugcuuuag 20

<210> 98

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 98

ggcaugcccu uagugcuuua 20

<210> 99

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 99

gggcaugccc uuagugcuuu 20

<210> 100

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 100

gcgcuccccu guuucaccga 20

<210> 101

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 101

agcgcucccc uguuucaccg 20

<210> 102

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 102

ugcgcccacc uuacccucag 20

<210> 103

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 103

agagccggcg guagcggcag 20

<210> 104

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 104

ggcgguagcg gcaguggcag 20

<210> 105

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 105

caguggcagc ggcgagagcu 20

<210> 106

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 106

aguggcagcg gcgagagcuu 20

<210> 107

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 107

ggcagcggcg agagcuuggg 20

<210> 108

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 108

ccucgcgagc gccgcgcgcc 20

<210> 109

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 109

cucgcgagcg ccgcgcgccc 20

<210> 110

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 110

gcaagucacg uccgcccccu 20

<210> 111

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 111

ucacguccgc ccccucggcg 20

<210> 112

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 112

gcgcggccgc cccgagacgc 20

<210> 113

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 113

cugccuuaug aauauugaug 20

<210> 114

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 114

ccuuaugaau auugaugcgg 20

<210> 115

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 115

ugaauauuga ugcgggaggcu 20

<210> 116

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 116

ugcuuucgua gagaagcaga 20

<210> 117

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 117

agaagcagaa ggaagcaaga 20

<210> 118

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 118

aagcaagaug gcugcccuuu 20

<210> 119

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 119

cccuuuagga uuuguuagaa 20

<210> 120

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 120

ggagacccga cugcaacugc 20

<210> 121

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 121

gcaacugcug gauugcugca 20

<210> 122

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 122

gcuggauugc ugcaaggcug 20

<210> 123

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 123

cuggauugcu gcaaggcuga 20

<210> 124

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 124

aaggcugagg gacgagaacg 20

<210> 125

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 125

gcugccacug ccgcuaccgc 20

<210> 126

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 126

cgcucgcgag gaggcggcgg 20

<210> 127

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 127

cggcgcucgc gaggaggcgg 20

<210> 128

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 128

gcgcggcgcu cgcgaggagg 20

<210> 129

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 129

ggcgcgcggc gcucgcgagg 20

<210> 130

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 130

ccgggcgcgc ggcgcucgcg 20

<210> 131

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 131

gcgagcggga ccggggcgcg 20

<210> 132

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 132

cuugcaugcg agcgggaccc 20

<210> 133

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 133

acuugcaugc gagcgggacc 20

<210> 134

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 134

gacgugacuu gcaugcgagc 20

<210> 135

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 135

ggacgugacu ugcaugcgag 20

<210> 136

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 136

gggcggccgc gccgaggggg 20

<210> 137

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 137

ucggggcggc cgcgccgagg 20

<210> 138

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 138

cucggggcgg ccgcgccgag 20

<210> 139

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 139

ucucggggcg gccgcgccga 20

<210> 140

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 140

gucucggggc ggccgcgccg 20

<210> 141

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 141

gcggggccgg cgucucgggg 20

<210> 142

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 142

ucagcggggc cggcgucucg 20

<210> 143

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 143

cucagcgggg ccggcgucuc 20

<210> 144

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 144

acucagcggggccggcgucu 20

<210> 145

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 145

uucucauacac ucagcggggc 20

<210> 146

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 146

ucuguucuca ucacucagcg 20

<210> 147

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 147

gucuguucuc aucacucagc 20

<210> 148

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 148

cgucuguucu caucacucag 20

<210> 149

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 149

ccuccgcauc aauauucaua 20

<210> 150

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 150

ccuuucuaac aaauccuaaa 20

<210> 151

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 151

uccuuucuaa caaauccuaa 20

<210> 152

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 152

gcaauccagc aguugcaguc 20

<210> 153

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 153

agcaauccag caguugcagu 20

<210> 154

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 154

aaacucuguc uucucuaggc 20

<210> 155

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 155

uccuguugag uuacaacgcu 20

<210> 156

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 156

gaguuacaac gcuuggaagc 20

<210> 157

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 157

caacgcuugg aagcaggaga 20

<210> 158

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 158

aacgcuugga agcaggagau 20

<210> 159

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 159

ugggcucagc agcagccaau 20

<210> 160

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 160

cagccaauag gacaugaucc 20

<210> 161

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 161

augauccagg aagagcagua 20

<210> 162

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 162

ugaucgga agagcaguaa 20

<210> 163

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 163

gcaguaagggacugagcugc 20

<210> 164

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 164

cugagcugcu gguaagacag 20

<210> 165

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 165

gcaaguaaac aaucuugaga 20

<210> 166

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 166

ggcaaguaaa caaucuugag 20

<210> 167

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 167

uuguaacuca acaggagcaa 20

<210> 168

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 168

uccaagcguu guaacucaac 20

<210> 169

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 169

cuuccuggau cauguccuau 20

<210> 170

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 170

cagucccuua cugcucuucc 20

<210> 171

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 171

gcucuuuaga auucaacuag 20

<210> 172

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 172

cucuuuagaa uucaacuaga 20

<210> 173

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 173

ucaacuagag ggcagccuug 20

<210> 174

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 174

cuaggggca gccuugugga 20

<210> 175

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 175

uggccccgaa gcaagccuga 20

<210> 176

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 176

cgaagcaagc cugauggaac 20

<210> 177

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 177

ggauagaacc aaccaugugug 20

<210> 178

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 178

gaugaacca accauguuga 20

<210> 179

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 179

cauuugccag acagaaccuc 20

<210> 180

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 180

cuggcuacaa agcuccagaa 20

<210> 181

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 181

agagcucauc cagaaguaaa 20

<210> 182

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 182

aaguaaaugg agacaccaag 20

<210> 183

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 183

cacucuuuca aaaguuauua 20

<210> 184

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 184

uuauggaaua cccuguauga 20

<210> 185

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 185

uuggaauac ccuguaugaa 20

<210> 186

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 186

gacuuuacac aagaaaguag 20

<210> 187

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 187

acuuuacaca agaaaguaga 20

<210> 188

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 188

uauuccaagu guuugcaaaa 20

<210> 189

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 189

uccaaguguu ugcaaaaugg 20

<210> 190

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 190

guuagugaac cuucucucuc 20

<210> 191

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 191

uuugugaacc uucucucucu 20

<210> 192

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 192

gaaauugaaa caagaccaaa 20

<210> 193

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 193

aaaaagacc aaaaggcuaa 20

<210> 194

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 194

aauggagaaa gacguaacuu 20

<210> 195

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 195

aggagaaag acguaacuuc 20

<210> 196

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 196

uggagaaaga cguaacuucg 20

<210> 197

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 197

guaagccaag aaagaaaucc 20

<210> 198

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 198

uuuucaacac auaacugcag 20

<210> 199

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 199

uuucaacaca uaacugcagu 20

<210> 200

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 200

gcuucagauu cugaaugagc 20

<210> 201

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 201

ucagauucug augagcagg 20

<210> 202

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 202

cagauucuga augagcagga 20

<210> 203

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 203

agauucugaa ugagcaggag 20

<210> 204

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 204

cauuguauua cuuaaaaaca 20

<210> 205

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 205

aaggcagugc uaaugccuaa 20

<210> 206

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 206

uacaguuucu gccucuuccg 20

<210> 207

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 207

ucuuccgugg aacacacaca 20

<210> 208

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 208

acacacacau ggugaacucc 20

<210> 209

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 209

uccagauuguguuuccauug 20

<210> 210

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 210

cauaaaugcc auuaacaguc 20

<210> 211

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 211

acucacccau cgcauaccuc 20

<210> 212

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 212

cucacccauc gcauaccuca 20

<210> 213

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 213

gccuccaaag ccagcugcag 20

<210> 214

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 214

gccagcugca guggugagug 20

<210> 215

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 215

uccuggagaa aauaacaucc 20

<210> 216

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 216

ccugcagaaa auaacaucca 20

<210> 217

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 217

ggaaccacaa agcuagcguc 20

<210> 218

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 218

ucggugaag aauucuguuc 20

<210> 219

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 219

agcagcaauu ugcaagcucc 20

<210> 220

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 220

agcaauuugc aagcuccugg 20

<210> 221

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 221

cuccuggugg cagcucugaa 20

<210> 222

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 222

uuaaaacaaaaugaaaugaa 20

<210> 223

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 223

gcaaagcuca guguucacua 20

<210> 224

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 224

ucccccuccu ccucuuccac 20

<210> 225

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 225

guuccucagc uuccuucaga 20

<210> 226

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 226

gaaggaaaaa gcacucugaa 20

<210> 227

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 227

ggaaaaagca cucugaaugg 20

<210> 228

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 228

aaaguaacac aacacuuuua 20

<210> 229

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 229

aaguaacaca acacuuuuaa 20

<210> 230

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 230

uuuaagggaa gugaaaauag 20

<210> 231

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 231

uuaagggaag ugaaaauaga 20

<210> 232

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 232

gaaaauagag gguaaaccug 20

<210> 233

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 233

cuucuccgau gcuuucugaa 20

<210> 234

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 234

cucagaauaa uugugugaac 20

<210> 235

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 235

aggaaugaca uacagacugc 20

<210> 236

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 236

ggaaugacauacagacugca 20

<210> 237

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 237

aagcauaacc caccaauuuu 20

<210> 238

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 238

ccaccaauuu uugguagcag 20

<210> 239

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 239

ugguagcagu ggagagcuac 20

<210> 240

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 240

caaaagcaa gagauucuga 20

<210> 241

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 241

aaagagcaagagauucugaa 20

<210> 242

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 242

gauucugaag ggucgagaca 20

<210> 243

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 243

acacagcacu aucugaaacc 20

<210> 244

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 244

agcacuaucu gaaaccagga 20

<210> 245

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 245

accaggaugg auugaauuga 20

<210> 246

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 246

ggccccucgu uuucaccaag 20

<210> 247

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 247

aucccaucua aaacguaaug 20

<210> 248

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 248

augaccucca aacaauacac 20

<210> 249

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 249

acuggaaauu ccaacaugcc 20

<210> 250

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 250

cuggaaauuc caacaugccu 20

<210> 251

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 251

uggaaauucc aacaugccug 20

<210> 252

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 252

ggaaauucca acaugccugg 20

<210> 253

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 253

gaaauuccaa caugccuggg 20

<210> 254

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 254

acaugccuggggggcuccca 20

<210> 255

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 255

cacccagaaa acaacacagc 20

<210> 256

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 256

uaccaaguug aaaugaauca 20

<210> 257

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 257

accaaguugaaaugaaucaa 20

<210> 258

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 258

augaaucaag ggcaguccca 20

<210> 259

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 259

agggcagucc caagguacag 20

<210> 260

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 260

guuccaaaaa cccucacacc 20

<210> 261

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 261

gcucaugugc agucacugug 20

<210> 262

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 262

gaaacagcac uugaaucaac 20

<210> 263

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 263

gcaacauaag ccucauaaac 20

<210> 264

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 264

auuacaaaua aagaauaaag 20

<210> 265

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 265

aacaugauc agcaaagaga 20

<210> 266

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 266

caaagagaag gaucauucuu 20

<210> 267

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 267

auucuuuggc cagacuaaag 20

<210> 268

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 268

aaauggaag aauguuuuca 20

<210> 269

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 269

cgagacucau aauguccaaa 20

<210> 270

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 270

gagacucaua auguccaaau 20

<210> 271

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 271

ucauaauguc caaaugggac 20

<210> 272

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 272

uaauguccaa augggacugg 20

<210> 273

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 273

aucaagugca ugcaaaauac 20

<210> 274

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 274

acacauccug aacuuuuugc 20

<210> 275

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 275

caaagcaaga ucuucuucac 20

<210> 276

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 276

uuucaagaac 20

<210> 277

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 277

acaacaagcu ucaguucuac 20

<210> 278

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 278

caacaagcuu caguucuaca 20

<210> 279

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 279

aauagaaacc aagauauguc 20

<210> 280

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 280

cugcgcaacu ugcucagcaa 20

<210> 281

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 281

uguuuuuccu gugccugacc 20

<210> 282

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 282

guuuuuccug ugccugacca 20

<210> 283

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 283

uuuccugugc cugaccaggg 20

<210> 284

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 284

cacucagacc ccuccccaga 20

<210> 285

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 285

cucaaaagca ugcugcucua 20

<210> 286

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 286

aaaagcaugc ugcucuaagg 20

<210> 287

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 287

cuugccaauag ucagaugcac 20

<210> 288

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 288

ucagaugcac aggccaauua 20

<210> 289

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 289

gaugcacagg ccaauuaagg 20

<210> 290

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 290

aggccaauua agguggaacc 20

<210> 291

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 291

caccaccaga 20

<210> 292

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 292

agaaaacaaa acauggaaaa 20

<210> 293

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 293

aaagagcauc auugagacca 20

<210> 294

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 294

caagucguua uuugaccaua 20

<210> 295

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 295

caaguaaaag uugaaauguc 20

<210> 296

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 296

aaguaaaagu ugaaauguca 20

<210> 297

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 297

uacucuaua aaaaauuuau 20

<210> 298

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 298

uucccaucuu gcagaugugugu 20

<210> 299

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 299

cagaaaugua cugagacaca 20

<210> 300

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 300

agcaaauuua ucuucagaua 20

<210> 301

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 301

gcaaauuuau cuucagaauau 20

<210> 302

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 302

cuuuuuuuaa aucuugaguc 20

<210> 303

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 303

agucuggcag caauuuguaa 20

<210> 304

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 304

gcucuuugua uauuuaucucc 20

<210> 305

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 305

uaucuccugg agagacagcu 20

<210> 306

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 306

augagaaaa uaacgaccau 20

<210> 307

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 307

uuuuuuuuuuuuuuuuca 20

<210> 308

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 308

uauuaguuuc acaagauuuc 20

<210> 309

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 309

ucacaagauu ucuggcuaau 20

<210> 310

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 310

cacagaauuu cuggcuaaua 20

<210> 311

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 311

uaucuucagu cuucaugagu 20

<210> 312

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 312

aucuucaguc uucaugaguu 20

<210> 313

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 313

ucuucagucu ucaugaguug 20

<210> 314

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 314

cuucagucuu caugaguugg 20

<210> 315

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 315

cuuuucucca uuuuuacauu 20

<210> 316

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 316

aaagcuuuuu guuaaaauuc 20

<210> 317

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 317

uaaaauucag gauauguaau 20

<210> 318

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 318

aggauaugua auaggucugu 20

<210> 319

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 319

uugugaaaua uuuuugcuga 20

<210> 320

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 320

gauggaugua gauauauacg 20

<210> 321

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 321

agacaaaugu uaaauuagug 20

<210> 322

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 322

gauaccccac acuguguaga 20

<210> 323

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 323

ccccacacug uguagaagga 20

<210> 324

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 324

cacacugugu agaaggaugg 20

<210> 325

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 325

acacugugua gaaggaugga 20

<210> 326

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 326

cuguguagaa ggauggaggg 20

<210> 327

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 327

cuacuguccc ucuuugcgug 20

<210> 328

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 328

ugguuauuaa guugccucac 20

<210> 329

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 329

gguuauuaag uugccucacu 20

<210> 330

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 330

cacaucucau agauaauauu 20

<210> 331

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 331

ucccacuuuu ccaucuuugu 20

<210> 332

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 332

uucuuuuugc cugacucucc 20

<210> 333

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 333

uucuaaagua cauacuaaua 20

<210> 334

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 334

ucuaaaguac auacuaauau 20

<210> 335

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 335

aguacauacu aauauggguc 20

<210> 336

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 336

aaacagcaau uaaauguuau 20

<210> 337

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 337

aacagcaauu aaauguuaua 20

<210> 338

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 338

auuaaauguu auagggaagu 20

<210> 339

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 339

auagggaagu aggaagaaaa 20

<210> 340

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 340

uagggaagua ggaagaaaaa 20

<210> 341

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 341

agggaaguag gaagaaaaag 20

<210> 342

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 342

caauaaacca agcaauauuc 20

<210> 343

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 343

aauaaaccaa gcaauauucu 20

<210> 344

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 344

auaaaccaag caauauucug 20

<210> 345

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 345

uaaaccaagc aauauucugg 20

<210> 346

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 346

accaagcaau auucuggggg 20

<210> 347

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 347

ccaagcaaua uucugggggu 20

<210> 348

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 348

uucugggggu gggauagagc 20

<210> 349

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 349

ucuuuuaaaa uccaaguaau 20

<210> 350

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 350

uuaaaaucca aguaauaggu 20

<210> 351

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 351

uuuuuuccag cucaaaaaau 20

<210> 352

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 352

uuuguuuagu uucauuuuuu 20

<210> 353

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 353

uguacauaua cuuaauuaug 20

<210> 354

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 354

uagagcccuu aauguguagu 20

<210> 355

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 355

agagcccuua auguguaguu 20

<210> 356

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 356

gagcccuuaa uguguaguug 20

<210> 357

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 357

agcccuuaau guguaguugg 20

<210> 358

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 358

uaguugggg uuaagcuuug 20

<210> 359

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 359

cuuuauauuu aguauaauug 20

<210> 360

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 360

caaauuauug aaaaagauga 20

<210> 361

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 361

gguccuuuuu auacccaucu 20

<210> 362

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 362

uuuuauaccc aucuaggagc 20

<210> 363

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 363

ucuaggagca gguccuaaug 20

<210> 364

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 364

ggcagcuauu agagaaauca 20

<210> 365

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 365

uuagagaaau cauggaagaa 20

<210> 366

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 366

aagaaaggua auuaacgcaa 20

<210> 367

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 367

gguaauuaac gcaaaggcac 20

<210> 368

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 368

guaauuaacg caaaggcaca 20

<210> 369

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 369

uaaauugagu aauuauuagu 20

<210> 370

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 370

uuaguaggcu uagcuauucu 20

<210> 371

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 371

uaguaggcuu agcuauucua 20

<210> 372

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 372

agagagucac aauauuugac 20

<210> 373

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 373

aggacuaua gucugcuagc 20

<210> 374

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 374

aauagucugc uagcuggcac 20

<210> 375

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 375

acaggcugcc cacuuugcga 20

<210> 376

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 376

cgauggaugc cagaaaaccc 20

<210> 377

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 377

cagaaaaccc aggcaugaac 20

<210> 378

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 378

acccaggcau gaacaggaau 20

<210> 379

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 379

ugaacaggaa ucggccagcc 20

<210> 380

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 380

cagccaggcu gccagccaca 20

<210> 381

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 381

ggcugccagc cacaagguac 20

<210> 382

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 382

cagccacaag guacuggcac 20

<210> 383

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 383

cuggcacagg cuccaacgag 20

<210> 384

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 384

uccaacgaga gguccacuc 20

<210> 385

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 385

aagugucaaa gcagaaagac 20

<210> 386

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 386

gcagaaagac ugguaaagug 20

<210> 387

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 387

uuuuuuucgc uaucaaucac 20

<210> 388

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 388

ugagcgagau aaugcagaga 20

<210> 389

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 389

cucugagcug uucuucuucu 20

<210> 390

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 390

ucugagcugu ucuucuucua 20

<210> 391

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 391

uuucauuaag 20

<210> 392

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 392

gugccuuuuc auuaagaggu 20

<210> 393

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 393

guauuauuau uaaaguacuu 20

<210> 394

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 394

uuaaaguacu uaggauacau 20

<210> 395

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 395

uaaaguacuu aggauacauu 20

<210> 396

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 396

aaaguacuua ggauacauug 20

<210> 397

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 397

uaggauacau uggggcagcu 20

<210> 398

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 398

uucacuaaau aaucaucuag 20

<210> 399

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 399

uaaauaauca ucuaguggcc 20

<210> 400

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 400

uuguuuuuua aacaagcagu 20

<210> 401

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 401

uuuuuuaaac aagcaguagg 20

<210> 402

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 402

acaagcagua gguggugcuu 20

<210> 403

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 403

uagguggugc uuuggucaua 20

<210> 404

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 404

agguggugcu uuggucauaa 20

<210> 405

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 405

ggaagauaua gucuauuucu 20

<210> 406

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 406

acuauuccau auuuuccaug 20

<210> 407

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 407

uuccauauuu uccauguggc 20

<210> 408

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 408

ucuaaauugu gagacauucu 20

<210> 409

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 409

aaauugugag acauucuugg 20

<210> 410

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 410

uaaaauagcu aaauuuagua 20

<210> 411

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 411

aaaauagcua aauuuaguaa 20

<210> 412

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 412

aucuguacau uuugauauug 20

<210> 413

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 413

uuugauauug aggaaaaaca 20

<210> 414

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 414

aaaccauuau ccaguuugcu 20

<210> 415

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 415

uauaaaccg uucauuucuc 20

<210> 416

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 416

accguucauu ucucaggaug 20

<210> 417

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 417

uaguugaauu cuaaagagca 20

<210> 418

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 418

uugcuucggg gccauccaca 20

<210> 419

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 419

guuccaucag gcuugcuucg 20

<210> 420

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 420

uguuccauca ggcuugcuuc 20

<210> 421

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 421

cuguuccauc aggcuugcuu 20

<210> 422

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 422

ugguucuauc cuguuccauc 20

<210> 423

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 423

ucuguugccc ucaacauggu 20

<210> 424

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 424

uuagucuguu gcccucaaca 20

<210> 425

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 425

ggaggugaug guaucaggaa 20

<210> 426

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 426

aaaugggagg ugaugguauc 20

<210> 427

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 427

gucggcaaa ugggagguga 20

<210> 428

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 428

gguucugucu ggcaaauggg 20

<210> 429

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 429

agagguucug ucuggcaaau 20

<210> 430

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 430

cagagguucu gucuggcaaa 20

<210> 431

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 431

uuguagccag agguucuguc 20

<210> 432

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 432

uucuggagcu uuguagccag 20

<210> 433

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 433

caggcagugg gcuuccauuc 20

<210> 434

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 434

gaugagcucu cucaggcagu 20

<210> 435

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 435

ggaugagcuc ucucaggcag 20

<210> 436

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 436

acuucuggau gagcucucuc 20

<210> 437

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 437

uuggugucuc cauuuacuuc 20

<210> 438

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 438

acuuuugaaa gagugccacu 20

<210> 439

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 439

uucuggcuuc ccuucauaca 20

<210> 440

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 440

auucuggcuu cccuucauac 20

<210> 441

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 441

caggacucac acgacuauuc 20

<210> 442

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 442

cuacuuucuu guguaaaguc 20

<210> 443

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 443

uccuccauuu ugcaaacacu 20

<210> 444

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 444

ugaaggagcc cagagagaga 20

<210> 445

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 445

guuucaauuu cuugaucuga 20

<210> 446

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 446

gucuuucucc auuagccuuuu 20

<210> 447

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 447

uuucaccugg auuucuuucu 20

<210> 448

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 448

uuugguugac ugcuuucacc 20

<210> 449

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 449

ucacucaaau cggagacauu 20

<210> 450

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 450

uucuuucuua ucacucaaau 20

<210> 451

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 451

aucuuuaacu gcauuuucuu 20

<210> 452

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 452

aaucuuuaac ugcauuuucu 20

<210> 453

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 453

gcaguuauguguugaaaaac 20

<210> 454

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 454

aucugaagcu cuggauuuuc 20

<210> 455

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 455

ucauucagaa ucugaagcuc 20

<210> 456

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 456

guaauacau guucuuguca 20

<210> 457

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 457

gcagaaacug uagcaccauu 20

<210> 458

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 458

auguggugugu uccacggaag 20

<210> 459

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 459

uucaccauguguguguucca 20

<210> 460

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 460

auugagacag uguuuuuucc 20

<210> 461

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 461

accgcaaugg aaacacaauc 20

<210> 462

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 462

ugugguuuuc ugcaccgcaa 20

<210> 463

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 463

aauggcauuu augugagaug 20

<210> 464

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 464

auuaguagcc ugacuguuaa 20

<210> 465

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 465

cgauggguga gugaucucac 20

<210> 466

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 466

ucugccuga gguaugcgau 20

<210> 467

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 467

aucugcccug agguaugcga 20

<210> 468

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 468

ugcggaauug aucugccug 20

<210> 469

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 469

cucagaguua gaggucugug 20

<210> 470

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 470

uggaggcagc ucagaguuag 20

<210> 471

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 471

accacugcag cuggcuuugg 20

<210> 472

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 472

cucaccacug cagcuggcuu 20

<210> 473

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 473

gccucacuca cccugcagc 20

<210> 474

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 474

aucagcauca ucagcaucac 20

<210> 475

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 475

uagcauugca gcuaguuuac 20

<210> 476

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 476

uucugguuuc ugaaaggaac 20

<210> 477

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 477

uaguuguucu gguuucugaa 20

<210> 478

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 478

uuuuguuguu guaguuguuc 20

<210> 479

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 479

uguuauuuuc ugcaggagau 20

<210> 480

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 480

auguuauuuu cugcaggaga 20

<210> 481

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 481

cccggaugu uauuuucugc 20

<210> 482

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 482

acgcuagcuu ugugguuccc 20

<210> 483

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 483

uucaccagac gcuagcuuug 20

<210> 484

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 484

aggagcuugc aaauugcugc 20

<210> 485

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 485

uaccguucag agcugccacc 20

<210> 486

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 486

accacaccaau cacccagaaa 20

<210> 487

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 487

accuguggaa gaggaggagg 20

<210> 488

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 488

aaccugga agaggaggag 20

<210> 489

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 489

gaaccugugg aagaggagga 20

<210> 490

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 490

gaaccugug gaagaggagg 20

<210> 491

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 491

guggaagagg 20

<210> 492

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 492

agcuggaa ccuguggaag 20

<210> 493

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 493

gaaggaagcu gaggaaccug 20

<210> 494

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 494

uuuccuucug aaggaagcug 20

<210> 495

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 495

agagugcuuu uuccuucuga 20

<210> 496

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 496

uacuuugguu gggguagugg 20

<210> 497

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 497

uguuacuuug guugggguag 20

<210> 498

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 498

guguuguguu acuuugguug 20

<210> 499

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 499

aguguugugu uacuuugguu 20

<210> 500

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 500

aaguguugug uuacuuuggu 20

<210> 501

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 501

uuaaaagugu uguguuacuu 20

<210> 502

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 502

cucugggaag guggugccuc 20

<210> 503

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 503

ggauuaggac ucgggaagg 20

<210> 504

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 504

guggauuag gacucuggga 20

<210> 505

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 505

Uguagaugga uuaggacucu 20

<210> 506

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 506

guguagaugg auuaggacuc 20

<210> 507

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 507

cauacaugug uagauggauu 20

<210> 508

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 508

gggcugcaua cauguguaga 20

<210> 509

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 509

uuucagaaag caucggagaa 20

<210> 510

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 510

cuuucagaaa gcaucggaga 20

<210> 511

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 511

ugaggccuuu cagaaagcau 20

<210> 512

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 512

cuguucacac aauuauucug 20

<210> 513

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 513

cuuguuuucu cagaacacaa 20

<210> 514

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 514

ugcuugaggu guucugacau 20

<210> 515

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 515

aaauuggugg guuaugcuug 20

<210> 516

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 516

cacugcuacc aaaaauuggu 20

<210> 517

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 517

ccacugcuac caaaaauugg 20

<210> 518

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 518

ucuccacugc uaccaaaaau 20

<210> 519

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 519

cuuuguuucu caucaacugc 20

<210> 520

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 520

uucagauagu gcuguguugg 20

<210> 521

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 521

uuucagauag ugcuguguug 20

<210> 522

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 522

guuucagaua gugcuguguu 20

<210> 523

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 523

gguuucagau agugcugugu 20

<210> 524

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 524

gccuucaauu caauccaucc 20

<210> 525

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 525

uuccgcuugg ugaaaacgag 20

<210> 526

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 526

auuccgcuug gugaaaacga 20

<210> 527

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 527

gauuccgcuu ggugaaaacg 20

<210> 528

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 528

guuuuagaug ggauuccgcu 20

<210> 529

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 529

ugccucauua cguuuuagau 20

<210> 530

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 530

augccucauu acguuuuaga 20

<210> 531

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 531

gguugauacu gaagaauuga 20

<210> 532

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 532

gucauuugau uggagagauu 20

<210> 533

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 533

ggucauuuga uuggagagau 20

<210> 534

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 534

uuguuuggag gucauuugau 20

<210> 535

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 535

auuuccagug uauuguuugg 20

<210> 536

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 536

ggaauuucca guguauuguu 20

<210> 537

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 537

ugggagcccc ccaggcaugu 20

<210> 538

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 538

gcuugccuug ggagcccccc 20

<210> 539

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 539

ucugggugua agcuugccuu 20

<210> 540

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 540

uucugggugu aagcuugccu 20

<210> 541

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 541

cuccagcugu guuguuuucu 20

<210> 542

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 542

gcuccagcug uguuguuuuc 20

<210> 543

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 543

gcccuugauu cauuucaacu 20

<210> 544

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 544

auguuggucc acuguaccuu 20

<210> 545

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 545

gauguugguc cacuguaccu 20

<210> 546

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 546

guuuuuggaa cuggagaugu 20

<210> 547

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 547

ggugugaggg uuuuuggaac 20

<210> 548

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 548

gcaccuggug ugaggguuuu 20

<210> 549

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 549

gagaagugca ccugguguga 20

<210> 550

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 550

ggagaagugc accuggugug 20

<210> 551

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 551

cuguuuugga gaagugcacc 20

<210> 552

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 552

uuuugguaaa uggucuguuu 20

<210> 553

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 553

gcacaugagc uuuugguaaa 20

<210> 554

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 554

agugacugca caugagcuuu 20

<210> 555

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 555

ggacauaagu uuuucaguuu 20

<210> 556

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 556

gggacauaag uuuuucaguu 20

<210> 557

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 557

caagugcugu uucaacacug 20

<210> 558

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 558

uuucaacacu 20

<210> 559

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 559

uucaagugcu guuucaacac 20

<210> 560

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 560

aaaaggugug aguuugaaaa 20

<210> 561

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 561

uaugaggcuu auguugcaaa 20

<210> 562

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 562

guuugugcug ccuguuuaug 20

<210> 563

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 563

gggagaugug aacucuggga 20

<210> 564

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 564

uugagggaga ugugaacucu 20

<210> 565

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 565

uuugagggag augugaacuc 20

<210> 566

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 566

gcugcuguug cugguuuuga 20

<210> 567

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 567

ugcugcuguu gcugguuuug 20

<210> 568

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 568

guaauuuuug cugcuguugc 20

<210> 569

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 569

uuugggggg aggaaaaguc 20

<210> 570

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 570

ucauuguugc uuuggggggug 20

<210> 571

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 571

gcugaucauu guugcuuugg 20

<210> 572

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 572

ugcugaucau uguugcuuug 20

<210> 573

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 573

uugcugauca uuguugcuuu 20

<210> 574

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 574

uuugcugauc auuguugcuu 20

<210> 575

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 575

aacauucuuc cacuuuaguc 20

<210> 576

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 576

guacuuccuc cagucccauu 20

<210> 577

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 577

uuucaugguc ugacuauaag 20

<210> 578

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 578

auuucauggu cugacuauaa 20

<210> 579

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 579

gauuucaugg ucugacuaua 20

<210> 580

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 580

uuugcaugca cuugauuuca 20

<210> 581

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 581

guucuuuauu cucugaaacu 20

<210> 582

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 582

uuguuuccug caaaaaguuc 20

<210> 583

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 583

uugcauguga ugcaaguuuu 20

<210> 584

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 584

augcaugug augcaaguuu 20

<210> 585

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 585

ugcuuuggga ucacauuauuu 20

<210> 586

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 586

ugugaagaag aucuugcuuu 20

<210> 587

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 587

cugugaagaa gaucuugcuu 20

<210> 588

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 588

cuuguugacc agacauaucu 20

<210> 589

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 589

gcacaggaaa aacauuugca 20

<210> 590

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 590

cuuccucccu ggucaggcac 20

<210> 591

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 591

gugugacuuc cucccugguc 20

<210> 592

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 592

ucugagugug acuucccccc 20

<210> 593

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 593

uugagugucc uucuggggag 20

<210> 594

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 594

uuugaguguc cuucugggga 20

<210> 595

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 595

uuuugagugu ccuucugggg 20

<210> 596

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 596

ugcuuuugag uguccuucug 20

<210> 597

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 597

augcuuuuga guguccuucu 20

<210> 598

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 598

caugcuuuug aguguccuuc 20

<210> 599

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 599

cuauggcaag acucaguuug 20

<210> 600

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 600

acuauggcaa gacucaguuu 20

<210> 601

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 601

gacuauggca agacucaguu 20

<210> 602

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 602

uuggcugug caucugacua 20

<210> 603

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 603

cauccagguu ccaccuuaau 20

<210> 604

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 604

caggcaugug gcuugcaucc 20

<210> 605

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 605

gcugugugca uacaggcaug 20

<210> 606

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 606

uggggugcu gugugcauac 20

<210> 607

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 607

uuccauguuu uguuuucugg 20

<210> 608

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 608

uuuuuccaug uuuuguuuuc 20

<210> 609

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 609

acauuaucac agcuugcagg 20

<210> 610

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 610

ugcacauuau cacagcuugc 20

<210> 611

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 611

cugcuucaga ugcugcucca 20

<210> 612

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 612

cuuaugguca aauaacgacu 20

<210> 613

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 613

auuugagagu aagagccuua 20

<210> 614

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 614

ugucuaguca aaacugugac 20

<210> 615

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 615

gcuaucaagu ucugcagcag 20

<210> 616

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 616

gcugcucuaa agcuggggug 20

<210> 617

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 617

uguuugcugc ucuaaagcug 20

<210> 618

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 618

uuguuugcug cucuaaagcu 20

<210> 619

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 619

guuguuugcu gcucuaaagc 20

<210> 620

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 620

gaagcagcug uucuuuuggu 20

<210> 621

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 621

aacagaagca gcuguucuuu 20

<210> 622

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 622

ggaguaucua guaauuugga 20

<210> 623

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 623

uauaggagua ucuaguaauu 20

<210> 624

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 624

guauccaaua aauuuuuuau 20

<210> 625

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 625

aaucauauu gagucuugac 20

<210> 626

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 626

uaccuacaca ucugcaagau 20

<210> 627

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 627

uuaccuacac aucugcaaga 20

<210> 628

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 628

caugugucuc aguacauuuc 20

<210> 629

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 629

gaagauaaau uugcuaauuc 20

<210> 630

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 630

acucaagauu uaaaaaaaga 20

<210> 631

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 631

cuuucacaag acacaagcau 20

<210> 632

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 632

gcacgauuau uuaauucuuu 20

<210> 633

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 633

uuuuuacagga ucugaagaga 20

<210> 634

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 634

auuuuacagg aucugaagag 20

<210> 635

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 635

cagauacauu caaauuuuuac 20

<210> 636

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 636

uaauauacaa agagcuaaau 20

<210> 637

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 637

ugcugccuag cugucucucc 20

<210> 638

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 638

uucguaacauu agacugccua 20

<210> 639

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 639

aauggagaaaaggaaacuuu 20

<210> 640

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 640

caaauguaua aauggagaaa 20

<210> 641

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 641

caacauucca aauguauaaaa 20

<210> 642

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 642

agaugaaauu uuagagaaaa 20

<210> 643

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 643

aagaugaaau uuuagagaaa 20

<210> 644

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 644

agggaaaaca uggcacgggu 20

<210> 645

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 645

caagagggaa aacauggcac 20

<210> 646

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 646

gcaagaggga aaacauggca 20

<210> 647

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 647

ucauugcaag agggaaaaca 20

<210> 648

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 648

uggguaucu cauugcaaga 20

<210> 649

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 649

gugggguauc ucauugcaag 20

<210> 650

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 650

ccauccuucu acacagugug 20

<210> 651

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 651

uccauccuuc uacacagugu 20

<210> 652

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 652

cuccauccuu cuacacagug 20

<210> 653

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 653

cacacgcaaa gagggacagu 20

<210> 654

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 654

uuaauaacca cacgcaaaga 20

<210> 655

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 655

cuuaauaacc acacgcaaag 20

<210> 656

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 656

ugugguguuu uagcccagug 20

<210> 657

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 657

aauauuaucu augagaugug 20

<210> 658

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 658

aaaagggga agauaggggu 20

<210> 659

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 659

gaaaagggg aagauagggg 20

<210> 660

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 660

auggaaaagu gggaagauag 20

<210> 661

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 661

guggaaaag ugggaagaua 20

<210> 662

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 662

agauggaaaa gugggagau 20

<210> 663

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 663

accaaaaag auggaaaagu 20

<210> 664

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 664

aaccaacaaa guggaaaag 20

<210> 665

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 665

cuguugcaaa ccaacaaaga 20

<210> 666

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 666

gagagucagg caaaaagaag 20

<210> 667

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 667

ggagagucag gcaaaaagaa 20

<210> 668

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 668

uggagaguca ggcaaaaaga 20

<210> 669

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 669

agagaaaauc cuggagaguc 20

<210> 670

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 670

uuuaugauga gagaaaaucc 20

<210> 671

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 671

uuuugcuugg uuuuuguca 20

<210> 672

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 672

cccaccccca gaauauugcu 20

<210> 673

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 673

cuggaagccu accuauuacu 20

<210> 674

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 674

aaaaaacauu uaaagcuaac 20

<210> 675

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 675

uacaauccaa uuuuuugagc 20

<210> 676

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 676

cuaaacaaag aauacaguga 20

<210> 677

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 677

acuaaacaaa gaauacagug 20

<210> 678

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 678

auauauuaca uuucagauau 20

<210> 679

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 679

aauauauuac auuucagaua 20

<210> 680

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 680

uauauguaca ugcugguugu 20

<210> 681

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 681

aauuaaguauauguacaugc 20

<210> 682

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 682

cuuuaaaaug aguagauuga 20

<210> 683

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 683

aacccccaac uacacauuaa 20

<210> 684

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 684

uaacccccaa cuacacauua 20

<210> 685

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 685

cucaauuaua cuaaauuaaa 20

<210> 686

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 686

gcuccuagau ggguauaaaa 20

<210> 687

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 687

auuaggaccu gcuccuagau 20

<210> 688

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 688

cauuaggacc ugcuccuaga 20

<210> 689

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 689

ucucuaauag cugccacauu 20

<210> 690

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 690

aaaauucuga cauauacaaa 20

<210> 691

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 691

acugcuuugugugugaaggc 20

<210> 692

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 692

guuuacugcu uuguguguga 20

<210> 693

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 693

aauagcacag uguguagugugu 20

<210> 694

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 694

ugucaaauau ugugacucuc 20

<210> 695

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 695

ucggcaucc ucgcaaagu 20

<210> 696

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 696

uucuggcauc caucgcaaag 20

<210> 697

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 697

cuguucaugc cuggguuuuc 20

<210> 698

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 698

gccgauuccu guucaugccu 20

<210> 699

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 699

ggccgauucc uguucaugcc 20

<210> 700

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 700

cuuguggcug gcagccuggc 20

<210> 701

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 701

guaccuugug gcuggcagcc 20

<210> 702

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 702

cugugccagu accuuguggc 20

<210> 703

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 703

gagccugugc caguaccuug 20

<210> 704

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 704

gccagagugg gaccucucgu 20

<210> 705

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 705

ucagguggga aagccagagu 20

<210> 706

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 706

aucaggggg aaagccagag 20

<210> 707

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 707

uugacacuuu auuaucaggu 20

<210> 708

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 708

uuugacacuu uauuaucagg 20

<210> 709

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 709

ugcuuugaca cuuuauuauc 20

<210> 710

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 710

acuaggugaa uuuaauucag 20

<210> 711

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 711

aaguaacucau uugcaacacu 20

<210> 712

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 712

ucacacuugc ucucuuuuua 20

<210> 713

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 713

auagcgaaaaaaaaaaaaaa 20

<210> 714

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 714

ucuucuacau gcaggaguaa 20

<210> 715

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 715

cauaagaguc uucuacaugc 20

<210> 716

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 716

gcuguauaaa uuuauaugaa 20

<210> 717

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 717

cugccuaccu cuuaaugaaa 20

<210> 718

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 718

agaaaugaau aauuuggaaa 20

<210> 719

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 719

uaauuuagaa augaauaauu 20

<210> 720

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 720

ggaaauucac uauuucugcc 20

<210> 721

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 721

guuguuuuuu uuggcacuua 20

<210> 722

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 722

uguuguuuuu uuuggcacuu 20

<210> 723

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 723

uguuuuuuug uuguuuuuuu 20

<210> 724

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 724

auccagcac auggaaaaua 20

<210> 725

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 725

auaguuagua uccagccaca 20

<210> 726

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 726

cacaauuuag aaaagggaggc 20

<210> 727

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 727

gucucacaau uuagaaaagg 20

<210> 728

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 728

aaugucucac aauuuagaaa 20

<210> 729

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 729

uuuagcuauu uuaaaacuug 20

<210> 730

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 730

auuuagcuau uuuaaaacuu 20

<210> 731

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 731

aauuuagcua uuuuaaaacu 20

<210> 732

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 732

uuucacaaag cacaaaauuc 20

<210> 733

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 733

aauuacaugu gggugaaaau 20

<210> 734

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 734

aaauuacaug ugggugaaaa 20

<210> 735

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 735

cuauuuugua aauuacaugugu 20

<210> 736

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 736

acuauuuugu aaauuacaug 20

<210> 737

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 737

acgccaagca aacuggauaa 20

<210> 738

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 738

caggucuacg ccaagcaaac 20

<210> 739

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 739

cgguuuauua uuuuuuaaac 20

<210> 740

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 740

accacauccu gagaaaugaa 20

<210> 741

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 741

cuguggguuu cuuuaagguu 20

<210> 742

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 742

uucuuuaagg uuuggacaga 20

<210> 743

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 743

ucuuuaaggu uuggacagaa 20

<210> 744

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 744

gacagaaggg uaaagcuauu 20

<210> 745

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 745

auugaaagag ucaucuauac 20

<210> 746

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 746

gucaucuaua cugguaaaga 20

<210> 747

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 747

uaaagaaggc aaaaguucuc 20

<210> 748

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 748

aaagaaggca aaaguucuca 20

<210> 749

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 749

agggaugucc uauugcuaag 20

<210> 750

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 750

gggauguccu auugcuaagu 20

<210> 751

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 751

acacuuaccc acuuagcaau 20

<210> 752

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 752

ggaaugguga uccacgcagg 20

<210> 753

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 753

ugaagaag cuacuguguu 20

<210> 754

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 754

agaagcuacu guguuuggug 20

<210> 755

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 755

gaagcuacug uguuuggugc 20

<210> 756

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 756

uguuuggugc gggagcgagc 20

<210> 757

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 757

gcgagcuggc cacaccugug 20

<210> 758

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 758

agugauugug auucucaucc 20

<210> 759

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 759

uugugauucu cauccuggug 20

<210> 760

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 760

ugugauucuc auccuggugu 20

<210> 761

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 761

auucucaucc ugguguggga 20

<210> 762

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 762

ggaaggaauc ccgcugucuc 20

<210> 763

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 763

ucuggcugac aaacucuacu 20

<210> 764

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 764

cggagcuuac cgagacgcug 20

<210> 765

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 765

accgagacgc ugaggaaaua 20

<210> 766

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 766

acggcacgcu caccaaucgc 20

<210> 767

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 767

augaagagua agugaagccc 20

<210> 768

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 768

ugaagaguaa gugaagccca 20

<210> 769

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 769

gcuucugcga accaccugcg 20

<210> 770

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 770

ucacugcagc cucacaggug 20

<210> 771

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 771

cacaaucacu gcagccucac 20

<210> 772

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 772

gcgggauucc uucccacacc 20

<210> 773

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 773

guuugucagc cagagacagc 20

<210> 774

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 774

aguuugucag ccagagacag 20

<210> 775

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 775

gccguauuuc cucagcgucu 20

<210> 776

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 776

auucaaggca caccggcgau 20

<210> 777

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 777

acucuucauu caaggcacac 20

<210> 778

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 778

cuucacuuac ucuucauuca 20

<210> 779

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 779

caggagaacu ugcgccuguc 20

<210> 780

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 780

aggagaacuu gcgccuguca 20

<210> 781

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 781

ggagaacuug cgccugucag 20

<210> 782

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 782

aacuugcgcc ugucaggggc 20

<210> 783

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 783

gggcuggauc cagaaaccug 20

<210> 784

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 784

ugggugccu ccuucucuuu 20

<210> 785

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 785

ccuucucuuu ugguuguuca 20

<210> 786

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 786

ucauggagca uguacuacaa 20

<210> 787

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 787

uugccagaag caagauccca 20

<210> 788

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 788

ccaaggaagu uuaagcugcu 20

<210> 789

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 789

caaggaaguu uaagcugcuu 20

<210> 790

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 790

aaggaaguuu aagcugcuug 20

<210> 791

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 791

gcuuggggau gacccaaaag 20

<210> 792

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 792

uucuggaucc agccccugac 20

<210> 793

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 793

aaggaggcac cacagguuuc 20

<210> 794

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 794

aaaagagaag gaggcaccac 20

<210> 795

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 795

ugaacaacca aaagagaagg 20

<210> 796

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 796

ccaugaacaa ccaaaagaga 20

<210> 797

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 797

cuuccuuggg aucuugcuuc 20

<210> 798

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 798

caagcagcuu aaacuuccuu 20

<210> 799

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 799

ccaagcagcu uaaacuuccu 20

<210> 800

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 800

gaaguaaaca aaccucuuuu 20

<210> 801

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 801

ggaaguaaac aaaccucuuu 20

<210> 802

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 802

cuuuauacag gaagagaaac 20

<210> 803

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 803

gcaaaaccug uccacucuua 20

<210> 804

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 804

accugaugca uauaauaauc 20

<210> 805

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 805

uuggugccau aagaguggac 20

<210> 806

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 806

auauguuggu gccauaagag 20

<210> 807

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 807

ggugcaaguu ucuuauaugu 20

<210> 808

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 808

accugauuau uauaugcauc 20

<210> 809

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 809

cagagcacca gagugccguc 20

<210> 810

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 810

agagcaccag agugccgucu 20

<210> 811

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 811

agagugccgu cugggucuga 20

<210> 812

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 812

ugccgucugg gucugaagga 20

<210> 813

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 813

aaggaaggcc guccauucuc 20

<210> 814

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 814

aggaaggccg uccauucuca 20

<210> 815

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 815

ggaaggccgu ccauucucag 20

<210> 816

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 816

cucagggguc acugcauguu 20

<210> 817

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 817

gacuugcaca acaugcagaa 20

<210> 818

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 818

caugcagaau ggcagcacau 20

<210> 819

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 819

auggcagcac auugguaagu 20

<210> 820

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 820

uggcagcaca uugguaaguu 20

<210> 821

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 821

cacauuggua aguugggcug 20

<210> 822

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 822

uucagaccca gacggcacuc 20

<210> 823

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 823

ggccuuccuu cagacccaga 20

<210> 824

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 824

cagugacccc ugagaaugga 20

<210> 825

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 825

caugcaguga ccccugagaa 20

<210> 826

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 826

cauguugugc aagucucugu 20

<210> 827

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 827

gcauguugug caagucucug 20

<210> 828

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 828

agagaagaca aucgagaauu 20

<210> 829

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 829

gaagacaauc gagaauuugg 20

<210> 830

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 830

agaauuugga ggaaaaccug 20

<210> 831

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 831

uuuauacaaa gucucugacg 20

<210> 832

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 832

gucucugacg uggaugaguu 20

<210> 833

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 833

ucucugacgu ggaugaguuu 20

<210> 834

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 834

cguggaugag uuugggagug 20

<210> 835

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 835

guugggagu guggaagcuc 20

<210> 836

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 836

ugggagugug gaagcucagg 20

<210> 837

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 837

aagcucagga ggagaaaaaa 20

<210> 838

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 838

caggaggaga aaaaacggag 20

<210> 839

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

<400> 839

aaaacggagu ggugccauuc 20

<210> 840

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 840

uucagguacu gaguucuuuu 20

<210> 841

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 841

guucuuuucg gcgaaaaguc 20

<210> 842

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 842

cagucaagac uugccgacaa 20

<210> 843

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 843

agcugaaaag cuuuccuccc 20

<210> 844

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 844

cagcucaaau aaaaaugaaa 20

<210> 845

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 845

acaaacugaa aacgcaagcc 20

<210> 846

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 846

cgcaagccag gcuaaacagu 20

<210> 847

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 847

agccaggcua aacaguuggc 20

<210> 848

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 848

gugagagugc auaccuggua 20

<210> 849

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 849

agugagagug cauaccuggu 20

<210> 850

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 850

cucuagugagagugcauacc 20

<210> 851

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 851

acgugaagcu gcucauccuc 20

<210> 852

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 852

acgucagaga cuuuguauaaa 20

<210> 853

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 853

aaaagaacuc aguaccugaa 20

<210> 854

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 854

cuuugucggc aagucuugac 20

<210> 855

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 855

uggcuucuag uuuccuuugu 20

<210> 856

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 856

cuuuucagcu gcagcuuucu 20

<210> 857

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 857

auuugagcug uucuccaggg 20

<210> 858

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 858

uuuuuugag cuguucucca 20

<210> 859

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 859

uuuuauuuga gcuguucucc 20

<210> 860

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 860

aguuuguuuu guacgugaug 20

<210> 861

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 861

caguuuguuu uguacgugau 20

<210> 862

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 862

ucaguuuguu uuguacguga 20

<210> 863

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 863

uaccugccaa cuguuuagcc 20

<210> 864

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 864

gucaacucuu auucugcuuc 20

<210> 865

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 865

ccaccaaucc auacaugaga 20

<210> 866

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 866

ucacacacuu cagauaucua 20

<210> 867

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 867

uccaccucau cucaagcugc 20

<210> 868

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 868

aaucccauga acccuuaccc 20

<210> 869

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 869

aucccaugaa cccuuacccu 20

<210> 870

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 870

uauccaucau aucaaugcaa 20

<210> 871

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

\oligonucleotide"

<400> 871

augcaaugga aaccuaucag 20

<210> 872

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 872

ggacaacugc uccccauauc 20

<210> 873

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 873

gacaacugcu ccccaauaucu 20

<210> 874

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 874

uucuccccag ucucagccga 20

<210> 875

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 875

cucagccgau ggaucuguau 20

<210> 876

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 876

uccauacacu uuaccagcca 20

<210> 877

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 877

acacuuuacc agccaagguu 20

<210> 878

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 878

aguuuuacau cuaaauacuu 20

<210> 879

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 879

acaucuaaau acuuagguua 20

<210> 880

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 880

uuuuggaaac caaaauauugc 20

<210> 881

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 881

uuggaaacc aaaauauugca 20

<210> 882

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 882

aaccaaaaua ugcagggaga 20

<210> 883

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 883

agaccaaaug uacaucaugu 20

<210> 884

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 884

gaccaaaugu acaucaugua 20

<210> 885

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 885

uccuuauccc acucaugaga 20

<210> 886

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 886

uucccacuc augagaugga 20

<210> 887

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 887

ugagauggau ggccacuuca 20

<210> 888

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 888

gagauggaug gccacuucau 20

<210> 889

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 889

caauucugagc aauccaaaca 20

<210> 890

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 890

ccaaacaugg acuauaaaaa 20

<210> 891

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 891

ccauaacuac agugcagcuc 20

<210> 892

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 892

cauaacuaca gugcagcucc 20

<210> 893

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 893

ugcccucau cuccaaaaca 20

<210> 894

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 894

augcuuuccc acacagcuaa 20

<210> 895

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 895

ugcuuuccca cacagcuaau 20

<210> 896

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 896

gaugaacug cuugugucca 20

<210> 897

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 897

agaacugcuu guguccaagg 20

<210> 898

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 898

cacaaauuaa gugaugcuaa 20

<210> 899

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 899

auuaagugau gcuaaugguc 20

<210> 900

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 900

uggucaggaa aagcagccau 20

<210> 901

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 901

gcagccauug gcacuagucc 20

<210> 902

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 902

cagccauugg cacuagucca 20

<210> 903

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 903

auuggcacua guccagggg 20

<210> 904

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 904

cuaguccagg guguggcuuc 20

<210> 905

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 905

ggguguggcu ucggugcag 20

<210> 906

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 906

uggugcagag gacaacgaug 20

<210> 907

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 907

cagaggacaa cgaugagguc 20

<210> 908

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 908

agacagcgag cagagcuuuc 20

<210> 909

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 909

agcuuucugg auccugacau 20

<210> 910

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 910

gcuuucugga uccugacauu 20

<210> 911

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 911

cuuucuggau ccugacauug 20

<210> 912

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 912

uuucuggauc cugacauugg 20

<210> 913

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 913

ggauccuac auugggggag 20

<210> 914

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 914

ugacauugggggaguggccg 20

<210> 915

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 915

guggccgugg cuccaacuca 20

<210> 916

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 916

uggccguggc uccaacucau 20

<210> 917

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 917

ccccuuuaaa gaaucccaau 20

<210> 918

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 918

ccaauaggaa ucaccccacc 20

<210> 919

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 919

agcaugaaug agccaaaaca 20

<210> 920

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 920

gaaugagcca aaacauggcu 20

<210> 921

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 921

caaaacaugg cuuggcucuu 20

<210> 922

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 922

aaaacauggc uuggcucuuu 20

<210> 923

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 923

ggcucuuugg gaagccaaaa 20

<210> 924

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 924

ugaaaaagcc cgugagaaag 20

<210> 925

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 925

gaggaagagu gugaaaagua 20

<210> 926

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 926

uaugugccuc agaaauccca 20

<210> 927

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 927

cccauggcaa aaaagugaaa 20

<210> 928

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 928

ccauggcaaa aaagugaaac 20

<210> 929

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 929

ucaucaaguc ucuugccgaa 20

<210> 930

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 930

caucuccaua ugccuucacu 20

<210> 931

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 931

aucuccaauau gccuucacuc 20

<210> 932

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 932

uaugccuuca cucgggucac 20

<210> 933

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 933

augccuucac ucgggucaca 20

<210> 934

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 934

augauaucac ccccuuuugugu 20

<210> 935

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 935

guaguauagu ucucaugacg 20

<210> 936

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 936

uaguauaguu cucaugacgu 20

<210> 937

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 937

aguucucaug acguggggcag 20

<210> 938

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 938

guucucauga cgugggcagu 20

<210> 939

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 939

uucucaugac gugggcagug 20

<210> 940

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 940

augacgugggcaguggggaa 20

<210> 941

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 941

cacaguauuc augacaaaug 20

<210> 942

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 942

aguauucaug acaaaugugg 20

<210> 943

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 943

guauucauga caaauguggu 20

<210> 944

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 944

cagcucacca gcaacaaaag 20

<210> 945

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 945

ccaauagcacu uaauuuucac 20

<210> 946

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 946

aauuuucacu ggcucccaag 20

<210> 947

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 947

ggcuccaag uggucacaga 20

<210> 948

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 948

aguggucaca gauggcaucu 20

<210> 949

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 949

aagcauucua ugcaaaaaga 20

<210> 950

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 950

cauucuaugc aaaaagaagg 20

<210> 951

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 951

auucuaugca aaaagaaggu 20

<210> 952

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 952

uucuaugcaa aaagaaggug 20

<210> 953

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 953

caauuuacau uuuuaaacac 20

<210> 954

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 954

uuaaacacug guucuauuau 20

<210> 955

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 955

auaucaaguu ugcauaguca 20

<210> 956

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 956

uacuguagua uuacagugac 20

<210> 957

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 957

aggaaucuua aaauaccauc 20

<210> 958

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 958

uauaugaugu acugaaauac 20

<210> 959

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 959

guacugaaau acuggaauua 20

<210> 960

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 960

uuauuuauca aaauagcuac 20

<210> 961

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 961

cuacaggaaa caugaauagc 20

<210> 962

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 962

aggaaaacac ugaauuuguu 20

<210> 963

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 963

uguuuggaug uucuaagaaa 20

<210> 964

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 964

aagaaauggu gcuaagaaaa 20

<210> 965

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 965

cuccagugcc cuugaauauau 20

<210> 966

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 966

uccagugccc uugaauaaua 20

<210> 967

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 967

ccagugcccu ugaauaauag 20

<210> 968

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 968

caagcuuagu uuuuaaaaug 20

<210> 969

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 969

uuaaaaugug gacauuuuaa 20

<210> 970

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 970

guggacauuu uaaaggccuc 20

<210> 971

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 971

ucauccagug aaguccuugu 20

<210> 972

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 972

ugacaacuug aacaaugcua 20

<210> 973

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 973

augcaaaguu gauuuuuuua 20

<210> 974

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 974

acagccaguu aaauccacca 20

<210> 975

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 975

cagccaguua aauccaccau 20

<210> 976

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 976

agccaguuaa auccaccaug 20

<210> 977

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 977

aaauccacca uggggcuuac 20

<210> 978

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 978

cauggggcuu acuggauuca 20

<210> 979

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 979

aggggcuua cuggauucaa 20

<210> 980

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 980

aguccacaaa acauguuuuc 20

<210> 981

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 981

aagaauuuuc uauuaacugc 20

<210> 982

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 982

cugaagccua ugcuauuuua 20

<210> 983

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 983

uaugcuauuu uuggaucau 20

<210> 984

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 984

aggcucuuca gagaacugaa 20

<210> 985

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 985

uaaguguccu cuuuaacaag 20

<210> 986

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 986

ccugcauaag augaauaaac 20

<210> 987

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 987

cugcauaaga ugaauaaaca 20

<210> 988

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 988

aguuaaaaag aaaaaaaac 20

<210> 989

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 989

aagaaacaaaaacaggcagc 20

<210> 990

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 990

aacaggcagc ugguuugcug 20

<210> 991

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 991

aggcagcugg uuugcugg 20

<210> 992

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 992

aagcagaauu cacaucauga 20

<210> 993

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 993

cauauaccuc aacacuaguu 20

<210> 994

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 994

cucaacacua guuuggcaau 20

<210> 995

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 995

ccuuuuuguu cuaaaaauuc 20

<210> 996

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 996

cuuuuuguuc uaaaaauuca 20

<210> 997

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 997

uguuuaugua 20

<210> 998

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 998

uaauaaauauauucuuuguc 20

<210> 999

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 999

aauaaauaua uucuuuguca 20

<210> 1000

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1000

aacuaauuuu guaaaucugu 20

<210> 1001

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1001

aaaagcauuu uaaaaguuug 20

<210> 1002

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1002

aucuuuugac uguuucaagc 20

<210> 1003

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1003

agaaugcacu gaguugauaa 20

<210> 1004

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1004

gaaugcacug aguugauaaa 20

<210> 1005

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1005

ugauaaaggg aaaaauugua 20

<210> 1006

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1006

aaagggaaaa auuguaaggc 20

<210> 1007

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1007

aaaauuguaa ggcaggaguu 20

<210> 1008

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1008

uaaggcagga guuuggcaag 20

<210> 1009

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1009

ggaguuuggc aaguggcugu 20

<210> 1010

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1010

uuugauccug uaauucacuga 20

<210> 1011

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1011

cugaagguac auacuccaug 20

<210> 1012

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1012

cauguggacu ucccuuaaac 20

<210> 1013

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1013

uuaaacaggc aaacaccuac 20

<210> 1014

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1014

ccuacaggua 20

<210> 1015

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1015

cagauuguac aauuacauuuu 20

<210> 1016

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1016

uaaaauaaau ucuuaaucag 20

<210> 1017

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1017

aauaaauucu uaaucagagg 20

<210> 1018

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1018

ucuuaaucag aggaggccuu 20

<210> 1019

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1019

cuuaaucaga ggaggccuuuu 20

<210> 1020

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1020

aggaggccuu uggguuuuau 20

<210> 1021

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1021

uuggucaaau cuuuguaagc 20

<210> 1022

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1022

uaaaaaauuuu cuugaauuug 20

<210> 1023

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1023

uuugauuacu acaugugcau 20

<210> 1024

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1024

acugucauuu guuaaacugc 20

<210> 1025

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1025

aacugcuggc caacaagaac 20

<210> 1026

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1026

caagaacagg aaguauaguu 20

<210> 1027

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1027

aagaacagga aguauaguuu 20

<210> 1028

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1028

agaacaggaa guauaguuug 20

<210> 1029

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1029

gaacaggaag uauaguuugg 20

<210> 1030

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1030

aacaggaagu auaguuuggg 20

<210> 1031

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1031

ggaaguauag uuugggggggu 20

<210> 1032

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1032

gaaguauagu uuggggggguu 20

<210> 1033

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1033

aaguauaguu uggggguug 20

<210> 1034

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1034

gguuggggag aguuuacaua 20

<210> 1035

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1035

aaggaagaga agaaauugag 20

<210> 1036

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1036

ccugccucag uuagaaugaa 20

<210> 1037

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1037

gaucuacaau uugcuaauau 20

<210> 1038

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1038

uuugcuaaua uaggaauauuc 20

<210> 1039

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1039

uacuugaaaa ugcuucugag 20

<210> 1040

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1040

cugaagcuag 20

<210> 1041

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1041

agcuaguggu uaacuugugu 20

<210> 1042

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1042

uuucauuuuc augagauguu 20

<210> 1043

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1043

uguuugguuu auaagaucug 20

<210> 1044

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1044

ugguuuauaa gaucugagga 20

<210> 1045

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1045

uauuguaaug uuaugaaugc 20

<210> 1046

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1046

ucgcaaaagu ucuguggaca 20

<210> 1047

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1047

gucgcaaaag uucuguggac 20

<210> 1048

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1048

acaaagucgc aaaaguucug 20

<210> 1049

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1049

aguugacaga cucugucuga 20

<210> 1050

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1050

gaguugacag acucugucug 20

<210> 1051

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1051

ccgucucaug uauggauugg 20

<210> 1052

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1052

gggccgucuc auguauggau 20

<210> 1053

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1053

ggauugggcc gucucaugua 20

<210> 1054

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1054

ggauaaggac uaacuggauu 20

<210> 1055

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1055

uggauaagga cuaacuggau 20

<210> 1056

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1056

gaguuuggau aggacuaac 20

<210> 1057

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1057

gugugugaag aguuuggaua 20

<210> 1058

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1058

ucugaagugugugaagaguu 20

<210> 1059

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1059

ggaauagaag uucauagggc 20

<210> 1060

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1060

agguggaaua gaaguucaua 20

<210> 1061

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1061

gagguggaau agaaguucau 20

<210> 1062

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1062

accugcagcu ugagaugagg 20

<210> 1063

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1063

ugaaccugca gcuugagaug 20

<210> 1064

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1064

agcccagggu aaggguucau 20

<210> 1065

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1065

aagcccaggg uaaggguuca 20

<210> 1066

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1066

gauucaaaag cccaggguaa 20

<210> 1067

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1067

ugauucaaaa gcccagggua 20

<210> 1068

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1068

uauucugauu caaaagccca 20

<210> 1069

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1069

guauucugau ucaaaagccc 20

<210> 1070

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1070

gcauugauaugauggauauu 20

<210> 1071

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1071

ugcauugaua ugauggauau 20

<210> 1072

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1072

uuuccauugc auugauauga 20

<210> 1073

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1073

gggagcaguu guccacugau 20

<210> 1074

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1074

agaauaggaa cccagauaug 20

<210> 1075

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1075

gagaauagga acccagauau 20

<210> 1076

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1076

ggagaauagg aacccagaua 20

<210> 1077

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1077

cggcugagac uggggagaau 20

<210> 1078

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1078

agauccaucg gcugagacug 20

<210> 1079

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1079

cagauccauc ggcugagacu 20

<210> 1080

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1080

acagauccau cggcugagac 20

<210> 1081

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1081

ggauaccuau acagauccau 20

<210> 1082

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1082

uuagacagag ggucuuggcu 20

<210> 1083

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1083

ugaggcuuaga cagagggucu 20

<210> 1084

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1084

guagacugag cuuagacaga 20

<210> 1085

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1085

gguagacuga gcuuagacag 20

<210> 1086

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1086

ugguaaagug uuggaugg 20

<210> 1087

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1087

ggcugguaaa guguauggau 20

<210> 1088

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1088

uggcugguaa aguguaugga 20

<210> 1089

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1089

accuuggcug guaaagugua 20

<210> 1090

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1090

ggcuauuucc aaaccuuggc 20

<210> 1091

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1091

cucuggcuau uuccaaaccu 20

<210> 1092

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1092

aguauuuaga uguaaaacuc 20

<210> 1093

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1093

aaccaucucc cugcauauuu 20

<210> 1094

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1094

augauguaca uuuggucuaa 20

<210> 1095

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1095

uucccuacau gauguacauu 20

<210> 1096

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1096

aucucaugag ugggauaagg 20

<210> 1097

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1097

uccaucucau gagugggaua 20

<210> 1098

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1098

uggccaucca ucucaugagu 20

<210> 1099

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1099

guggccaucc aucucaugag 20

<210> 1100

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1100

uagagguggc ucccaugaag 20

<210> 1101

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1101

auggguggu aaucuagagg 20

<210> 1102

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1102

cagauugggu gguaaucuag 20

<210> 1103

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1103

uuuggauugc ucagauuggg 20

<210> 1104

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1104

auguuuggau ugcucagauu 20

<210> 1105

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1105

cauguuugga uugcucagau 20

<210> 1106

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1106

ccauuuuuau aguccauguu 20

<210> 1107

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1107

uuaugugaga 20

<210> 1108

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1108

ccggagcugc acuguaguua 20

<210> 1109

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1109

agagagcugu ugaacaugcc 20

<210> 1110

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1110

cuccuuguuu uggagaugca 20

<210> 1111

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1111

ucuccuuguu uuggagaugc 20

<210> 1112

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1112

gcaugucauu cuccuuguuu 20

<210> 1113

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1113

ugauaaccca uuagcugugu 20

<210> 1114

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1114

uugauaaccc auuagcugug 20

<210> 1115

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1115

guucuaucau gguuaagagc 20

<210> 1116

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1116

ggacacaagc aguucauca 20

<210> 1117

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1117

uuaauuugug uaagccuccu 20

<210> 1118

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1118

acaccugga cuagugccaa 20

<210> 1119

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1119

cugcaccaga agccacaccc 20

<210> 1120

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1120

acggccacuc ccccaauguc 20

<210> 1121

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1121

ugacccauga guuggagcca 20

<210> 1122

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1122

augagaauug acccaugagu 20

<210> 1123

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1123

gggauucuuu aaagggguug 20

<210> 1124

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1124

ccuauuggga uucuuuaaag 20

<210> 1125

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1125

uccuauuggg auucuuuaaa 20

<210> 1126

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1126

uuccuauugg gauucuuuaa 20

<210> 1127

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1127

cugguggggu gauuccuauu 20

<210> 1128

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1128

ccugggggg ugauuccuau 20

<210> 1129

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1129

agacgaggga gauccuggug 20

<210> 1130

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1130

agauccugggu 20

<210> 1131

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1131

aaagacgagg gagauccugg 20

<210> 1132

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1132

guaaaagacg agggaucc 20

<210> 1133

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1133

cuuaugcugg uaaaagacga 20

<210> 1134

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1134

ucuuaugcug guaaaagacg 20

<210> 1135

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1135

gcucauucau gcucuuaugc 20

<210> 1136

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1136

caaaagcca agccauguuu 20

<210> 1137

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1137

acgggcuuuu ucagccauuu 20

<210> 1138

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1138

acacucuucc ucuuucucac 20

<210> 1139

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1139

cacacucuuc cucuuucuca 20

<210> 1140

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1140

auuucugagg cacauagucu 20

<210> 1141

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1141

gauuucugag gcacaauaguc 20

<210> 1142

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1142

uuuuugccau gggauuucug 20

<210> 1143

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1143

ccguuucacu uuuuugccau 20

<210> 1144

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1144

cccguuucac uuuuuugcca 20

<210> 1145

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1145

gaaguuucau guggcucagc 20

<210> 1146

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1146

gugggcucug aaguuucaug 20

<210> 1147

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1147

uugaugaaac gcagguaagu 20

<210> 1148

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1148

cuugaugaaa cgcagguaag 20

<210> 1149

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1149

caagagacuu gaugaaacgc 20

<210> 1150

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1150

ggucacggac ugguccuuuu 20

<210> 1151

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1151

ggagucugug gucacggaca 20

<210> 1152

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1152

uacuguggag ucugugguca 20

<210> 1153

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1153

uguaguuacu guggagucug 20

<210> 1154

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1154

auauggagau guaguuacug 20

<210> 1155

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1155

gugacccgag ugaaggcaua 20

<210> 1156

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1156

aggccugug acccgaguga 20

<210> 1157

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1157

uaucauauauaucuguugua 20

<210> 1158

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1158

gugagguaac caacaaaagg 20

<210> 1159

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1159

agugagguaa ccaacaaaag 20

<210> 1160

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1160

aagugaggua accaacaaaa 20

<210> 1161

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1161

caagugaggu aaccaacaaa 20

<210> 1162

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1162

gguuggguc uuuucaagug 20

<210> 1163

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1163

uacuacugac agguugguug 20

<210> 1164

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1164

gaacuauacu acugacaggu 20

<210> 1165

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1165

augagaacua uacuacugac 20

<210> 1166

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1166

cuuuuguugc uggugagcug 20

<210> 1167

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1167

aagauaaccu cuuuuguugc 20

<210> 1168

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1168

ccagugaaaa uuaagugcua 20

<210> 1169

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1169

gugccaucu gugaccacuu 20

<210> 1170

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1170

agaugccauc ugugaccacu 20

<210> 1171

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1171

ucuuuuugca uagaaugcuu 20

<210> 1172

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1172

guguuuaaaa auguaaauug 20

<210> 1173

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1173

agaguuguaa gcgggggggg 20

<210> 1174

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1174

uagaguugua agcggggggg 20

<210> 1175

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1175

guagaguugu aagcgggggg 20

<210> 1176

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1176

uguagaguug uaagcggggg 20

<210> 1177

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1177

guguagaguu guaagcgggg 20

<210> 1178

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1178

uguguagagu uguaagcggg 20

<210> 1179

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1179

uuguaagcgg 20

<210> 1180

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1180

gauguguaga guuguaagcg 20

<210> 1181

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1181

agauguguag aguuguaagc 20

<210> 1182

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1182

cagaugugua gaguuguaag 20

<210> 1183

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1183

aaacuugaua uuauuaaaag 20

<210> 1184

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1184

aucauauauu cagcaccaga 20

<210> 1185

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1185

auagcaucuu gaugauauaa 20

<210> 1186

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1186

ccccuauuau ucaagggcac 20

<210> 1187

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1187

aagguacccc uauuauucaa 20

<210> 1188

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1188

aaagguaccc cuauuauuca 20

<210> 1189

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1189

ugauaaaaac uugaaugaaa 20

<210> 1190

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1190

aacuaagcuu guguaagaau 20

<210> 1191

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1191

acuggaugg caaaauccag 20

<210> 1192

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1192

uuguccuaca aggacuucac 20

<210> 1193

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1193

auaucguuua uuguccuaca 20

<210> 1194

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1194

uguucaaguu gucaaagcuu 20

<210> 1195

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1195

auugcucauc agcagaugca 20

<210> 1196

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1196

uuaacuggcu guguuaaaaa 20

<210> 1197

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1197

agccccaugg uggauuuaac 20

<210> 1198

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1198

gaauccagua agccccaugg 20

<210> 1199

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1199

cuugaaucca guaagcccca 20

<210> 1200

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1200

gcaccagaaa acauguuuug 20

<210> 1201

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1201

uaugauccau aaaauagcau 20

<210> 1202

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1202

guacauaauu aucaacacaa 20

<210> 1203

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1203

gcucaauccu cuuguuaaag 20

<210> 1204

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1204

ccuguuuauu caucuuaugc 20

<210> 1205

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1205

uuuuaacuga cagauucaca 20

<210> 1206

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1206

cauuaugaua uauuuguagc 20

<210> 1207

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1207

uguugaggua uaugacaagu 20

<210> 1208

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1208

cuauugccaa acuaguguug 20

<210> 1209

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1209

aaggacuugg aaaaaaauga 20

<210> 1210

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1210

uaacaauaaa aaaaaggacu 20

<210> 1211

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1211

uuuuuuuaac aauaaaaaaa 20

<210> 1212

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1212

agaaaucaag uauugaaaaa 20

<210> 1213

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1213

ccugaauuuu uagaacaaaa 20

<210> 1214

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1214

acaggugaca uguuggcaua 20

<210> 1215

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1215

cacaggugac auguuggcau 20

<210> 1216

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1216

cauaaacaca ggugacaugu 20

<210> 1217

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1217

caacaauuuu acauaaacac 20

<210> 1218

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1218

agaagggauu caaaauaaaa 20

<210> 1219

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1219

uagaagggau ucaaaauaaa 20

<210> 1220

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1220

cauguacaag uaaaauagaa 20

<210> 1221

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1221

acauguacaa guaaaauaga 20

<210> 1222

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1222

gagaguuaca aguaagucuc 20

<210> 1223

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1223

uauguaccuu cagugauuac 20

<210> 1224

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1224

cuguuuaagg gaaguccaca 20

<210> 1225

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1225

guagguguuu gccuguuuaa 20

<210> 1226

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1226

uguagguguu ugccuguuua 20

<210> 1227

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1227

cuguugcaca ccauaccugu 20

<210> 1228

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1228

uaguaagcaa aaauguauuuu 20

<210> 1229

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1229

auuugaccaa uaaaacccaa 20

<210> 1230

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1230

uuuuggaaau guuugcaaau 20

<210> 1231

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1231

guaagcaaag caaacauuuuu 20

<210> 1232

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1232

caaaaaacau uaaaaucaug 20

<210> 1233

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1233

auguuugggg cuagauauua 20

<210> 1234

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1234

auguaguaau caaauguuug 20

<210> 1235

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1235

cauguaguaa ucaaauguuu 20

<210> 1236

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1236

acauguagua aucaaauguu 20

<210> 1237

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1237

cagaaaucaa auauuaagaa 20

<210> 1238

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1238

gcaguuuaac aaaugacagu 20

<210> 1239

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1239

acuauacuuc cuguucuugu 20

<210> 1240

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1240

ccauucauuc uaacugaggc 20

<210> 1241

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1241

cuuuccauuc auucuaacug 20

<210> 1242

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1242

cucagaagca uuuucaagua 20

<210> 1243

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1243

aacacucaca uagcauuauc 20

<210> 1244

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1244

cacatggcgt ttatccagaa t 21

<210> 1245

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1245

cagatgcaca ggccaattaa g 21

<210> 1246

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1246

gagctgctga attcaactag a 21

<210> 1247

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1247

cagatcgcca taacataaat a 21

<210> 1248

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1248

gaccatggag cagcatctga a 21

<210> 1249

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1249

gccaagtcat tattgacca t 21

<210> 1250

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1250

ccctcagagat attgtgggtt t 21

<210> 1251

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1251

gggtaagcca agaaagaaa 19

<210> 1252

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1252

gggtaagcca agaaagaaa 19

<210> 1253

<211> 112

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 1253

cacatggcgt ttatccagaa tctcgagatt ctggataaac gccatgtgtt ttttgaattc 60

gcaccagcac gctacgcaca cacagtacac acactgacgt ttcgccgtct tc 112

<210> 1254

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 1254

gaagacgcac cggcagatgt acaggctaat taaggttaat attcatagcc ttaattggcc 60

tgtgcatctg ttttttgaat tcgcaccagc acgctacgca acacgtcaac cagtgtcagt 120

gtttcgccgt 130

<210> 1255

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 1255

gaagacgcac cgggagctgc tgaattcaat tagagttaat attcatagct ctagttgaat 60

tcagcagctc ttttttgaat tcgcaccagc acgctacgca tgcagtcaac cagtgtcaac 120

cattcgccgt 130

<210> 1256

<211> 112

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 1256

cagatcgcca taacataaat actcgagtat ttatgttatg gcgatctgtt ttttgaattc 60

gcaccagcac gctacgcatg accagtacac acactgcatg ttcgccgtct tc 112

<210> 1257

<211> 130

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 1257

gaagacgcac cgggaccatg gagtagcatt tgaagttaat attcatagct tcagatgctg 60

ctccatggtc ttttttgaat tcgcaccagc acgctacgca tggtgtcaac cagtgtcagt 120

tgttcgccgt 130

<210> 1258

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1258

gccaagtcat tatttgacca tctcgagatg gtcaaataat gacttggctt ttttga 56

<210> 1259

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1259

cctcagagat attgtgggtt tctcgagaaa cccacaatat ctctgaggtt ttttga 56

<210> 1260

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1260

gggtaagcca agaaagaaac tcgagtttct ttcttggctt accctttttt ga 52

<210> 1261

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 1261

gaagacgcac cgggggtaag ccaagaaaga aagttaatat tcatagcttt ctttcttggc 60

ttaccctttt ttgaattcgc accagcacgc tacgcaacac gtcaaccagt gtcagtgttt 120

cgccgt 126

<210> 1262

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 1262

Arg Gly Asp Ser

1

<210> 1263

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1263

catgtacgtt gctatccagg c 21

<210> 1264

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1264

ctccttaatg tcacgcacgat 21

<210> 1265

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1265

ctcaccatgg atgatgatat cgc 23

<210> 1266

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1266

ccacatagga atccttctga ccc 23

<210> 1267

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1267

cagaacctaa accacccgtg 20

<210> 1268

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1268

tgcttcgtag cgccattgta a 21

<210> 1269

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1269

ataccctgta tgaagggaag cc 22

<210> 1270

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1270

cttaccccga agttacgtct ttc 23

<210> 1271

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1271

cacccggctc tatgaaacct t 21

<210> 1272

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

primer"

<400> 1272

ccagccactc gaggtagtca 20

<210> 1273

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1273

cagagcacca gagugccguc 20

<210> 1274

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1274

agagcaccag agugccgucu 20

<210> 1275

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1275

uucagaccca gacggcacuc 20

<210> 1276

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1276

auggcagcac auugguaagu 20

<210> 1277

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1277

cacauuggua aguugggcug 20

<210> 1278

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1278

gacuugcaca acaugcagaa 20

<210> 1279

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1279

ucauggagca uguacuacaa 20

<210> 1280

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1280

aacuugcgcc ugucaggggc 20

<210> 1281

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1281

ccaaggaagu uuaagcugcu 20

<210> 1282

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1282

ccaagcagcu uaaacuuccu 20

<210> 1283

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1283

uuggugccau aagaguggac 20

<210> 1284

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1284

gcaaaaccug uccacucuua 20

<210> 1285

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1285

auauguuggu gccauaagag 20

<210> 1286

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1286

aaaacggagu ggugccauuc 20

<210> 1287

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1287

gucucugacg uggaugaguu 20

<210> 1288

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1288

uuuauacaaa gucucugacg 20

<210> 1289

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1289

agagaagaca aucgagaauu 20

<210> 1290

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1290

acgucagaga cuuuguauaaa 20

<210> 1291

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1291

ggauagaacc aaccaugugug 20

<210> 1292

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1292

uuguagccag agguucuguc 20

<210> 1293

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1293

ucuguugccc ucaacauggu 20

<210> 1294

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1294

gaugaacca accauguuga 20

<210> 1295

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1295

uucuggagcu uuguagccag 20

<210> 1296

<211> 73

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1296

aacagcauag caaguuaaaa uaaggcuagu ccguuaucaa cuugaaaaag uggcaccgag 60

ucgguguuuu uuu 73

<210> 1297

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 1297

Cys Leu Val Gly Ala Asn Arg Asp Asp Lys Ile Ile Phe

1 5 10

<210> 1298

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 1298

Cys Ala Ser Ser Leu Asp Gly Ser Gly Gln Gly Ser Asp Tyr Gly Tyr

1 5 10 15

Thr Phe

<210> 1299

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1299

tgtgcagcaa gtagggaggc gacaccgaca agctcatctt t 41

<210> 1300

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1300

tgcctcgtgg gtgcgaacag agatgacaag atcatcttt 39

<210> 1301

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1301

gcgccagcag cttggacggt tcgggacagg gatcggacta tggcta 46

<210> 1302

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1302

aagcaagcct gatggaacag actaagtcca ttcctgatac catcacctcc c 51

<210> 1303

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1303

aagcaagcct gatggaacag gatagaacca acagactaag tccattcctg ataccatcac 60

ctccc 65

<210> 1304

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1304

aagcaagcct gatggaacag gatagaacca accattgagg gcaacagact aagtccattc 60

ctgataccat cacctccc 78

<210> 1305

<211> 77

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1305

aagcaagcct gatggaacag gatagaacca accatgaggg caacagacta agtccattcc 60

tgataccatc acctccc 77

<210> 1306

<211> 61

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1306

aagcaagcct gatggaacag gatagaacag actaagtcca ttcctgatac catcacctcc 60

c 61

<210> 1307

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1307

actaagtcca ttcctgatac catcacctcc catttgccag acagaacgtc tggctacaaa 60

gctccagaat ggaagcccac 80

<210> 1308

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1308

actaagtcca ttcctgatac catcacctcc catttgccag acaagaacct ctggctacaa 60

agctccagaa tggaagccc 79

<210> 1309

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1309

actaagtcca ttcctgatac catcacctcc catttgccag acaagaacgt ctggctacaa 60

agctccagaa tggaagccc 79

<210> 1310

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1310

actaagtcca ttcctgatac catcacctcc catttgcctc tggctacaaa gctccagaat 60

ggaagcccac 70

<210> 1311

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1311

actaagtcca ttcctgatac catcacctcc catttgccag acctctggct acaaagctcc 60

agaatggaag cccac 75

<210> 1312

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1312

atggccccga agcaagcctg atggaacagg atagaaccaa ccaatgttga gggcaacaga 60

ctaagtccat tcctgatac 79

<210> 1313

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1313

atggccccga agcaagcctg atggaacagg atagaaccaa cctgttgagg gcaacagact 60

aagtccattc ctgatacca 79

<210> 1314

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1314

atggccccga agcaagcctg atggaacaga ctaagtccat tcctgatacc a 51

<210> 1315

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1315

atggccccga agcaagcctg atggaacagg atagaaccaa ccgttgaggg caacagacta 60

agtccattcc tgatacca 78

<210> 1316

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1316

atggccccga agcaagcctg atggaacagg atagaaccaa cagactaagt ccattcctga 60

tacca 65

<210> 1317

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1317

agcaagcctg atggaacagg atagaaccaa ccatgtttga gggcaacaga ctaagtccat 60

tcctgatacc atcacctcc 79

<210> 1318

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1318

agcaagcctg atggaacagg atagaaccaa ccatgtgagg gcaacagact aagtccattc 60

ctgataccat cacctccca 79

<210> 1319

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1319

agcaagcctg atggaacaga ctaagtccat tcctgatacc atcacctccc a 51

<210> 1320

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1320

agcaagcctg atggaacagg atagaaccaa cagactaagt ccattcctga taccatcacc 60

tcca 65

<210> 1321

<211> 77

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1321

agcaagcctg atggaacagg atagaaccaa ccatgagggc aacagactaa gtccattcct 60

gataccatca cctccca 77

<210> 1322

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1322

ttcctgatac catcacctcc catttgccag acagaacgtc tggctacaaa gctccagaat 60

ggaagcccac tgcctgagag 80

<210> 1323

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1323

ttcctgatac catcacctcc catttgccag acagaacctc tgggctacaa agctccagaa 60

tggaagccca ctgcctgag 79

<210> 1324

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1324

ttcctgatac catcacctcc catttgccag acagaacctc tgctacaaag ctccagaatg 60

gaagcccact gcctgagag 79

<210> 1325

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1325

ttcctgatac catcacctcc catttgccag acagaatgga agcccactgc ctgagag 57

<210> 1326

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1326

ttcctgatac catcacctcc catttgccag acagaacctc cagaatggaa gcccactgcc 60

tgagag 66

<210> 1327

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1327

ttcagattga atatgaacac agagcaccag agtgcgtctg ggtctgaagg aaggccgtcc 60

attctcaggg gtcactgca 79

<210> 1328

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1328

ttcagattga atatgaacac agagcaccag agtgcccgtc tgggtctgaa ggaaggccgt 60

ccattctcag gggtcactg 79

<210> 1329

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1329

ttcagattga atatgaacac agagcaccag agtgccgtct gggtccgaag gaaggccgtc 60

cattctcagg ggtcactgca 80

<210> 1330

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1330

ttcagattga atatgagcac agagcaccag agtgccgtct gggtctgaag gaaggccgtc 60

cattctcagg ggtcactgca 80

<210> 1331

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1331

ttcagattga atatgaacac agagcaccag agtgccgtct gggtctgaag gagggccgtc 60

cattctcagg ggtcactgca 80

<210> 1332

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1332

tcagattgaa tatgaacaca gagcaccaga gtgcctctgg gtctgaagga aggccgtcca 60

ttctcagggg tcactgcat 79

<210> 1333

<211> 73

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1333

tcagattgaa tatgaacaca gagcaccaga gtgggtctga aggaaggccg tccattctca 60

ggggtcactg cat 73

<210> 1334

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1334

tcagattgaa tatgaacaca gagcaccaga gtctgaagga aggccgtcca ttctcagggg 60

tcactgcat 69

<210> 1335

<211> 76

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1335

tcagattgaa tatgaacaca gagcaccaga gtgctgggtc tgaaggaagg ccgtccattc 60

tcaggggtca ctgcat 76

<210> 1336

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1336

tcagattgaa tatgaacaca gagcaccaga gtgctgaagg aaggccgtcc attctcaggg 60

gtcactgcat 70

<210> 1337

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1337

acttttattt ttcagattga atatgaacac agagcaccgt ctgggtctga aggaaggccg 60

tccattctca gg 72

<210> 1338

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1338

actttttttttcagattga atatgaacac agagcaccag agttgccgtc tgggtctgaa 60

ggaaggccgt ccattctca 79

<210> 1339

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1339

actttttttttcagattga atatgaacac agagccgtct gggtctgaag gaaggccgtc 60

cattctcagg 70

<210> 1340

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1340

actttttttttcagattga atatgaacac agagcaccag agccgtctgg gtctgaagga 60

aggccgtcca ttctcagg 78

<210> 1341

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1341

acttttattt ttcagattga atatgaacac agagcaccag tgccgtctgg gtctgaagga 60

aggccgtcca ttctcagg 78

<210> 1342

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1342

gacttgcaca acatgcagaa tggcagcaca ttgggctgag gacagcttag cagctgttga 60

gtctgttctc ac 72

<210> 1343

<211> 76

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1343

gacttgcaca acatgcagaa tggcagcaca ttggttgggc tgaggacagc ttagcagctg 60

ttgagtctgt tctcac 76

<210> 1344

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1344

gacttgcaca acatgcagaa tggcagcaca ttggtagttg ggctgaggac agcttagcag 60

ctgttgagtc tgttctcac 79

<210> 1345

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1345

gacttgcaca acatgcagaa tggcagctta gcagctgttg agtctgttct cac 53

<210> 1346

<211> 71

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1346

gacttgcaca acatgcagaa tggcagcaca ttggctgagg acagcttagc agctgttgag 60

tctgttctca c 71

<210> 1347

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1347

acaacatgca gaatggcagc acattggtaa gttggctgag gacagcttag cagctgttga 60

gtctgttctc acactgcta 79

<210> 1348

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1348

acaacatgca gaatggcagc acattggtaa gttggggagg acagcttagc agctgttgag 60

tctgttctca cactgcta 78

<210> 1349

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1349

acaacatgca gaatggcagc acattggtaa gtctgttctc acactgcta 49

<210> 1350

<211> 74

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1350

acaacatgca gaatggcagc acattggtaa gttgggacag cttagcagct gttgagtctg 60

ttctcacact gcta 74

<210> 1351

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1351

acaacatgca gaatggcagc acattggtaa gcttagcagc tgttgagtct gttctcacac 60

tgcta 65

<210> 1352

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1352

ctgtgctcat gcccacagag acttgcacaa catgcaagaa tggcagcaca ttggtaagtt 60

gggctgagga cagcttagc 79

<210> 1353

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1353

ctgtgctcat gccacagag acttgcacat tggtaagttg ggctgaggac agcttagcag 60

<210> 1354

<211> 73

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1354

ctgtgctcat gccccagag acttgcacaa catggcagca cattggtaag ttgggctgag 60

gacagcttag cag 73

<210> 1355

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1355

ctgtgctcat gcccacagag acttgcagca cattggtaag ttgggctgag gacagcttag 60

cag 63

<210> 1356

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

oligonucleotide"

<400> 1356

ctgtgctcat gcccacagag acttgcacaa catagaatgg cagcacattg gtaagttggg 60

ctgaggacag cttagcag 78

<210> 1357

<211> 2002

<212> PROTEIN

<213> Homo sapiens

<400> 1357

Met Glu Gln Asp Arg Thr Asn His Val Glu Gly Asn Arg Leu Ser Pro

1 5 10 15

Phe Leu Ile Pro Ser Pro Pro Ile Cys Gln Thr Glu Pro Leu Ala Thr

20 25 30

Lys Leu Gln Asn Gly Ser Pro Leu Pro Glu Arg Ala His Pro Glu Val

35 40 45

Asn Gly Asp Thr Lys Trp His Ser Phe Lys Ser Tyr Tyr Gly Ile Pro

50 55 60

Cys Met Lys Gly Ser Gln Asn Ser Arg Val Ser Pro Asp Phe Thr Gln

65 70 75 80

Glu Ser Arg Gly Tyr Ser Lys Cys Leu Gln Asn Gly Gly Ile Lys Arg

85 90 95

Thr Val Ser Glu Pro Ser Leu Ser Gly Leu Leu Gln Ile Lys Lys Leu

100 105 110

Lys Gln Asp Gln Lys Ala Asn Gly Glu Arg Arg Asn Phe Gly Val Ser

115 120 125

Gln Glu Arg Asn Pro Gly Glu Ser Ser Gln Pro Asn Val Ser Asp Leu

130 135 140

Ser Asp Lys Lys Glu Ser Val Ser Ser Val Ala Gln Glu Asn Ala Val

145 150 155 160

Lys Asp Phe Thr Ser Phe Ser Thr His Asn Cys Ser Gly Pro Glu Asn

165 170 175

Pro Glu Leu Gln Ile Leu Asn Glu Gln Glu Gly Lys Ser Ala Asn Tyr

180 185 190

His Asp Lys Asn Ile Val Leu Leu Lys Asn Lys Ala Val Leu Met Pro

195 200 205

Asn Gly Ala Thr Val Ser Ala Ser Ser Val Glu His Thr His Gly Glu

210 215 220

Leu Leu Glu Lys Thr Leu Ser Gln Tyr Tyr Pro Asp Cys Val Ser Ile

225 230 235 240

Ala Val Gln Lys Thr Thr Ser His Ile Asn Ala Ile Asn Ser Gln Ala

245 250 255

Thr Asn Glu Leu Ser Cys Glu Ile Thr His Pro Ser His Thr Ser Gly

260 265 270

Gln Ile Asn Ser Ala Gln Thr Ser Asn Ser Glu Leu Pro Pro Lys Pro

275 280 285

Ala Ala Val Val Ser Glu Ala Cys Asp Ala Asp Asp Ala Asp Asn Ala

290 295 300

Ser Lys Leu Ala Ala Met Leu Asn Thr Cys Ser Phe Gln Lys Pro Glu

305 310 315 320

Gln Leu Gln Gln Gln Lys Ser Val Phe Glu Ile Cys Pro Ser Pro Ala

325 330 335

Glu Asn Asn Ile Gln Gly Thr Thr Lys Leu Ala Ser Gly Glu Glu Phe

340 345 350

Cys Ser Gly Ser Ser Ser Asn Leu Gln Ala Pro Gly Gly Ser Ser Glu

355 360 365

Arg Tyr Leu Lys Gln Asn Glu Met Asn Gly Ala Tyr Phe Lys Gln Ser

370 375 380

Ser Val Phe Thr Lys Asp Ser Phe Ser Ala Thr Thr Thr Pro Pro Pro

385 390 395 400

Pro Ser Gln Leu Leu Leu Ser Pro Pro Pro Pro Leu Pro Gln Val Pro

405 410 415

Gln Leu Pro Ser Glu Gly Lys Ser Thr Leu Asn Gly Gly Val Leu Glu

420 425 430

Glu His His His Tyr Pro Asn Gln Ser Asn Thr Thr Leu Leu Arg Glu

435 440 445

Val Lys Ile Glu Gly Lys Pro Glu Ala Pro Pro Ser Gln Ser Pro Asn

450 455 460

Pro Ser Thr His Val Cys Ser Pro Ser Pro Met Leu Ser Glu Arg Pro

465 470 475 480

Gln Asn Asn Cys Val Asn Arg Asn Asp Ile Gln Thr Ala Gly Thr Met

485 490 495

Thr Val Pro Leu Cys Ser Glu Lys Thr Arg Pro Met Ser Glu His Leu

500 505 510

Lys His Asn Pro Pro Ile Phe Gly Ser Ser Gly Glu Leu Gln Asp Asn

515 520 525

Cys Gln Gln Leu Met Arg Asn Lys Glu Gln Glu Ile Leu Lys Gly Arg

530 535 540

Asp Lys Glu Gln Thr Arg Asp Leu Val Pro Pro Thr Gln His Tyr Leu

545 550 555 560

Lys Pro Gly Trp Ile Glu Leu Lys Ala Pro Arg Phe His Gln Ala Glu

565 570 575

Ser His Leu Lys Arg Asn Glu Ala Ser Leu Pro Ser Ile Leu Gln Tyr

580 585 590

Gln Pro Asn Leu Ser Asn Gln Met Thr Ser Lys Gln Tyr Thr Gly Asn

595 600 605

Ser Asn Met Pro Gly Gly Leu Pro Arg Gln Ala Tyr Thr Gln Lys Thr

610 615 620

Thr Gln Leu Glu His Lys Ser Gln Met Tyr Gln Val Glu Met Asn Gln

625 630 635 640

Gly Gln Ser Gln Gly Thr Val Asp Gln His Leu Gln Phe Gln Lys Pro

645 650 655

Ser His Gln Val His Phe Ser Lys Thr Asp His Leu Pro Lys Ala His

660 665 670

Val Gln Ser Leu Cys Gly Thr Arg Phe His Phe Gln Gln Arg Ala Asp

675 680 685

Ser Gln Thr Glu Lys Leu Met Ser Pro Val Leu Lys Gln His Leu Asn

690 695 700

Gln Gln Ala Ser Glu Thr Glu Pro Phe Ser Asn Ser His Leu Leu Gln

705 710 715 720

His Lys Pro His Lys Gln Ala Ala Gln Thr Gln Pro Ser Gln Ser Ser

725 730 735

His Leu Pro Gln Asn Gln Gln Gln Gln Gln Lys Leu Gln Ile Lys Asn

740 745 750

Lys Glu Glu Ile Leu Gln Thr Phe Pro His Pro Gln Ser Asn Asn Asp

755 760 765

Gln Gln Arg Glu Gly Ser Phe Phe Gly Gln Thr Lys Val Glu Glu Cys

770 775 780

Phe His Gly Glu Asn Gln Tyr Ser Lys Ser Ser Glu Phe Glu Thr His

785 790 795 800

Asn Val Gln Met Gly Leu Glu Glu Val Gln Asn Ile Asn Arg Arg Asn

805 810 815

Ser Pro Tyr Ser Gln Thr Met Lys Ser Ser Ala Cys Lys Ile Gln Val

820 825 830

Ser Cys Ser Asn Asn Thr His Leu Val Ser Glu Asn Lys Glu Gln Thr

835 840 845

Thr His Pro Glu Leu Phe Ala Gly Asn Lys Thr Gln Asn Leu His His

850 855 860

Met Gln Tyr Phe Pro Asn Asn Val Ile Pro Lys Gln Asp Leu Leu His

865 870 875 880

Arg Cys Phe Gln Glu Gln Glu Gln Lys Ser Gln Gln Ala Ser Val Leu

885 890 895

Gln Gly Tyr Lys Asn Arg Asn Gln Asp Met Ser Gly Gln Gln Ala Ala

900 905 910

Gln Leu Ala Gln Gln Arg Tyr Leu Ile His Asn His Ala Asn Val Phe

915 920 925

Pro Val Pro Asp Gln Gly Gly Ser His Thr Gln Thr Pro Pro Gln Lys

930 935 940

Asp Thr Gln Lys His Ala Ala Leu Arg Trp His Leu Leu Gln Lys Gln

945 950 955 960

Glu Gln Gln Gln Thr Gln Gln Pro Gln Thr Glu Ser Cys His Ser Gln

965 970 975

Met His Arg Pro Ile Lys Val Glu Pro Gly Cys Lys Pro His Ala Cys

980 985 990

Met His Thr Ala Pro Pro Glu Asn Lys Thr Trp Lys Lys Val Thr Lys

995 1000 1005

Gln Glu Asn Pro Pro Ala Ser Cys Asp Asn Val Gln Gln Lys Ser

1010 1015 1020

Ile Ile Glu Thr Met Glu Gln His Leu Lys Gln Phe His Ala Lys

1025 1030 1035

Ser Leu Phe Asp His Lys Ala Leu Thr Leu Lys Ser Gln Lys Gln

1040 1045 1050

Val Lys Val Glu Met Ser Gly Pro Val Thr Val Leu Thr Arg Gln

1055 1060 1065

Thr Thr Ala Ala Glu Leu Asp Ser His Thr Pro Ala Leu Glu Gln

1070 1075 1080

Gln Thr Thr Ser Ser Glu Lys Thr Pro Thr Lys Arg Thr Ala Ala

1085 1090 1095

Ser Val Leu Asn Asn Phe Ile Glu Ser Pro Ser Lys Leu Leu Asp

1100 1105 1110

Thr Pro Ile Lys Asn Leu Leu Asp Thr Pro Val Lys Thr Gln Tyr

1115 1120 1125

Asp Phe Pro Ser Cys Arg Cys Val Glu Gln Ile Ile Glu Lys Asp

1130 1135 1140

Glu Gly Pro Phe Tyr Thr His Leu Gly Ala Gly Pro Asn Val Ala

1145 1150 1155

Ala Ile Arg Glu Ile Met Glu Glu Arg Phe Gly Gln Lys Gly Lys

1160 1165 1170

Ala Ile Arg Ile Glu Arg Val Ile Tyr Thr Gly Lys Glu Gly Lys

1175 1180 1185

Ser Ser Gln Gly Cys Pro Ile Ala Lys Trp Val Val Arg Arg Ser

1190 1195 1200

Ser Ser Glu Glu Lys Leu Leu Cys Leu Val Arg Glu Arg Ala Gly

1205 1210 1215

His Thr Cys Glu Ala Ala Val Ile Val Ile Leu Ile Leu Val Trp

1220 1225 1230

Glu Gly Ile Pro Leu Ser Leu Ala Asp Lys Leu Tyr Ser Glu Leu

1235 1240 1245

Thr Glu Thr Leu Arg Lys Tyr Gly Thr Leu Thr Asn Arg Arg Cys

1250 1255 1260

Ala Leu Asn Glu Glu Arg Thr Cys Ala Cys Gln Gly Leu Asp Pro

1265 1270 1275

Glu Thr Cys Gly Ala Ser Phe Ser Phe Gly Cys Ser Trp Ser Met

1280 1285 1290

Tyr Tyr Asn Gly Cys Lys Phe Ala Arg Ser Lys Ile Pro Arg Lys

1295 1300 1305

Phe Lys Leu Leu Gly Asp Asp Pro Lys Glu Glu Glu Lys Leu Glu

1310 1315 1320

Ser His Leu Gln Asn Leu Ser Thr Leu Met Ala Pro Thr Tyr Lys

1325 1330 1335

Lys Leu Ala Pro Asp Ala Tyr Asn Asn Gln Ile Glu Tyr Glu His

1340 1345 1350

Arg Ala Pro Glu Cys Arg Leu Gly Leu Lys Glu Gly Arg Pro Phe

1355 1360 1365

Ser Gly Val Thr Ala Cys Leu Asp Phe Cys Ala His Ala His Arg

1370 1375 1380

Asp Leu His Asn Met Gln Asn Gly Ser Thr Leu Val Cys Thr Leu

1385 1390 1395

Thr Arg Glu Asp Asn Arg Glu Phe Gly Gly Lys Pro Glu Asp Glu

1400 1405 1410

Gln Leu His Val Leu Pro Leu Tyr Lys Val Ser Asp Val Asp Glu

1415 1420 1425

Phe Gly Ser Val Glu Ala Gln Glu Glu Lys Lys Arg Ser Gly Ala

1430 1435 1440

Ile Gln Val Leu Ser Ser Phe Arg Arg Lys Val Arg Met Leu Ala

1445 1450 1455

Glu Pro Val Lys Thr Cys Arg Gln Arg Lys Leu Glu Ala Lys Lys

1460 1465 1470

Ala Ala Ala Glu Lys Leu Ser Ser Leu Glu Asn Ser Ser Asn Lys

1475 1480 1485

Asn Glu Lys Glu Lys Ser Ala Pro Ser Arg Thr Lys Gln Thr Glu

1490 1495 1500

Asn Ala Ser Gln Ala Lys Gln Leu Ala Glu Leu Leu Arg Leu Ser

1505 1510 1515

Gly Pro Val Met Gln Gln Ser Gln Gln Pro Gln Pro Leu Gln Lys

1520 1525 1530

Gln Pro Pro Gln Pro Gln Gln Gln Gln Arg Pro Gln Gln Gln Gln

1535 1540 1545

Pro His His Pro Gln Thr Glu Ser Val Asn Ser Tyr Ser Ala Ser

1550 1555 1560

Gly Ser Thr Asn Pro Tyr Met Arg Arg Pro Asn Pro Val Ser Pro

1565 1570 1575

Tyr Pro Asn Ser Ser His Thr Ser Asp Ile Tyr Gly Ser Thr Ser

1580 1585 1590

Pro Met Asn Phe Tyr Ser Thr Ser Ser Gln Ala Ala Gly Ser Tyr

1595 1600 1605

Leu Asn Ser Ser Asn Pro Met Asn Pro Tyr Pro Gly Leu Leu Asn

1610 1615 1620

Gln Asn Thr Gln Tyr Pro Ser Tyr Gln Cys Asn Gly Asn Leu Ser

1625 1630 1635

Val Asp Asn Cys Ser Pro Tyr Leu Gly Ser Tyr Ser Pro Gln Ser

1640 1645 1650

Gln Pro Met Asp Leu Tyr Arg Tyr Pro Ser Gln Asp Pro Leu Ser

1655 1660 1665

Lys Leu Ser Leu Pro Pro Ile His Thr Leu Tyr Gln Pro Arg Phe

1670 1675 1680

Gly Asn Ser Gln Ser Phe Thr Ser Lys Tyr Leu Gly Tyr Gly Asn

1685 1690 1695

Gln Asn Met Gln Gly Asp Gly Phe Ser Ser Cys Thr Ile Arg Pro

1700 1705 1710

Asn Val His His Val Gly Lys Leu Pro Pro Tyr Pro Thr His Glu

1715 1720 1725

Met Asp Gly His Phe Met Gly Ala Thr Ser Arg Leu Pro Pro Asn

1730 1735 1740

Leu Ser Asn Pro Asn Met Asp Tyr Lys Asn Gly Glu His His Ser

1745 1750 1755

Pro Ser His Ile Ile His Asn Tyr Ser Ala Ala Pro Gly Met Phe

1760 1765 1770

Asn Ser Ser Leu His Ala Leu His Leu Gln Asn Lys Glu Asn Asp

1775 1780 1785

Met Leu Ser His Thr Ala Asn Gly Leu Ser Lys Met Leu Pro Ala

1790 1795 1800

Leu Asn His Asp Arg Thr Ala Cys Val Gln Gly Gly Leu His Lys

1805 1810 1815

Leu Ser Asp Ala Asn Gly Gln Glu Lys Gln Pro Leu Ala Leu Val

1820 1825 1830

Gln Gly Val Ala Ser Gly Ala Glu Asp Asn Asp Glu Val Trp Ser

1835 1840 1845

Asp Ser Glu Gln Ser Phe Leu Asp Pro Asp Ile Gly Gly Val Ala

1850 1855 1860

Val Ala Pro Thr His Gly Ser Ile Leu Ile Glu Cys Ala Lys Arg

1865 1870 1875

Glu Leu His Ala Thr Thr Pro Leu Lys Asn Pro Asn Arg Asn His

1880 1885 1890

Pro Thr Arg Ile Ser Leu Val Phe Tyr Gln His Lys Ser Met Asn

1895 1900 1905

Glu Pro Lys His Gly Leu Ala Leu Trp Glu Ala Lys Met Ala Glu

1910 1915 1920

Lys Ala Arg Glu Lys Glu Glu Glu Cys Glu Lys Tyr Gly Pro Asp

1925 1930 1935

Tyr Val Pro Gln Lys Ser His Gly Lys Lys Val Lys Arg Glu Pro

1940 1945 1950

Ala Glu Pro His Glu Thr Ser Glu Pro Thr Tyr Leu Arg Phe Ile

1955 1960 1965

Lys Ser Leu Ala Glu Arg Thr Met Ser Val Thr Thr Asp Ser Thr

1970 1975 1980

Val Thr Thr Ser Pro Tyr Ala Phe Thr Arg Val Thr Gly Pro Tyr

1985 1990 1995

Asn Arg Tyr Ile

2000

<210> 1358

<211> 9796

<212> DNA

<213> Homo sapiens

<400> 1358

ggcagtggca gcggcgagag cttgggcggc cgccgccgcc tcctcgcgag cgccgcgcgc 60

ccgggtcccg ctcgcatgca agtcacgtcc gccccctcgg cgcggccgcc ccgagacgcc 120

ggccccgctg agtgatgaga acagacgtca aactgcctta tgaatattga tgcgggaggct 180

aggctgcttt cgtagagaag cagaaggaag caagatggct gccctttagg atttgttaga 240

aaggagaccc gactgcaact gctggattgc tgcaaggctg agggacgaga acgaggctgg 300

caaacattca gcagcacacc ctctcaagat tgtttacttg cctttgctcc tgttgagtta 360

caacgcttgg aagcaggaga tgggctcagc agcagccaat aggacatgat cggaagag 420

cagtaaggga ctgagctgct gaattcaact agagggcagc cttgtggatg gccccgaagc 480

aagcctgatg gaacaggata gaaccaacca tgttgagggc aacagactaa gtccattcct 540

gataccatca cctcccattt gccagacaga acctctggct acaaagctcc agaatggaag 600

cccactgcct gagagagctc atccagaagt aaatggagac accaagtggc actctttcaa 660

aagttattat ggaataccct gtatgaaggg aagccagaat agtcgtgtga gtcctgactt 720

tacacaagaa agtagagggt attccaagtg tttgcaaaat ggaggaataa aacgcacagt 780

tagtgaacct tctctctctg ggctccttca gatcaagaaa ttgaaacaag accaaaaggc 840

taatggagaa agacgtaact tcggggtaag ccaagaaaga aatccaggtg aaagcagtca 900

accaaatgtc tccgatttga gtgataagaa agaatctgtg agttctgtag cccaagaaaa 960

tgcagttaaa gatttcacca gtttttcaac acataactgc agtgggcctg aaaatccaga 1020

gcttcagatt ctgaatgagc aggaggggaa aagtgctaat taccatgaca agaacattgt 1080

attacttaaa aacaaggcag tgctaatgcc taatggtgct acagtttctg cctcttccgt 1140

ggaacacaca catggtgaac tcctggaaaa aacactgtct caatattatc cagattgtgt 1200

ttccattgcg gtgcagaaaa ccacatctca cataaatgcc attaacagtc aggctactaa 1260

tgagttgtcc tgtgagatca ctcacccatc gcatacctca gggcagatca attccgcaca 1320

gacctctaac tctgagctgc ctccaaagcc agctgcagtg gtgagtgagg cctgtgatgc 1380

1440

accagaacaa ctacaacaac aaaaatcagt ttttgagata tgcccatctc ctgcagaaaa 1500

taacatccag ggaaccacaa agctagcgtc tggtgaagaa ttctgttcag gttccagcag 1560

caatttgcaa gctcctggtg gcagctctga acggtattta aaacaaaatg aaatgaatgg 1620

tgcttacttc aagcaaagct cagtgttcac taaggattcc ttttctgcca ctaccacacc 1680

accaccacca tcacaattgc ttctttctcc ccctcctcct cttccacagg ttcctcagct 1740

tccttcagaa ggaaaaagca ctctgaatgg tggagtttta gaagaacacc accactaccc 1800

caaccaaagt aacacaacac ttttaaggga agtgaaaata gagggtaaac ctgaggcacc 1860

accttcccag agtcctaatc catctacaca tgtatgcagc ccttctccga tgctttctga 1920

aaggcctcag aataattgtg tgaacaggaa tgacatacag actgcaggga caatgactgt 1980

tccattgtgt tctgagaaaa caagaccaat gtcagaacac ctcaagcata acccaccaat 2040

2100

agagattctg aagggtcgag acaaggagca aacacgagat cttgtgcccc caacacagca 2160

ctatctgaaa ccaggatgga ttgaattgaa ggcccctcgt tttcaccaag cggaatccca 2220

tctaaaacgt aatgaggcat cactgccatc aattcttcag tatcaaccca atctctccaa 2280

tcaaatgacc tccaaacaat acactggaaa ttccaacatg cctggggggc tcccaaggca 2340

agcttacacc cagaaaacaa cacagctgga gcacaagtca caaatgtacc aagttgaaat 2400

gaatcaaggg cagtcccaag gtacagtgga ccaacatctc cagttccaaa aaccctcaca 2460

ccaggtgcac ttctccaaaa cagaccattt accaaaagct catgtgcagt cactgtgtgg 2520

cactagattt cattttcaac aaagagcaga ttcccaaact gaaaaactta tgtccccagt 2580

gttgaaacag cacttgaatc aacaggcttc agagactgag ccattttcaa actcacacct 2640

tttgcaacat aagcctcata aacaggcagc acaaacacaa ccatcccaga gttcacatct 2700

ccctcaaaac cagcaacagc agcaaaaatt acaaataaag aataaagagg aaatactcca 2760

gacttttcct cacccccaaa gcaacaatga tcagcaaaga gaaggatcat tctttggcca 2820

gactaaagtg gaagaatgtt ttcatggtga aaatcagtat tcaaaatcaa gcgagttcga 2880

gactcataat gtccaaatgg gactggagga agtacagaat ataaatcgta gaaattcccc 2940

ttatagtcag accatgaaat caagtgcatg caaaatacag gtttcttgtt caaacaatac 3000

acacctagtt tcagagaata aagaacagac tacacatcct gaactttttg caggaaacaa 3060

gacccaaaac ttgcatcaca tgcaatattt tccaaataat gtgatcccaa agcaagatct 3120

3180

atataaaaat agaaaccaag atatgtctgg tcaacaagct gcgcaacttg ctcagcaaag 3240

gtacttgata cataaccatg caaatgtttt tcctgtgcct gaccagggag gaagtcacac 3300

tcagacccct ccccagaagg acactcaaaa gcatgctgct ctaaggtggc atctcttaca 3360

gaagcaagaa cagcagcaaa cacagcaacc ccaaactgag tcttgccata gtcagatgca 3420

caggccaatt aaggtggaac ctggatgcaa gccacatgcc tgtatgcaca cagcaccacc 3480

agaaaacaaa acatggaaaa aggtaactaa gcaagagaat ccacctgcaa gctgtgataa 3540

tgtgcagcaa aagagcatca ttgaccat ggagcagcat ctgaagcagt ttcacgccaa 3600

gtcgttattt gaccataagg ctcttactct caaatcacag aagcaagtaa aagttgaaat 3660

gtcaggggcca gtcacagttt tgactagaca aaccactgct gcagaacttg atagccacac 3720

cccagcttta gagcagcaaa caacttcttc agaaaagaca ccaaccaaaa gaacagctgc 3780

ttctgttctc aataatttta tagagtcacc ttccaaatta ctagatactc ctataaaaaa 3840

tttattggat acacctgtca agactcaata tgatttccca tcttgcagat gtgtagagca 3900

aattattgaa aaagatgaag gtccttttta tacccatcta ggagcaggtc ctaatgtggc 3960

agctattaga gaaatcatgg aagaaaggtt tggacagaag ggtaaagcta ttaggattga 4020

aagagtcatc tatactggta aagaaggcaa aagttctcag ggatgtccta ttgctaagtg 4080

ggtggttcgc agaagcagca gtgaagagaa gctactgtgt ttggtgcggg agcgagctgg 4140

ccacacctgt gaggctgcag tgattgtgat tctcatcctg gtgtgggaag gaatcccgct 4200

gtctctggct gacaaactct actcggagct taccgagacg ctgaggaaat acggcacgct 4260

caccaatcgc cggtgtgcct tgaatgaaga gagaacttgc gcctgtcagg ggctggatcc 4320

agaaacctgt ggtgcctcct tctcttttgg ttgttcatgg agcatgtact acaatggatg 4380

taagtttgcc agaagcaaga tcccaaggaa gtttaagctg cttggggatg acccaaaaga 4440

ggaagagaaa ctggagtctc atttgcaaaa cctgtccact cttatggcac caacatataa 4500

gaaacttgca cctgatgcat ataataatca gattgaatat gaacacagag caccagagtg 4560

ccgtctgggt ctgaaggaag gccgtccatt ctcaggggtc actgcatgtt tggacttctg 4620

tgctcatgcc cacagagact tgcacaacat gcagaatggc agcacattgg tatgcactct 4680

cactagagaa gacaatcgag aatttggagg aaaacctgag gatgagcagc ttcacgttct 4740

gcctttatac aaagtctctg acgtggatga gtttgggagt gtggaagctc aggaggagaa 4800

aaaacggagt ggtgccattc aggtactgag ttcttttcgg cgaaaagtca ggatgttagc 4860

agagccagtc aagacttgcc gacaaaggaa actagaagcc aagaaagctg cagctgaaaa 4920

gctttcctcc ctggagaaca gctcaaataa aaatgaaaag gaaaagtcag ccccatcacg 4980

tacaaaacaa actgaaaacg caagccaggc taaacagttg gcagaacttt tgcgactttc 5040

aggaccagtc atgcagcagt cccagcagcc ccagcctcta cagaagcagc caccacagcc 5100

ccagcagcag cagagacccc agcagcagca gccacatcac cctcagacag agtctgtcaa 5160

ctcttattct gcttctggat ccaccaatcc atacatgaga cggcccaatc cagttagtcc 5220

ttatccaaac tcttcacaca cttcagatat ctatggaagc accagcccta tgaacttcta 5280

ttccacctca tctcaagctg caggttcata tttgaattct tctaatccca tgaaccctta 5340

ccctgggctt ttgaatcaga atacccaata tccatcatat caatgcaatg gaaacctatc 5400

agtggacaac tgctccccat atctgggttc ctattctccc cagtctcagc cgatggatct 5460

gtataggtat ccaagccaag accctctgtc taagctcagt ctaccaccca tccatacact 5520

ttaccagcca aggtttggaa atagccagag ttttacatct aaatacttag gttatggaaa 5580

ccaaaatatg cagggatg gtttcagcag ttgtaccatt agaccaaatg tacatcatgt 5640

agggaaattg cctccttatc ccactcatga gatggatggc cacttcatgg gagccacctc 5700

tagattacca cccaatctga gcaatccaaa catggactat aaaaatggtg aacatcattc 5760

accttctcac ataatccata actacagtgc agctccggggc atgttcaaca gctctcttca 5820

tgccctgcat ctccaaaaca aggagaatga catgctttcc cacacagcta atgggttatc 5880

aaagatgctt ccagctctta accatgatag aactgcttgt gtccaaggag gcttacacaa 5940

attaagtgat gctaatggtc aggaaaagca gccattggca ctagtccagg gtgtggcttc 6000

tggtgcagag gacaacgatg aggtctggtc agacagcgag cagagctttc tggatcctga 6060

cattggggga gtggccgtgg ctccaactca tgggtcaatt ctcattgagt gtgcaaagcg 6120

tgagctgcat gccacaaccc ctttaaagaa tcccaatagg aatcacccca ccaggatctc 6180

6240

agccaaaatg gctgaaaaag cccgtgagaa agaggaagag tgtgaaaagt atggcccaga 6300

ctatgtgcct cagaaatccc atggcaaaaa agtgaaacgg gagcctgctg agccacatga 6360

aacttcagag cccacttacc tgcgtttcat caagtctctt gccgaaagga ccatgtccgt 6420

gaccacagac tccacagtaa ctacatctcc atatgccttc actcgggtca cagggcctta 6480

caacagatat atatgatatc accccctttt gttggttacc tcacttgaaa agaccacaac 6540

caacctgtca gtagtatagt tctcatgacg tgggcagtgg ggaaaggtca cagtattcat 6600

6660

cacttaatttcactggctc ccaagtggtc acagatggca tctaggaaaa gaccaaagca 6720

ttctatgcaa aaagaaggtg gggaagaaag tgttccgcaa tttacatttt taaacactgg 6780

ttctattatt ggacgagatg atatgtaaat gtgatccccc ccccccgctt acaactctac 6840

acatctgtga ccacttttaa taatatcaag tttgcatagt catggaacac aaatcaaaca 6900

agtactgtag tattacagtg acaggaatct taaaatacca tctggtgctg aatatatgat 6960

gtactgaaat actggaatta tggctttttg aaatgcagtt tttactgtaa tcttaacttt 7020

tatttatcaa aatagctaca ggaaacatga atagcaggaa aacactgaat ttgtttggat 7080

gttctaagaa atggtgctaa gaaaatggtg tctttaatag ctaaaaattt aatgccttta 7140

tatcatcaag atgctatcag tgtactccag tgcccttgaa taataggggt accttttcat 7200

tcaagttttt atcataatta cctattctta cacaagctta gtttttaaaa tgtggacatt 7260

ttaaaggcct ctggattttg ctcatccagt gaagtccttg taggacaata aacgtatata 7320

7380

atggtgtttt agaagcactt tgtctaccta agctttgaca acttgaacaa tgctaaggta 7440

ctgagatgtt taaaaaacaa gtttactttc attttagaat gcaaagttga tttttttaag 7500

gaaacaaaga aagcttttaa aatatttttg cttttagcca tgcatctgct gatgagcaat 7560

tgtgtccatt tttaacacag ccagttaaat ccaccatggg gcttactgga ttcaagggaa 7620

tacgttagtc cacaaaacat gttttctggt gctcatctca catgctatac tgtaaaacag 7680

ttttatacaa aattgtatga caagttcatt gctcaaaaat gtacagtttt aagaattttc 7740

tattaactgc aggtaataat tagctgcatg ctgcagactc aacaaagcta gttcactgaa 7800

gcctatgcta ttttatggat cataggctct tcagagaact gaatggcagt ctgcctttgt 7860

gttgataatt atgtacattg tgacgttgtc atttcttagc ttaagtgtcc tctttaacaa 7920

gaggattgag cagactgatg cctgcataag atgaataaac agggttagtt ccatgtgaat 7980

ctgtcagtta aaaagaaaca aaaacaggca

8100

8160

atatatatat atatatatat gagtttgaag cagaattcac atcatgatgg tgctactcag 8220

cctgctacaa atatatcata atgtgagcta agaattcatt aaatgtttga gtgatgttcc 8280

tacttgtcat atacctcaac actagtttgg caataggata ttgaactgag agtgaaagca 8340

ttgtgtacca tcattttttt ccaagtcctt tttttttattg ttaaaaaaaa aagcatacct 8400

tttttcaata cttgatttct tagcaagtat aacttgaact tcaacctttt tgttctaaaa 8460

attcagggat atttcagctc atgctctccc tatgccaaca tgtcacctgt gtttatgtaa 8520

aattgttgta ggttaataaa tatattcttt gtcagggatt taaccctttt attttgaatc 8580

8640

ctctttttat tgtaaagaaa agcattttaa aagtttgagg aatcttttga ctgtttcaag 8700

caggaaaaaa aaattacatg aaaatagaat gcactgagtt gataaaggga aaaattgtaa 8760

ggcaggagtt tggcaagtgg ctgttggcca gagacttact tgtaactctc taaatgaagt 8820

ttttttgatc ctgtaatcac tgaaggtaca tactccatgt ggacttccct taaacaggca 8880

aacacctaca ggtatggtgt gcaacagatt gtacaattac attttggcct aaatacattt 8940

ttgcttacta gtatttaaaa taaattctta atcagaggag gccttttgggt tttattggtc 9000

aaatctttgt aagctggctt ttgtcttttt aaaaaatttc ttgaatttgt ggttgtgtcc 9060

aatttgcaaa catttccaaa aatgtttgct ttgcttacaa accacatgat tttaatgttt 9120

tttgtatacc ataatatcta gccccaaaca tttgattact acatgtgcat tggtgatttt 9180

gatcatccat tcttaatatt tgatttctgt gtcacctact gtcatttgtt aaactgctgg 9240

ccaacaagaa caggaagtat agtttggggg gttggggaga gtttacataa ggaagagaag 9300

aaattgagtg gcatattgta aatatcagat ctataattgt aaattaaaa cctgcctcag 9360

ttagaatgaa tggaaagcag atctacaatt tgctaatata ggaatatcag gttgactata 9420

tagccatact tgaaaatgct tctgagtggt gtcaacttta cttgaatgaa tttttcatct 9480

tgattgacgc acagtgatgt acagttcact tctgaagcta gtggttaact tgtgtaggaa 9540

acttttgcag tttgacacta agataacttc tgtgtgcatt tttctatgct tttttaaaaa 9600

ctagtttcat ttcattttca tgagatgttt ggtttataag atctgaggat ggttataaat 9660

actgtaagta ttgtaatgtt atgaatgcag gttatttgaa agctgtttat tattatatca 9720

ttcctgataa tgctatgtga gtgtttttaa taaaatttat atttatttaa tgcactctaa 9780

aaaaaaaaaaaaaaaa 9796

<210> 1359

<211> 9437

<212> DNA

<213> Homo sapiens

<400> 1359

aagcagaagg aagcaagatg gctgcccttt aggatttgtt agaaaggaga cccgactgca 60

actgctggat tgctgcaagg ctgagggacg agaacgagaa ttcaactaga gggcagcctt 120

gtggatggcc ccgaagcaag cctgatggaa caggatagaa ccaaccatgt tgagggcaac 180

agactaagtc cattcctgat accatcacct cccatttgcc agacagaacc tctggctaca 240

aagctccaga atggaagccc actgcctgag agagctcatc cagaagtaaa tggagacacc 300

360

cgtgtgagtc ctgactttac acaagaaagt agagggtatt ccaagtgttt gcaaaatgga 420

ggaataaaac gcacagttag tgaaccttct ctctctgggc tccttcagat caagaaattg 480

aaaaagacc aaaaggctaa tggagaaaga cgtaacttcg gggtaagcca agaaagaaat 540

ccaggtgaaa gcagtcaacc aaatgtctcc gatttgagtg ataagaaaga atctgtgagt 600

tctgtagccc aagaaaatgc agttaaagat ttcaccagtt tttcaacaca taactgcagt 660

gggcctgaaa atccagagct tcagattctg aatgagcagg aggggaaaag tgctaattac 720

catgacaaga acattgtatt acttaaaaac aaggcagtgc taatgcctaa tggtgctaca 780

gtttctgcct cttccgtgga acacacacat ggtgaactcc tggaaaaaac actgtctcaa 840

tattatccag attgtgtttc cattgcggtg cagaaaacca catctcacat aaatgccatt 900

aacagtcagg ctactaatga gttgtcctgt gagatcactc acccatcgca tacctcaggg 960

cagatcaatt ccgcacagac ctctaactct gagctgcctc caaagccagc tgcagtggtg 1020

agtgaggcct gtgatgctga tgatgctgat aatgccagta aactagctgc aatgctaaat 1080

acctgttcct ttcagaaacc agaacaacta caacaacaaa aatcagtttt tgagatatgc 1140

ccatctcctg cagaaaataa catccaggga accacaaagc tagcgtctgg tgaagaattc 1200

1260

caaaatgaaa tgaatggtgc ttacttcaag caaagctcag tgttcactaa ggattccttt 1320

tctgccacta ccacaccacc acaccatca caattgcttc

ccacaggttc ctcagcttcc ttcagaagga aaaagcactc tgaatggtgg agttttagaa 1440

1500

ggtaaacctg aggcaccacc ttcccagagt cctaatccat ctacacatgt atgcagccct 1560

tctccgatgc tttctgaaag gcctcagaat aattgtgtga acaggaatga catacagact 1620

gcagggacaa tgactgttcc attgtgttct gagaaaacaa gaccaatgtc agaacacctc 1680

aagcataacc caccaatttt tggtagcagt ggagagctac aggacaactg ccagcagttg 1740

1800

gtgcccccaa cacagcacta tctgaaacca ggatggattg aattgaaggc ccctcgtttt 1860

caccaagcgg aatcccatct aaaacgtaat gaggcatcac tgccatcaat tcttcagtat 1920

caacccaatc tctccaatca aatgacctcc aaacaataca ctggaaattc caacatgcct 1980

ggggggctcc caaggcaagc ttacacccag aaaacaacac agctggagca caagtcacaa 2040

atgtaccaag ttgaaatgaa tcaagggcag tcccaaggta cagtggacca acatctccag 2100

ttccaaaaac cctcacacca ggtgcacttc tccaaaacag accatttacc aaaagctcat 2160

gtgcagtcac tgtgtggcac tagatttcat tttcaacaaa gagcagattc ccaaactgaa 2220

aaacttatgt ccccagtgtt gaaacagcac ttgaatcaac aggcttcaga gactgagcca 2280

ttttcaaact cacacctttt gcaacataag cctcataaac aggcagcaca aacacaacca 2340

2400

aaagaggaaa tactccagac ttttcctcac ccccaaagca acaatgatca gcaaagagaa 2460

ggatcattct ttggccagac taaagtggaa gaatgttttc atggtgaaaa tcagtattca 2520

2580

aatcgtagaa attcccctta tagtcagacc atgaaatcaa gtgcatgcaa aatacaggtt 2640

tcttgttcaa acaatacaca cctagtttca gagaataaag aacagactac acatcctgaa 2700

ctttttgcag gaaacaagac ccaaaacttg catcacatgc aatattttcc aaataatgtg 2760

atcccaaagc aagatcttct tcacaggtgc tttcaagaac aggagcagaa gtcacaacaa 2820

gcttcagttc tacagggata taaaaataga aaccaagata tgtctggtca acaagctgcg 2880

caacttgctc agcaaaggta cttgatacat aaccatgcaa atgtttttcc tgtgcctgac 2940

cagggaggaa gtcacactca gacccctccc cagaaggaca ctcaaaagca tgctgctcta 3000

aggtggcatc tcttacagaa gcaagaacag cagcaaacac agcaacccca aactgagtct 3060

tgccatagtc agatgcacag gccaattaag gtggaacctg gatgcaagcc acatgcctgt 3120

3180

cctgcaagct gtgataatgt gcagcaaaag agcatcattg agaccatgga gcagcatctg 3240

aagcagtttc acgccaagtc gttatttgac cataaggctc ttactctcaa atcacagaag 3300

3360

gaacttgata gccacacccc agctttagag cagcaaacaa cttcttcaga aaagacacca 3420

accaaaagaa cagctgcttc tgttctcaat aattttatag agtcaccttc caaattacta 3480

gatactccta taaaaaattt attggataca cctgtcaaga ctcaatatga tttcccatct 3540

tgcagatgtg tagagcaaat tattgaaaaa gatgaaggtc ctttttatac ccatctagga 3600

3660

aaagctatta ggattgaaag agtcatctat actggtaaag aaggcaaaag ttctcaggga 3720

tgtcctattg ctaagtgggt ggttcgcaga agcagcagtg aagagaagct actgtgtttg 3780

gtgcgggagc gagctggcca cacctgtgag gctgcagtga ttgtgattct catcctggtg 3840

tgggaaggaa tcccgctgtc tctggctgac aaactctact cggagcttac cgagacgctg 3900

aggaaatacg gcacgctcac caatcgccgg tgtgccttga atgaagagag aacttgcgcc 3960

tgtcaggggc tggatccaga aacctgtggt gcctccttct cttttggttg ttcatggagc 4020

atgtactaca atggatgtaa gtttgccaga agcaagatcc caaggaagtt taagctgctt 4080

ggggatgacc caaaagagga agagaaactg gagtctcatt tgcaaaacct gtccactctt 4140

atggcaccaa catataagaa acttgcacct gatgcatata ataatcagat tgaatatgaa 4200

cacagagcac cagagtgccg tctgggtctg aaggaaggcc gtccattctc aggggtcact 4260

gcatgtttgg acttctgtgc tcatgcccac agagacttgc acaacatgca gaatggcagc 4320

acattggtat gcactctcac tagagaagac aatcgagaat ttggaggaaa acctgaggat 4380

gagcagcttc acgttctgcc tttatacaaa gtctctgacg tggatgagtt tgggagtgtg 4440

gaagctcagg aggagaaaaa acggagtggt gccattcagg tactgagttc ttttcggcga 4500

aaagtcagga tgttagcaga gccagtcaag acttgccgac aaaggaaact agaagccaag 4560

aaagctgcag ctgaaaagct ttcctccctg gagaacagct caaataaaaa tgaaaaggaa 4620

aagtcagccc catcacgtac aaaacaaact gaaaacgcaa gccaggctaa acagttggca 4680

gaacttttgc gactttcagg accagtcatg cagcagtccc agcagcccca gcctctacag 4740

aagcagccac cacagcccca gcagcagcag agaccccagc agcagcagcc acatcaccct 4800

cagacagagt ctgtcaactc ttattctgct tctggatcca ccaatccata catgagacgg 4860

cccaatccag ttagtcctta tccaaactct tcacacactt cagatatcta tggaagcacc 4920

agccctatga acttctattc cacctcatct caagctgcag gttcatattt gaattcttct 4980

aatcccatga acccttaccc tgggcttttg aatcagaata cccaatatcc atcatatcaa 5040

tgcaatggaa acctatcagt ggacaactgc tccccatatc tgggttccta ttctccccag 5100

tctcagccga tggatctgta taggtatcca agccaagacc ctctgtctaa gctcagtcta 5160

ccacccatcc atacacttta ccagccaagg ttttggaaata gccagagttt tacatctaaa 5220

tacttaggtt atggaaacca aaatatgcag ggagatggtt tcagcagttg taccattaga 5280

ccaaatgtac atcatgtagg gaaattgcct ccttatccca ctcatgagat ggatggccac 5340

ttcatgggag ccacctctag attaccaccc aatctgagca atccaaacat ggactataaa 5400

aatggtgaac atcattcacc ttctcacata atccataact acagtgcagc tccgggcatg 5460

ttcaacagct ctcttcatgc cctgcatctc caaaacaagg agaatgacat gctttcccac 5520

acagctaatg ggttatcaaa gatgcttcca gctcttaacc atgatagaac tgcttgtgtc 5580

caaggaggct tacacaaatt aagtgatgct aatggtcagg aaaagcagcc attggcacta 5640

gtccagggtg tggcttctgg tgcagaggac aacgatgagg tctggtcaga cagcgagcag 5700

agctttctgg atcctgacat tgggggagtg gccgtggctc caactcatgg gtcaattctc 5760

attgagtgtg caaagcgtga gctgcatgcc acaacccctt taaagaatcc caataggaat 5820

caccccacca ggatctccct cgtcttttac cagcataaga gcatgaatga gccaaaacat 5880

ggcttggctc tttgggaagc caaaatggct gaaaaagccc gtgagaaaga ggaagagtgt 5940

gaaaagtatg gccagacta tgtgcctcag aaatcccatg gcaaaaaagt gaaacgggag 6000

cctgctgagc cacatgaaac ttcagagccc acttacctgc gtttcatcaa gtctcttgcc 6060

gaaaggacca tgtccgtgac cacagactcc acagtaacta catctccata tgccttcact 6120

6180

cttgaaaaga ccacaaccaa cctgtcagta gtatagttct catgacgtgg gcagtgggga 6240

aaggtcacag tattcatgac aaatgtggtg ggaaaaacct cagctcacca gcaacaaaag 6300

aggttatctt accatagcac ttaattttca ctggctccca agtggtcaca gatggcatct 6360

aggaaaagac caaagcattc tatgcaaaaa gaaggtgggg aagaaagtgt tccgcaattt 6420

acatttttaa acactggttc tattattgga cgagatgata tgtaaatgtg atcccccccc 6480

cccgcttaca actctacaca tctgtgacca cttttaataa tatcaagttt gcatagtcat 6540

6600

ggtgctgaat atatgatgta ctgaaatact ggaattatgg ctttttgaaa tgcagttttt 6660

actgtaatct taacttttat ttatcaaaat agctacagga aacatgaata gcaggaaaac 6720

actgaatttg tttggatgtt ctaagaaatg gtgctaagaa aatggtgtct ttaatagcta 6780

aaaatttaat gcctttatat catcaagatg ctatcagtgt actccagtgc ccttgaataa 6840

taggggtacc ttttcattca agtttttatc ataattacct attcttacac aagcttagtt 6900

tttaaaatgt ggacatttta aaggcctctg gattttgctc atccagtgaa gtccttgtag 6960

7020

atgtataaat attttaaatg gtgttttaga agcactttgt ctacctaagc tttgacaact 7080

tgaacaatgc taaggtactg agatgtttaa aaaacaagtt tactttcatt ttagaatgca 7140

aagttgattt ttttaaggaa acaaagaaag cttttaaaat atttttgctt ttagccatgc 7200

atctgctgat gagcaattgt gtccattttt aacacagcca gttaaatcca ccatggggct 7260

tactggattc aagggaatac gttagtccac aaaacatgtt ttctggtgct catctcacat 7320

gctatactgt aaaacagttt tatacaaaat tgtatgacaa gttcattgct caaaaatgta 7380

cagttttaag aattttctat taactgcagg taataattag ctgcatgctg cagactcaac 7440

aaagctagtt cactgaagcc tatgctattt tatggatcat aggctcttca gagaactgaa 7500

tggcagtctg cctttgtgtt gataattatg tacattgtga cgttgtcatt tcttagctta 7560

agtgtcctct ttaacaagag gattgagcag actgatgcct gcataagatg aataaacagg 7620

gttagttcca tgtgaatctg tcagttaaaa agaaacaaaa acaggcagct ggtttgctgt 7680

ggtggtttta aatcattaat ttgtataaag aagtgaaaga gttgtatagt aaattaaatt 7740

gtaaacaaaa cttttttaat gcaatgcttt agtattttag tactgtaaaa aaattaaata 7800

tatacatata tatatatata tatatatata tatatatgag tttgaagcag aattcacatc 7860

atgatggtgc tactcagcct gctacaaata tatcataatg tgagctaaga attcattaaa 7920

tgtttgagtg atgttcctac ttgtcatata cctcaacact agtttggcaa taggatattg 7980

8040

aaaaaaaaag catacctttt ttcaatactt gatttcttag caagtataac ttgaacttca 8100

acctttttgt tctaaaaatt cagggatatt tcagctcatg ctctccctat gccaacatgt 8160

cacctgtgtt tatgtaaaat tgttgtaggt taataaatat attctttgtc agggatttaa 8220

cccttttatt ttgaatccct tctattttac ttgtacatgt gctgatgtaa ctaaaactaa 8280

ttttgtaaat ctgttggctc tttttattgt aaagaaaagc attttaaaag tttgaggaat 8340

cttttgactg tttcaagcag gaaaaaaaaa ttacatgaaa atagaatgca ctgagttgat 8400

aaagggaaaa attgtaaggc aggagtttgg caagtggctg ttggccagag acttacttgt 8460

aactctctaa atgaagtttt tttgatcctg taatcactga aggtacatac tccatgtgga 8520

cttcccttaa acaggcaaac acctacaggt atggtgtgca acagattgta caattacatt 8580

ttggcctaaa tacatttttg cttactagta tttaaaataa attcttaatc agaggaggcc 8640

tttgggtttt attggtcaaa tctttgtaag ctggcttttg tctttttaaa aaatttcttg 8700

aatttgtggt tgtgtccaat ttgcaaacat ttccaaaaat gtttgctttg cttacaaacc 8760

acatgatttt aatgtttttt gtataccata atatctagcc ccaaacattt gattactaca 8820

tgtgcattgg tgattttgat catccattct taatatttga tttctgtgtc acctactgtc 8880

atttgttaaa ctgctggcca acaagaacag gaagtatagt ttggggggtt ggggagagtt 8940

tacataagga agagaagaaa ttgagtggca tattgtaaat atcagatcta taattgtaaa 9000

tataaaacct gcctcagtta gaatgaatgg aaagcagatc tacaatttgc taatatagga 9060

atatcaggtt gactatatag ccatacttga aaatgcttct gagtggtgtc aactttactt 9120

gaatgaattt ttcatcttga ttgacgcaca gtgatgtaca gttcacttct gaagctagtg 9180

9240

ctatgctttt ttaaaaacta gtttcatttc attttcatga gatgtttggt ttataagatc 9300

tgaggatggt tataaatact gtaagtattg taatgttatg aatgcaggtt atttgaaagc 9360

tgtttattat tatatcattc ctgataatgc tatgtgagtg tttttaataa aatttatatt 9420

tatttaatgc actctaa 9437

<210> 1360

<211> 9289

<212> DNA

<213> Homo sapiens

<400> 1360

gtagagaagc agaaggaagc aagatggctg ccctttagga tttgttagaa aggagacccg 60

actgcaactg ctggattgct gcaaggctga gggacgagaa cgaggctggc aaacattcag 120

cagcacaccc tctcaagatt gtttacttgc ctttgctcct gttgagttac aacgcttgga 180

agcaggagat gggctcagca gcagccaata ggacatgatc caggaagagc agtaagggac 240

tgagctgctg aattcaacta gagggcagcc ttgtggatgg ccccgaagca agcctgatgg 300

aacaggatag aaccaaccat gttgagggca acagactaag tccattcctg ataccatcac 360

ctcccatttg ccagacagaa ccctggcta caaagctcca gaatggaagc ccactgcctg 420

agagagctca tccagaagta aatggagaca ccaagtggca ctctttcaaa agttattatg 480

gaataccctg tatgaaggga agccagaata gtcgtgtgag tcctgacttt acacaagaaa 540

gtagagggta ttccaagtgt ttgcaaaatg gaggaataaa acgcacagtt agtgaacctt 600

ctctctctgg gctccttcag atcaagaaat tgaaacaaga ccaaaaggct aatggagaaa 660

gacgtaactt cggggtaagc caagaaagaa atccaggtga aagcagtcaa ccaaatgtct 720

ccgatttgag tgataagaaa gaatctgtga gttctgtagc ccaagaaaat gcagttaaag 780

atttcaccag tttttcaaca cataactgca gtgggcctga aaatccagag cttcagattc 840

tgaatgagca ggaggggaaa agtgctaatt accatgacaa gaacattgta ttacttaaaa 900

acaaggcagt gctaatgcct aatggtgcta cagtttctgc ctcttccgtg gaacacacac 960

atggtgaact cctggaaaaa acactgtctc aatattatcc agattgtgtt tccattgcgg 1020

tgcagaaaac cacatctcac ataaatgcca ttaacagtca ggctactaat gagttgtcct 1080

gtgagatcac tcacccatcg catacctcag ggcagatcaa ttccgcacag acctctaact 1140

ctgagctgcc tccaaagcca gctgcagtgg tgagtgaggc ctgtgatgct gatgatgctg 1200

ataatgccag taaactagct gcaatgctaa atacctgttc ctttcagaaa ccagaacaac 1260

1320

gaaccacaaa gctagcgtct ggtgaagaat tctgttcagg ttccagcagc aatttgcaag 1380

ctcctggtgg cagctctgaa cggtatttaa aacaaaatga aatgaatggt gcttacttca 1440

agcaaagctc agtgttcact aaggattcct tttctgccac taccacacca ccaccaccat 1500

1560

1620

acacaacact tttaagggaa gtgaaaatag agggtaaacc tgaggcacca ccttcccaga 1680

gtcctaatcc atctacacat gtatgcagcc cttctccgat gctttctgaa aggcctcaga 1740

ataattgtgt gaacaggaat gacatacaga ctgcagggac aatgactgtt ccattgtgtt 1800

ctgagaaaac aagaccaatg tcagaacacc tcaagcataa cccaccaatt tttggtagca 1860

gtggagagct acaggacaac tgccagcagt tgatgagaaa caaagagcaa gagattctga 1920

agggtcgaga caaggagcaa acacgagatc ttgtgccccc aacacagcac tatctgaaac 1980

caggatggat tgaattgaag gcccctcgtt ttcaccaagc ggaatcccat ctaaaacgta 2040

atgaggcatc actgccatca attcttcagt atcaacccaa tctctccaat caaatgacct 2100

ccaaacaata cactggaaat tccaacatgc ctggggggct cccaaggcaa gcttacaccc 2160

agaaaacaac acagctggag cacaagtcac aaatgtacca agttgaaatg aatcaagggc 2220

agtcccaagg tacagtggac caacatctcc agttccaaaa accctcacac caggtgcact 2280

tctccaaaac agaccattta ccaaaagctc atgtgcagtc actgtgtggc actagatttc 2340

attttcaaca aagagcagat tcccaaactg aaaaacttat gtccccagtg ttgaaacagc 2400

acttgaatca acaggcttca gagactgagc cattttcaaa ctcacacctt ttgcaacata 2460

agcctcataa acaggcagca caaacacaac catcccagag ttcacatctc cctcaaaacc 2520

agcaacagca gcaaaaatta caaataaaga ataaagagga aatactccag acttttcctc 2580

acccccaaag caacaatgat cagcaaaag aaggatcatt ctttggccag actaaagtgg 2640

aagaatgttt tcatggtgaa aatcagtatt caaaatcaag cgagttcgag actcataatg 2700

tccaaatggg actggaggaa gtacagaata taaatcgtag aaattcccct tatagtcaga 2760

2820

cagagaataa agaacagact acacatcctg aactttttgc aggaaacaag acccaaaact 2880

tgcatcacat gcaatatttt ccaaataatg tgatcccaaa gcaagatctt cttcacaggt 2940

gctttcaaga acaggagcag aagtcacaac aagcttcagt tctacaggga tataaaaata 3000

3060

ataaccatgc aaatgttttt cctgtgcctg accagggagg aagtcacact cagacccctc 3120

cccagaagga cactcaaaag catgctgctc taaggtggca tctcttacag aagcaagaac 3180

agcagcaaac acagcaaccc caaactgagt cttgccatag tcagatgcac aggccaatta 3240

aggtggaacc tggatgcaag ccacatgcct gtatgcacac agcaccacca gaaaacaaaa 3300

3360

agagcatcat tgagaccatg gagcagcatc tgaagcagtt tcacgccaag tcgttatttg 3420

accataaggc tcttactctc aaatcacaga agcaagtaaa agttgaaatg tcagggccag 3480

tcacagtttt gactagacaa accactgctg cagaacttga tagccacacc ccagctttag 3540

agcagcaaac aacttcttca gaaaagacac caaccaaaag aacagctgct tctgttctca 3600

ataattttat agagtcacct tccaaattac tagatactcc tataaaaaat ttattggata 3660

cacctgtcaa gactcaatat gatttcccat cttgcagatg tgtaggtttg gacagaaggg 3720

taaagctatt aggattgaaa gagtcatcta tactggtaaa gaaggcaaaa gttctcaggg 3780

atgtcctatt gctaagtggg agaacttgcg cctgtcaggg gctggatcca gaaacctgtg 3840

gtgcctcctt ctcttttggt tgttcatgga gcatgtacta caatggatgt aagtttgcca 3900

gaagcaagat cccaaggaag tttaagctgc ttggggatga cccaaaagag gaagagaaac 3960

tggagtctca tttgcaaaac ctgtccactc ttatggcacc aacatataag aaacttgcac 4020

ctgatgcata taataatcag attgaatatg aacacagagc accagagtgc cgtctgggtc 4080

tgaaggaagg ccgtccattc tcaggggtca ctgcatgttt ggacttctgt gctcatgccc 4140

acagagactt gcacaacatg cagaatggca gcacattggt atgcactctc actagagaag 4200

acaatcgaga atttggagga aaacctgagg atgagcagct tcacgttctg cctttataca 4260

aagtctctga cgtggatgag tttgggagtg tggaagctca ggaggagaaa aaacggagtg 4320

gtgccattca ggtactgagt tcttttcggc gaaaagtcag gatgttagca gagccagtca 4380

agacttgccg acaaaggaaa ctagaagcca agaaagctgc agctgaaaag ctttcctccc 4440

tggagaacag ctcaaataaa aatgaaaagg aaaagtcagc cccatcacgt acaaaacaaa 4500

ctgaaaacgc aagccaggct aaacagttgg cagaactttt gcgactttca ggaccagtca 4560

tgcagcagtc ccagcagccc cagcctctac agaagcagcc accacagccc cagcagcagc 4620

agagacccca gcagcagcag ccacatcacc ctcagacaga gtctgtcaac tcttattctg 4680

cttctggatc caccaatcca tacatgagac ggcccaatcc agttagtcct tatccaaact 4740

cttcacacac ttcagatatc tatggaagca ccagccctat gaacttctat tccacctcat 4800

ctcaagctgc aggttcatat ttgaattctt ctaatcccat gaacccttac cctgggcttt 4860

tgaatcagaa tacccaatat ccatcatatc aatgcaatgg aaacctatca gtggacaact 4920

gctccccata tctgggttcc tattctcccc agtctcagcc gatggatctg tataggtatc 4980

caagccaaga ccctctgtct aagctcagtc taccacccat ccatacactt taccagccaa 5040

ggtttggaaa tagccagagt tttacatcta aatacttagg ttatggaaac caaaatatgc 5100

agggagatgg tttcagcagt tgtaccatta gaccaaatgt acatcatgta gggaaattgc 5160

ctccttatcc cactcatgag atggatggcc acttcatggg agccacctct agattaccac 5220

ccaatctgag caatccaaac atggactata aaaatggtga acatcattca ccttctcaca 5280

taatccataa ctacagtgca gctccgggca tgttcaacag ctctcttcat gccctgcatc 5340

tccaaaacaa ggagaatgac atgctttccc acacagctaa tgggttatca aagatgcttc 5400

cagctcttaa ccatgataga actgcttgtg tccaaggagg cttacacaaa ttaagtgatg 5460

ctaatggtca ggaaaagcag ccattggcac tagtccaggg tgtggcttct ggtgcagagg 5520

acaacgatga ggtctggtca gacagcgagc agagctttct ggatcctgac attgggggag 5580

tggccgtggc tccaactcat gggtcaattc tcattgagtg tgcaaagcgt gagctgcatg 5640

ccacaacccc tttaaagaat cccaatagga atcaccccac caggatctcc ctcgtctttt 5700

accagcataa gagcatgaat gagccaaaac atggcttggc tctttgggaa gccaaaatgg 5760

ctgaaaaagc ccgtgagaaa gaggaagagt gtgaaaagta tggcccagac tatgtgcctc 5820

agaaatccca tggcaaaaaa gtgaaacggg agcctgctga gccacatgaa acttcagagc 5880

ccacttacct gcgtttcatc aagtctcttg ccgaaaggac catgtccgtg accacagact 5940

ccacagtaac tacatctcca tatgccttca ctcgggtcac agggccttac aacagatata 6000

tatgatatca cccccttttg ttggttacct cacttgaaaa gaccacaacc aacctgtcag 6060

tagtatagtt ctcatgacgt gggcagtggg gaaaggtcac agtattcatg acaaatgtgg 6120

6180

cactggctcc caagtggtca cagatggcat ctaggaaaag accaaagcat tctatgcaaa 6240

aagaaggtgg ggaagaaagt gttccgcaat ttacattttt aaacactggt tctattattg 6300

tgatcccccc cccccgctta caactctaca catctgtgac 6360

cacttttaat aatatcaagt ttgcatagtc atggaacaca aatcaaacaa gtactgtagt 6420

attacagtga caggaatctt aaaataccat ctggtgctga atatatgatg tactgaaata 6480

ctggaattat ggctttttga aatgcagttt ttactgtaat cttaactttt atttatcaaa 6540

tagctacag gaaacatgaa tagcaggaaa acactgaatt tgtttggatg ttctaagaaa 6600

tggtgctaag aaaatggtgt ctttaatagc taaaaattta atgcctttat atcatcaaga 6660

tgctatcagt gtactccagt gcccttgaat aataggggta ccttttcatt caagttttta 6720

tcataattac ctattcttac acaagcttag tttttaaaat gtggacattt taaaggcctc 6780

tggattttgc tcatccagtg aagtccttgt aggacaataa acgtatatat gtacatatatat 6840

acacaaacat gtatatgtgc acacacatgt atatgtataa atattttaaa tggtgtttta 6900

6960

7020

agcttttaaa atatttttgc ttttagccat gcatctgctg atgagcaatt gtgtccattt 7080

ttaacacagc cagttaaatc caccatgggg cttactggat tcaagggaat acgttagtcc 7140

acaaaacatg ttttctggtg ctcatctcac atgctatact gtaaaacagt tttatacaaa 7200

attgtatgac aagttcattg ctcaaaaatg tacagtttta agaattttct attaactgca 7260

ggtaataatt agctgcatgc tgcagactca acaaagctag ttcactgaag cctatgctat 7320

tttatggatc ataggctctt cagagaactg aatggcagtc tgcctttgtg ttgataatta 7380

tgtacattgt gacgttgtca tttcttagct taagtgtcct ctttaacaag aggattgagc 7440

agactgatgc ctgcataaga tgaataaaca gggttagttc catgtgaatc tgtcagttaa 7500

aaagaaacaa aaacaggcag ctggtttgct gtggtggttt taaatcatta atttgtataa 7560

agaagtgaaa gagttgtata gtaaattaaa ttgtaaacaa aactttttta atgcaatgct 7620

ttagtatttt agtactgtaa aaaaattaaa tatatacata tatatatata tatatatata 7680

tatatatatg agtttgaagc agaattcaca tcatgatggt gctactcagc ctgctacaaa 7740

tatatcataa tgtgagctaa gaattcatta aatgtttgag tgatgttcct acttgtcata 7800

tacctcaaca ctagtttggc aataggatat tgaactgaga gtgaaagcat tgtgtaccat 7860

catttttttc caagtccttt tttttattgt taaaaaaaaa agcatacctt ttttcaatac 7920

ttgatttctt agcaagtata acttgaactt caaccttttt gttctaaaaa ttcagggata 7980

tttcagctca tgctctccct atgccaacat gtcacctgtg tttatgtaaa attgttgtag 8040

gttaataaat atattctttg tcagggattt aaccctttta ttttgaatcc cttctatttt 8100

acttgtacat gtgctgatgt aactaaaact aattttgtaa atctgttggc tctttttatt 8160

8220

aattacatga aaatagaatg cactgagttg ataaagggaa aaattgtaag gcaggagttt 8280

ggcaagtggc tgttggccag agacttactt gtaactctct aaatgaagtt tttttgatcc 8340

tgtaatcact gaaggtacat actccatgtg gacttccctt aaacaggcaa acacctacag 8400

gtatggtgtg caacagattg tacaattaca ttttggccta aatacatttt tgcttactag 8460

tatttaaaat aaattcttaa tcagaggagg cctttggggtt ttattggtca aatctttgta 8520

agctggcttt tgtcttttta aaaaatttct tgaatttgtg gttgtgtcca atttgcaaac 8580

atttccaaaa atgtttgctt tgcttacaaa ccacatgatt ttaatgtttt ttgtatacca 8640

taatatctag ccccaaacat ttgattacta catgtgcatt ggtgattttg atcatccatt 8700

cttaatattt gatttctgtg tcacctactg tcatttgtta aactgctggc caacaagaac 8760

aggaagtata gtttgggggg ttggggagag tttacataag gaagagaaga aattgagtgg 8820

catattgtaa atatcagatc tataattgta aatataaaac ctgcctcagt tagaatgaat 8880

ggaaagcaga tctacaattt gctaatatag gaatatcagg ttgactatat agccatactt 8940

gaaaatgctt ctgagtggtg tcaactttac ttgaatgaat ttttcatctt gattgacgca 9000

9060

ttgacactaa gataacttct gtgtgcattt ttctatgctt ttttaaaaac tagtttcatt 9120

9180

tgtaatgtta tgaatgcagg ttatttgaaa gctgtttatt attatatcat tcctgataat 9240

gctatgtgag tgtttttaat aaaatttata tttatttaat gcactctaa 9289

<210> 1361

<211> 9236

<212> DNA

<213> Homo sapiens

<400> 1361

aaacagaagg tgggccgggg cggggagaaa cagaactcgg tcaatttccc agtttgtcgg 60

gtctttaaaa atacaggccc ctaaagcact aagggcatgc cctcggtgaa acaggggagc 120

gcttctgctg aatgagatta aagcgacaga aaagggaaag gagagcgcgg gcaacgggat 180

ctaaagggag atagagacgc gggcctctga gggctggcaa acattcagca gcacaccctc 240

tcaagattgt ttacttgcct ttgctcctgt tgagttacaa cgcttggaag caggagatgg 300

gctcagcagc agccaatagg acatgatcca ggaagagcag taagggactg agctgctgaa 360

ttcaactaga gggcagcctt gtggatggcc ccgaagcaag cctgatggaa caggatagaa 420

ccaaccatgt tgagggcaac agactaagtc cattcctgat accatcacct cccatttgcc 480

agacagaacc tctggctaca aagctccaga atggaagccc actgcctgag agagctcatc 540

cagaagtaaa tggagacacc aagtggcact ctttcaaaag ttattatgga ataccctgta 600

tgaagggaag ccagaatagt cgtgtgagtc ctgactttac acaagaaagt agagggtatt 660

ccaagtgttt gcaaaatgga ggaataaaac gcacagttag tgaaccttct ctctctgggc 720

tccttcagat caagaaattg aaaaagacc aaaaggctaa tggagaaaga cgtaacttcg 780

gggtaagcca agaaagaaat ccaggtgaaa gcagtcaacc aaatgtctcc gatttgagtg 840

ataagaaaga atctgtgagt tctgtagccc aagaaaatgc agttaaagat ttcaccagtt 900

tttcaacaca taactgcagt gggcctgaaa atccagagct tcagattctg aatgagcagg 960

aggggaaaag tgctaattac catgacaaga acattgtatt acttaaaaac aaggcagtgc 1020

taatgcctaa tggtgctaca gtttctgcct cttccgtgga acacacacat ggtgaactcc 1080

tggaaaaaac actgtctcaa tattatccag attgtgtttc cattgcggtg cagaaaacca 1140

catctcacat aaatgccatt aacagtcagg ctactaatga gttgtcctgt gagatcactc 1200

acccatcgca tacctcaggg cagatcaatt ccgcacagac ctctaactct gagctgcctc 1260

caaagccagc tgcagtggtg agtgaggcct gtgatgctga tgatgctgat aatgccagta 1320

aactagctgc aatgctaaat acctgttcct ttcagaaacc agaacaacta caacaacaaa 1380

aatcagtttt tgagatatgc ccatctcctg cagaaaataa catccaggga accacaaagc 1440

tagcgtctgg tgaagaattc tgttcaggtt ccagcagcaa tttgcaagct cctggtggca 1500

gctctgaacg gtatttaaaa caaaatgaaa tgaatggtgc ttacttcaag caaagctcag 1560

tgttcactaa ggattccttt tctgccacta ccacaccacc accaccatca caattgcttc 1620

tttctccccc tcctcctctt ccacaggttc ctcagcttcc ttcagaagga aaaagcactc 1680

tgaatggtgg agttttagaa gaacaccacc actaccccaa ccaaagtaac acaacacttt 1740

taagggaagt gaaaatagag ggtaaacctg aggcaccacc ttcccagagt cctaatccat 1800

ctacacatgt atgcagccct tctccgatgc tttctgaaag gcctcagaat aattgtgtga 1860

acaggaatga catacagact gcagggacaa tgactgttcc attgtgttct gagaaaacaa 1920

gaccaatgtc agaacacctc aagcataacc caccaatttt tggtagcagt ggagagctac 1980

aggacaactg ccagcagttg atgagaaaca aagagcaaga gattctgaag ggtcgagaca 2040

aggagcaaac acgagatctt gtgcccccaa cacagcacta tctgaaacca ggatggattg 2100

aattgaaggc ccctcgtttt caccaagcgg aatcccatct aaaacgtaat gaggcatcac 2160

tgccatcaat tcttcagtat caacccaatc tctccaatca aatgacctcc aaacaataca 2220

ctggaaattc caacatgcct ggggggctcc caaggcaagc ttacacccag aaaacaacac 2280

agctggagca caagtcacaa atgtaccaag ttgaaatgaa tcaagggcag tcccaaggta 2340

cagtggacca acatctccag ttccaaaaac cctcacacca ggtgcacttc tccaaaacag 2400

accatttacc aaaagctcat gtgcagtcac tgtgtggcac tagatttcat tttcaacaaa 2460

gagcagattc ccaaactgaa aaacttatgt ccccagtgtt gaaacagcac ttgaatcaac 2520

aggcttcaga gactgagcca ttttcaaact cacacctttt gcaacataag cctcataaac 2580

2640

aaaaattaca aataaagaat aaagggaaa tactccagac ttttcctcac ccccaaagca 2700

acaatgatca gcaaagagaa ggatcattct ttggccagac taaagtggaa gaatgttttc 2760

atggtgaaaa tcagtattca aaatcaagcg agttcgagac tcataatgtc caaatgggac 2820

tggaggaagt acagaatata aatcgtagaa attcccctta tagtcagacc atgaaatcaa 2880

gtgcatgcaa aatacaggtt tcttgttcaa acaatacaca cctagtttca gagaataaag 2940

aacagactac acatcctgaa ctttttgcag gaaacaagac ccaaaacttg catcacatgc 3000

3060

aggagcagaa gtcacaacaa gcttcagttc tacagggata taaaaataga aaccaagata 3120

tgtctggtca acaagctgcg caacttgctc agcaaaggta cttgatacat aaccatgcaa 3180

atgtttttcc tgtgcctgac cagggaggaa gtcacactca gacccctccc cagaaggaca 3240

ctcaaaagca tgctgctcta aggtggcatc tcttacagaa gcaagaacag cagcaaacac 3300

agcaacccca aactgagtct tgccatagtc agatgcacag gccaattaag gtggaacctg 3360

gatgcaagcc acatgcctgt atgcacacag caccaccaga aaacaaaaca tggaaaaagg 3420

taactaagca agagaatcca cctgcaagct gtgataatgt gcagcaaaag agcatcattg 3480

agaccatgga gcagcatctg aagcagtttc acgccaagtc gttatttgac cataaggctc 3540

ttactctcaa atcacagaag caagtaaaag ttgaaatgtc agggccagtc acagttttga 3600

ctagacaaac cactgctgca gaacttgata gccacacccc agctttagag cagcaaacaa 3660

cttcttcaga aaagacacca accaaaagaa cagctgcttc tgttctcaat aattttatag 3720

agtcaccttc caaattacta gatactccta taaaaaattt attggataca cctgtcaaga 3780

3840

atggcgttta tccagaatta gcaaatttat cttcagatat gggattttcc ttcttttttt 3900

3960

gtcaagttac cgatgcttgt gtcttgtgaa agagaacttc acttacatgc agtttttcca 4020

aaagaattaa ataatcgtgc atgtttattt ttccctctct tcagatcctg taaaatttga 4080

atgtatctgt tttagatcaa ttcgcctatt tagctctttg tatattatct cctggagaga 4140

cagctaggca gcaaaaaaac aatctattaa aatgagaaaa taacgaccat aggcagtcta 4200

atgtacgaac tttaaatatt ttttaattca aggtaaaata tattagtttc acaagatttc 4260

tggctaatag ggaaattatt atcttcagtc ttcatgagtt gggggaaatg ataatgctga 4320

cactcttagt gctcctaaag ttttccttttc tccatttata catttggaat gttgtgattt 4380

atattcattt tgattccctt ttctctaaaa tttcatcttt ttgattaaaa aatatgatac 4440

aggcatacct cagagatatt gtgggtttgg ctccatacca caataaaatg aatattacaa 4500

4560

tactgtaaca tactaagtgt gcaatagcat tgtgtctaaa aaatatatac tttaaaaata 4620

atttattgtt aaaaaaatgc caacaattat ctgggccttt agtgagtgct aatctttttg 4680

ctggtggagg gtcgtgcttc agtattgatc gctgtggact gatcatggtg gtagttgctg 4740

aaggttgctg ggatggctgt gtgtgtggca atttcttaaa ataagacaac agtgaagtgc 4800

tgtatcaatt gatttttcca ttcacaaaag atttctctgt agcatgcaat gctgtttgat 4860

agcatttaac ccacagcaga atttctttga aaattggact cagtcctctc aaactgtgct 4920

gctgctttat caactaagtt tttgtaattt tctgaatcct ttgttgtcat ttcagcagtt 4980

tacagcatct tcattggaag tatattccat ctcaaacatt ctttgttcat ccataagaag 5040

caacttctta tcaagttttt tcatgacatt gcagtaactc agccccatct tcaggctcta 5100

cttctaattc tggttctctt gctacatctc cctcatctgc agtgacctct ccacggaagt 5160

cttgaactcc tcaaagtaat ccatgagggt tggaatcaac ttctaaactc ctgttaatgt 5220

tgatatattg accccctccc atgaattatg aatgttctta ataacttcta aatggtgata 5280

cctttccaga aggctttcaa tgtactttgc ccggatccat cagaagacta tcttggcagc 5340

tgtagactaa caatatattt cttaaatgat aagacttgaa agtcaaaagt actccttaat 5400

ccataggctg cagaatcaat gttgtattaa caggcacgaa aacagcatta atcttgtgca 5460

tctccatcgg agctcttgggg tgactaggtg ccttgagcag taatattttg aaaggaggtt 5520

ttggttttgt tttttgtttt ttttttttgt tttttagcag taagtctcaa cactgggctt 5580

aaaatattca gtaaactatg ttgtaaaaag atgtgttatc atccagactt tgttgttcca 5640

ttactctaca caagcagggt acacttagca taattcttaa gggccttgga attttcagaa 5700

tggtaaatga gtatgggctt caacttaaaa tcatcaactg cattagcctg taacaagaga 5760

gtcagcctgt cctttgaagc aaggcattga cttctatcta tgaaagtctt agatggcacc 5820

ttgtttcaat agtaggctgt ttagtacagc caccttcatc agtgatctta gctagatctt 5880

ctgcataact tgctgcagct tctacatcag cacttgctgc ctcaccttgt ccttttatgt 5940

tatagagaca gctgcgcttc ttaaacttta taaaccaact tctgctagct tccaacttct 6000

cttctgcagc ttcctcattc tcttcataga actgaaggga gtcaaggcct tgctctggat 6060

taagctttgg cttaaggaat gttgtggctg acgtgatctt ctatccagac cactaaagcg 6120

ctctccatat cagcaataag gccgttttgc ttttcttacct ttcatgtgtt cactggagta 6180

atttccttca agaatttttc ctttacattc acaacttggc taactggcat gcaaggccta 6240

gctttcagcc tgtcttggct tttgacatgc cttcctcact tagctcgtca tatctagctt 6300

ttgatttaaa gtggcaggca tacaactctt cctttcactt gaacacttag aggccactgt 6360

agggttatta attggcctaa tttcaatatt gttgtgtttt agggaataga gaggcccagg 6420

6480

6540

tagaatagta acatcaaaga tcactgatca cagatcgcca taacataaat aataataaac 6600

tttaaaatac tgtgagaatt accaaaatgt gatacagaga catgaagtga gcacatgctg 6660

ttgaaaaaaa tgacactgat agacatactt aacacgtggg attgccacaa accttcagtt 6720

tgtaaaagtc acagtaactg tgactcacaa aagaacaaag cacaataaaa cgaggtatgc 6780

aaaaaaagct ttttgttaaa attcaggata tgtaataggt ctgtaggaat 6840

agtgaaatat ttttgctgat ggatgtagat atatacgtgg atagagatga agatcttaat 6900

6960

tggctaatga cctacccgtg ccatgttttc cctcttgcaa tgagataccc cacactgtgt 7020

agaaggatgg agggaggact cctactgtcc ctctttgcgt gtggttatta agttgcctca 7080

ctgggctaaa acaccacaca tctcatagat aatatttggt aagttgtaat cgtcttcact 7140

cttctcttat cacccacccc tatcttccca cttttccatc tttgttggtt tgcaacagcc 7200

ccttcttttt gcctgactct ccaggatttt ctctcatcat aaattgttct aaagtacata 7260

ctaatatggg tctggattga ctattcttat ttgcaaaaca gcaattaaat gttataggga 7320

agtaggaaga aaaaggggta tccttgacaa taaaccaagc aatattctgg gggtgggata 7380

gagcaggaaa ttttattttt aatcttttaa aatccaagta ataggtaggc ttccagttag 7440

ctttaaatgt ttttttttttc cagctcaaaa aattggattg tagttgatac tacatataat 7500

acattctaat tccctcactg tattctttgt ttagtttcat ttatttggtt taaaataatt 7560

ttttatccca tatctgaaat gtaatatatt tttatccaac aaccagcatg tacatatact 7620

taattatgtg gcacattttc taatagatca gtccatcaat ctactcattt taaagaaaaa 7680

aaaattttaa agtcactttt agagccctta atgtgtagtt gggggttaag ctttgtggat 7740

gtagccttta tatttagtat aattgaggtc taaaataata atcttctatt atctcaacag 7800

agcaaattat tgaaaaagat gaaggtcctt tttataccca tctaggagca ggtcctaatg 7860

tggcagctat tagagaaatc atggaagaaa ggtaattaac gcaaaggcac agggcagatt 7920

aacgtttatc cttttgtata tgtcagaatt tttccagcct tcacacacaa agcagtaaac 7980

aattgtaaat tgagtaatta ttagtaggct tagctattct agggttgcca acactacaca 8040

ctgtgctatt caccagagag tcacaatatt tgacaggact aatagtctgc tagctggcac 8100

aggctgccca ctttgcgatg gatgccagaa aacccaggca tgaacaggaa tcggccagcc 8160

aggctgccag ccacaaggta ctggcacagg ctccaacgag aggtcccact ctggctttcc 8220

cacctgataa taaagtgtca aagcagaaag actggtaaag tgtggtataa gaaaagaacc 8280

actgaattaa attcacctag tgttgcaaat gagtacttat ctctaagttt tcttttacca 8340

taaaaagaga gcaagtgtga tatgttgaat agaaagagaa acatactatt tacagctgcc 8400

tttttttttt tttttcgcta tcaatcacag gtatacaagt acttgccttt actcctgcat 8460

gtagaagact cttatgagcg agataatgca gagaaggcct ttcatataaa tttatacagc 8520

tctgagctgt tcttcttcta gggtgccttt tcattaagag gtaggcagta ttattatattaa 8580

agtacttagg atacattggg gcagctagga catattcagt atcattcttg ctccatttcc 8640

aaattattca tttctaaatt agcatgtaga agttcactaa ataatcatct agtggcctgg 8700

cagaaatagt gaatttccct aagtgccttt tttttgttgt ttttttgttt tgttttttaa 8760

acaagcagta ggtggtgctt tggtcataag ggaagatata gtctatttct aggactattc 8820

catattttcc atgtggctgg atactaacta tttgccagcc tccttttcta aattgtgaga 8880

cattcttgga ggaacagttc taactaaaat ctattatgac tccccaagtt ttaaaaatagc 8940

taaatttagt aagggaaaaa atagtttatg ttttagaaga ctgaacttag caaactaacc 9000

tgaattttgt gctttgtgaa attttatatc gaaatgagct ttcccatttt cacccacatg 9060

aaaaacaagg 9120

cttaaaaacc attatccagt ttgcttggcg tagacctgtt taaaaaataa taaaccgttc 9180

atttctcagg atgtggtcat agaataaagt tatgctcaaa tgttcaaata tttaaa 9236

<210> 1362

<211> 5969

<212> DNA

<213> Homo sapiens

<400> 1362

tcaggctcta cttctaattc tggttctctt gctacatctc cctcatctgc agtgacctct 60

ccacggaagt cttgaactcc tcaaaagcaa attattgaaa aagatgaagg tcctttttat 120

acccatctag gagcaggtcc taatgtggca gctattagag aaatcatgga agaaaggttt 180

ggacagaagg gtaaagctat taggattgaa agagtcatct atactggtaa agaaggcaaa 240

agttctcagg gatgtcctat tgctaagtgg gtggttcgca gaagcagcag tgaagagaag 300

ctactgtgtt tggtgcggga gcgagctggc cacacctgtg aggctgcagt gattgtgatt 360

ctcatcctgg tgtgggaagg aatcccgctg tctctggctg acaaactcta ctcggagctt 420

accgagacgc tgaggaaata cggcacgctc acccaatcgcc ggtgtgcctt gaatgaagag 480

agaacttgcg cctgtcaggg gctggatcca gaaacctgtg gtgcctcctt ctcttttggt 540

tgttcatgga gcatgtacta caatggatgt aagtttgcca gaagcaagat cccaaggaag 600

660

ctgtccactc ttatggcacc aacatataag aaacttgcac ctgatgcata taataatcag 720

attgaatatg aacacagagc accagagtgc cgtctgggtc tgaaggaagg ccgtccattc 780

tcaggggtca ctgcatgttt ggacttctgt gctcatgccc acagagactt gcacaacatg 840

cagaatggca gcacattggt atgcactctc actagagaag acaatcgaga atttggagga 900

aaacctgagg atgagcagct tcacgttctg cctttataca aagtctctga cgtggatgag 960

tttgggagtg tggaagctca ggaggagaaa aaacggagtg gtgccattca ggtactgagt 1020

1080

ctgaagcca agaaagctgc agctgaaaag ctttcctccc tggagaacag ctcaaataaa 1140

aatgaaaagg aaaagtcagc cccatcacgt acaaaacaaa ctgaaaacgc aagccaggct 1200

aaacagttgg cagaactttt gcgactttca ggaccagtca tgcagcagtc ccagcagccc 1260

cagcctctac agaagcagcc accacagccc cagcagcagc agagacccca gcagcagcag 1320

ccacatcacc ctcagacaga gtctgtcaac tcttattctg cttctggatc caccaatcca 1380

tacatgagac ggcccaatcc agttagtcct tatccaaact cttcacacac ttcagatatc 1440

tatggaagca ccagccctat gaacttctat tccacctcat ctcaagctgc aggttcatat 1500

ttgaattctt ctaatcccat gaacccttac cctgggcttt tgaatcagaa tacccaatat 1560

ccatcatatc aatgcaatgg aaacctatca gtggacaact gctccccata tctgggttcc 1620

tattctcccc agtctcagcc gatggatctg tataggtatc caagccaaga ccctctgtct 1680

aagctcagtc taccacccat ccatacactt taccagccaa ggtttggaaa tagccagagt 1740

tttacatcta aatacttagg ttatggaaac caaaatatgc agggagatgg tttcagcagt 1800

tgtaccatta gaccaaatgt acatcatgta gggaaattgc ctccttatcc cactcatgag 1860

atggatggcc acttcatgggg agccacctct agattaccac ccaatctgag caatccaaac 1920

atggactata aaaatggtga acatcattca ccttctcaca taatccataa ctacagtgca 1980

gctccgggca tgttcaacag ctctcttcat gccctgcatc tccaaaacaa ggagaatgac 2040

atgctttccc acacagctaa tgggttatca aagatgcttc cagctcttaa ccatgataga 2100

actgcttgtg tccaaggagg cttacacaaa ttaagtgatg ctaatggtca ggaaaagcag 2160

ccattggcac tagtccaggg tgtggcttct ggtgcagagg acaacgatga ggtctggtca 2220

gacagcgagc agagctttct ggatcctgac attgggggag tggccgtggc tccaactcat 2280

gggtcaattc tcattgagtg tgcaaagcgt gagctgcatg ccacaacccc tttaaagaat 2340

accagcataa gagcatgaat 2400

gagccaaaac atggcttggc tctttgggaa gccaaaatgg ctgaaaaagc ccgtgagaaa 2460

gaggaagagt gtgaaaagta tggcccagac tatgtgcctc agaaatccca tggcaaaaaa 2520

gtgaaacggg agcctgctga gccacatgaa acttcagagc ccacttacct gcgtttcatc 2580

aagtctcttg ccgaaaggac catgtccgtg accacagact ccacagtaac tacatctcca 2640

2700

ttggttacct cacttgaaaa gaccacaacc aacctgtcag tagtatagtt ctcatgacgt 2760

gggcagtggg gaaaggtcac agtattcatg acaaatgtgg tgggaaaaac ctcagctcac 2820

cagcaacaaa agaggttatc ttaccatagc acttaatttt cactggctcc caagtggtca 2880

cagatggcat ctaggaaaag accaaagcat tctatgcaaa aagaaggtgg ggaagaaagt 2940

gttccgcaat ttacattttt aaacactggt tctattattg gacgagatga tatgtaaatg 3000

tgatcccccc cccccgctta caactctaca catctgtgac cacttttaat aatatcaagt 3060

ttgcatagtc atggaacaca aatcaaacaa gtactgtagt attacagtga caggaatctt 3120

aaaataccat ctggtgctga atatatgatg tactgaaata ctggaattat ggctttttga 3180

aatgcagttt ttactgtaat cttaactttt atttatcaaa atagctacag gaaacatgaa 3240

3300

ctttaatagc taaaaattta atgcctttat atcatcaaga tgctatcagt gtactccagt 3360

gcccttgaat aataggggta ccttttcatt caagttttta tcataattac ctattcttac 3420

3480

aagtccttgt aggacaataa acgtatatat gtacatatat acacaaacat gtatatgtgc 3540

acacacatgt atatgtataa atattttaaa tggtgtttta gaagcacttt gtctacctaa 3600

3660

ttttagaatg caaagttgat tttttttaagg aaacaaagaa agcttttaaa atatttttgc 3720

ttttagccat gcatctgctg atgagcaatt gtgtccattt ttaacacagc cagttaaatc 3780

caccatgggg cttactggat tcaagggaat acgttagtcc acaaaacatg ttttctggtg 3840

ctcatctcac atgctatact gtaaaacagt tttatacaaa attgtatgac aagttcattg 3900

3960

tgcagactca acaaagctag ttcactgaag cctatgctat tttatggatc ataggctctt 4020

cagagaactg aatggcagtc tgcctttgtg ttgataatta tgtacattgt gacgttgtca 4080

tttcttagct taagtgtcct ctttaacaag aggattgagc agactgatgc ctgcataaga 4140

tgaataaaca gggttagttc catgtgaatc tgtcagttaa aaagaaacaa aaacaggcag 4200

ctggtttgct gtggtggttt taaatcatta atttgtataa agaagtgaaa gagttgtata 4260

gtaaattaaa ttgtaaacaa aactttttta atgcaatgct ttagtatttt agtactgtaa 4320

aaaaattaaa tatatacata tatatatata tatatatata tatatatatg agtttgaagc 4380

agaattcaca tcatgatggt gctactcagc ctgctacaaa tatatcataa tgtgagctaa 4440

gaattcatta aatgtttgag tgatgttcct acttgtcata tacctcaaca ctagtttggc 4500

aataggatat tgaactgaga gtgaaagcat tgtgtaccat catttttttc caagtccttt 4560

tttttattgt taaaaaaaaa agcatacctt ttttcaatac ttgatttctt agcaagtata 4620

acttgaactt caaccttttt gttctaaaaa ttcagggata tttcagctca tgctctccct 4680

atgccaacat gtcacctgtg tttatgtaaa attgttgtag gttaataaat atattctttg 4740

tcagggattt aacccttttta ttttgaatcc cttctatttt acttgtacat gtgctgatgt 4800

aactaaaact aattttgtaa atctgttggc tcttttttatt gtaaagaaaa gcattttaaa 4860

agtttgagga atcttttgac tgtttcaagc aggaaaaaaa aattacatga aaatagaatg 4920

cactgagttg ataaagggaa aaattgtaag gcaggagttt ggcaagtggc tgttggccag 4980

agacttactt gtaactctct aaatgaagtt tttttgatcc tgtaatcact gaaggtacat 5040

actccatgtg gacttccctt aaacaggcaa acacctacag gtatggtgtg caacagattg 5100

tacaattaca ttttggccta aatacatttt tgcttactag tatttaaaat aaattcttaa 5160

tcagaggagg cctttggggtt ttattggtca aatctttgta agctggcttt tgtcttttta 5220

aaaaatttct tgaatttgtg gttgtgtcca atttgcaaac atttccaaaa atgtttgctt 5280

tgcttacaaa ccacatgatt ttaatgtttt ttgtatacca taatatctag ccccaaacat 5340

ttgattacta catgtgcatt ggtgattttg atcatccatt cttaatattt gatttctgtg 5400

tcacctactg tcatttgtta aactgctggc caacaagaac aggaagtata gtttgggggg 5460

ttggggagag tttacataag gaagagaaga aattgagtgg catattgtaa atatcagatc 5520

tataattgta aatataaaac ctgcctcagt tagaatgaat ggaaagcaga tctacaattt 5580

gctaatatag gaatatcagg ttgactatat agccatactt gaaaatgctt ctgagtggtg 5640

tcaactttac ttgaatgaat ttttcatctt gattgacgca cagtgatgta cagttcactt 5700

ctgaagctag tggttaactt gtgtaggaaa cttttgcagt ttgacactaa gataacttct 5760

gtgtgcattt ttctatgctt ttttaaaaac tagtttcatt tcattttcat gagatgtttg 5820

gtttataaga tctgaggatg gttataaata ctgtaagtat tgtaatgtta tgaatgcagg 5880

ttatttgaaa gctgtttatt attatatcat tcctgataat gctatgtgag tgtttttaat 5940

aaaatttata tttatttaat gcactctaa 5969

<210> 1363

<211> 4594

<212> DNA

<213> Homo sapiens

<400> 1363

gtagagaagc agaaggaagc aagatggctg ccctttagga tttgttagaa aggagacccg 60

actgcaactg ctggattgct gcaaggctga gggacgagaa cgaggctggc aaacattcag 120

cagcacaccc tctcaagatt gtttacttgc ctttgctcct gttgagttac aacgcttgga 180

agcaggagat gggctcagca gcagccaata ggacatgatc caggaagagc agtaagggac 240

tgagctgctg aattcaacta gagggcagcc ttgtggatgg ccccgaagca agcctgatgg 300

aacaggatag aaccaaccat gttgagggca acagactaag tccattcctg ataccatcac 360

ctcccatttg ccagacagaa ccctggcta caaagctcca gaatggaagc ccactgcctg 420

agagagctca tccagaagta aatggagaca ccaagtggca ctctttcaaa agttattatg 480

gaataccctg tatgaaggga agccagaata gtcgtgtgag tcctgacttt acacaagaaa 540

gtagagggta ttccaagtgt ttgcaaaatg gaggaataaa acgcacagtt agtgaacctt 600

ctctctctgg gctccttcag atcaagaaat tgaaacaaga ccaaaaggct aatggagaaa 660

gacgtaactt cggggtaagc caagaaagaa atccaggtga aagcagtcaa ccaaatgtct 720

ccgatttgag tgataagaaa gaatctgtga gttctgtagc ccaagaaaat gcagttaaag 780

atttcaccag tttttcaaca cataactgca gtgggcctga aaatccagag cttcagattc 840

tgaatgagca ggaggggaaa agtgctaatt accatgacaa gaacattgta ttacttaaaa 900

acaaggcagt gctaatgcct aatggtgcta cagtttctgc ctcttccgtg gaacacacac 960

atggtgaact cctggaaaaa acactgtctc aatattatcc agattgtgtt tccattgcgg 1020

tgcagaaaac cacatctcac ataaatgcca ttaacagtca ggctactaat gagttgtcct 1080

gtgagatcac tcacccatcg catacctcag ggcagatcaa ttccgcacag acctctaact 1140

ctgagctgcc tccaaagcca gctgcagtgg tgagtgaggc ctgtgatgct gatgatgctg 1200

ataatgccag taaactagct gcaatgctaa atacctgttc ctttcagaaa ccagaacaac 1260

1320

gaaccacaaa gctagcgtct ggtgaagaat tctgttcagg ttccagcagc aatttgcaag 1380

ctcctggtgg cagctctgaa cggtatttaa aacaaaatga aatgaatggt gcttacttca 1440

agcaaagctc agtgttcact aaggattcct tttctgccac taccacacca ccaccaccat 1500

1560

1620

acacaacact tttaagggaa gtgaaaatag agggtaaacc tgaggcacca ccttcccaga 1680

gtcctaatcc atctacacat gtatgcagcc cttctccgat gctttctgaa aggcctcaga 1740

ataattgtgt gaacaggaat gacatacaga ctgcagggac aatgactgtt ccattgtgtt 1800

ctgagaaaac aagaccaatg tcagaacacc tcaagcataa cccaccaatt tttggtagca 1860

gtggagagct acaggacaac tgccagcagt tgatgagaaa caaagagcaa gagattctga 1920

agggtcgaga caaggagcaa acacgagatc ttgtgccccc aacacagcac tatctgaaac 1980

caggatggat tgaattgaag gcccctcgtt ttcaccaagc ggaatcccat ctaaaacgta 2040

atgaggcatc actgccatca attcttcagt atcaacccaa tctctccaat caaatgacct 2100

ccaaacaata cactggaaat tccaacatgc ctggggggct cccaaggcaa gcttacaccc 2160

agaaaacaac acagctggag cacaagtcac aaatgtacca agttgaaatg aatcaagggc 2220

agtcccaagg tacagtggac caacatctcc agttccaaaa accctcacac caggtgcact 2280

tctccaaaac agaccattta ccaaaagctc atgtgcagtc actgtgtggc actagatttc 2340

attttcaaca aagagcagat tcccaaactg aaaaacttat gtccccagtg ttgaaacagc 2400

acttgaatca acaggcttca gagactgagc cattttcaaa ctcacacctt ttgcaacata 2460

agcctcataa acaggcagca caaacacaac catcccagag ttcacatctc cctcaaaacc 2520

agcaacagca gcaaaaatta caaataaaga ataaagagga aatactccag acttttcctc 2580

acccccaaag caacaatgat cagcaaaag aaggatcatt ctttggccag actaaagtgg 2640

aagaatgttt tcatggtgaa aatcagtatt caaaatcaag cgagttcgag actcataatg 2700

tccaaatggg actggaggaa gtacagaata taaatcgtag aaattcccct tatagtcaga 2760

2820

cagagaataa agaacagact acacatcctg aactttttgc aggaaacaag acccaaaact 2880

tgcatcacat gcaatatttt ccaaataatg tgatcccaaa gcaagatctt cttcacaggt 2940

gctttcaaga acaggagcag aagtcacaac aagcttcagt tctacaggga tataaaaata 3000

3060

ataaccatgc aaatgttttt cctgtgcctg accagggagg aagtcacact cagacccctc 3120

cccagaagga cactcaaaag catgctgctc taaggtggca tctcttacag aagcaagaac 3180

agcagcaaac acagcaaccc caaactgagt cttgccatag tcagatgcac aggccaatta 3240

aggtggaacc tggatgcaag ccacatgcct gtatgcacac agcaccacca gaaaacaaaa 3300

3360

agagcatcat tgagaccatg gagcagcatc tgaagcagtt tcacgccaag tcgttatttg 3420

accataaggc tcttactctc aaatcacaga agcaagtaaa agttgaaatg tcagggccag 3480

tcacagtttt gactagacaa accactgctg cagaacttga tagccacacc ccagctttag 3540

agcagcaaac aacttcttca gaaaagacac caaccaaaag aacagctgct tctgttctca 3600

ataattttat agagtcacct tccaaattac tagatactcc tataaaaaat ttattggata 3660

cacctgtcaa gactcaatat gatttcccat cttgcagatg tgtagagcaa attattgaaa 3720

aagatgaagg tcctttttat acccatctag gagcaggtcc taatgtggca gctattagag 3780

aaatcatgga agaaaggtat acaagtactt gcctttactc ctgcatgtag aagactctta 3840

3900

cttctagggt gccttttcat taagaggtag gcagtattat tattaaagta cttaggatac 3960

attggggcag ctaggacata ttcagtatca ttcttgctcc atttccaaat tattcatttc 4020

taaattagca tgtagaagtt cactaaataa tcatctagtg gcctggcaga aatagtgaat 4080

ttccctaagt gccttttttt tgttgttttt ttgttttgtt ttttaaacaa gcagtaggtg 4140

gtgctttggt cataagggaa gatatagtct atttctagga ctattccata ttttccatgt 4200

ggctggatac taactatttg ccagcctcct tttctaaatt gtgagacatt cttggaggaa 4260

cagttctaac taaaatctat tatgactccc caagttttaa aatagctaaa tttagtaagg 4320

gaaaaaatag tttatgtttt agaagactga acttagcaaa ctaacctgaa ttttgtgctt 4380

tgtgaaattt tatatcgaaa tgagctttcc cattttcacc cacatgtaat ttacaaaata 4440

gttcattaca attatctgta cattttgata ttgaggaaaa acaaggctta aaaaccatta 4500

tccagtttgc ttggcgtaga cctgtttaaa aaataataaa ccgttcattt ctcaggatgt 4560

ggtcatagaa taaagttatg ctcaaatgtt caaa 4594

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Claims (69)

1. An immune effector cell engineered to express a chimeric antigen receptor (CAR), where the CAR contains an antigen-binding domain that binds to a tumor antigen, a transmembrane domain, and an intracellular signaling domain, where the immune effector cell is a T cell or NK cell and where the immune effector cell contains: (a) a Tet2 inhibitor, wherein said inhibitor is: (i) an siRNA or shRNA specific for Tet2 or a nucleic acid encoding said siRNA or shRNA; or (ii) (1) a gene editing system targeting one or more regions in a gene encoding Tet2 or in their regulatory elements; (2) a nucleic acid encoding one or more components of said gene editing system; or (3) combinations thereof; where the gene editing system is a CRISPR/Cas9 system; (b) disruption of the Tet2 gene. 2. The immune effector cell of claim 1 wherein the tumor antigen is selected from the group consisting of: CD19, TSHR, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA -4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, 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-1a, MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, protein sperm 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, protein, survivin and telomerase, PCTA-1/galectin 8, MelanA/MART1, Ras mutant , hTER T, breakpoints in sarcoma translocation, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75 , GPC3, FCRL5 and IGLL1. 3. An immune effector cell according to claim 2, wherein the tumor antigen is CD19. 4. An immune effector cell according to any one of the preceding claims, wherein the transmembrane domain comprises: an amino acid sequence having at least one, two or three modifications, but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 12; or the sequence of SEQ ID NO: 12. 5. An immune effector cell according to any one of the preceding claims, wherein the antigen-binding domain is linked to the transmembrane domain by a hinge region, wherein said hinge region contains the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6 or an amino acid sequence having 95-99% identity with them. 6. An immune effector cell according to any one of the preceding claims, wherein the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain, wherein the primary signaling domain comprises a functional signaling domain of a protein selected from CD3-zeta, CD3-gamma, CD3-delta, CD3, total FcR-gamma (FCER1G), FcR-beta (Fc-epsilon R1b), CD79a, CD79b, Fc-gamma RIIa, DAP10 or DAP12. 7. Immune effector cell according to claim 6, where the primary signaling domain contains: an amino acid sequence having at least one, two or three, but not more than 20, 10 or 5 amino acid sequence modifications of SEQ ID NO: 18 or SEQ ID NO: 20, or an amino acid sequence having 95-99% identity with the amino acid sequence SEQ ID NO: 18 or SEQ ID NO: 20; or the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20. 8. An immune effector cell according to any one of the preceding claims, wherein the intracellular signaling domain comprises a costimulatory signaling domain and/or a primary signaling domain, wherein the costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27, CD28, 4-1BB ( CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytic function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8-alpha, CD8-beta, IL2R-beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, 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, Ly108), SLAM (SLAM F1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46 and NKG2D 9. Immune effector cell according to any one of paragraphs. 6-8, wherein the co-stimulatory signaling domain of said CAR comprises an amino acid sequence having at least one, two, or three modifications, but no more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16, or an amino acid sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16. 10. Immune effector cell according to any one of paragraphs. 6-8, where the co-stimulatory signaling domain contains the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16. 11. An immune effector cell according to any one of the preceding claims, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences containing the intracellular signaling domain are expressed in one reading frame and as a single polypeptide chain. 12. An immune effector cell according to any one of the preceding claims, further comprising a leader sequence which comprises the amino acid sequence of SEQ ID NO: 2. 13. An immune effector cell according to any one of the preceding claims, wherein the immune effector cell is a T cell. 14. The immune effector cell of claim 13, wherein the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof. 15. An immune effector cell according to any one of the preceding claims, wherein the immune effector cell is a human cell. 16. The immune effector cell of any one of the preceding claims, wherein the gene editing system binds to a target sequence in an early exon or intron of a gene encoding Tet2. 17. The immune effector cell of claim 16, wherein the target sequence is upstream of exon 4 or in exon 1, exon 2, or exon 3. 18. The immune effector cell of any one of the preceding claims, wherein the gene editing system binds to a target sequence in the late exon or intron of a gene encoding Tet2. 19. The immune effector cell of claim 18, wherein the target sequence is located downstream of exon 8 or is located in exon 9, exon 10, or exon 11. 20. The immune effector cell of any one of the preceding claims, wherein the gene editing system is a CRISPR/Cas system comprising an gRNA molecule containing a targeting sequence that hybridizes to the target sequence of the Tet2 gene. 21. The immune effector cell of claim 20, wherein the targeting sequence is the targeting sequence shown in any of SEQ ID NOs: 73-1243. 22. The immune effector cell of claim 20, wherein the targeting sequence is the targeting sequence shown in any of SEQ ID NOs: 1273-1295. 23. The immune effector cell of any one of the preceding claims, wherein the siRNA or shRNA contains a sequence complementary to the Tet2 mRNA sequence or contains a shRNA target sequence as set forth in any of SEQ ID NOs: 1244-1252. 24. A method for enhancing the therapeutic efficacy of an immune effector cell according to any one of the preceding claims, comprising the step of contacting said immune effector cell with a Tet2 inhibitor, wherein the Tet inhibitor is selected from the group consisting of: (i) an siRNA or shRNA specific for Tet2 or a nucleic acid encoding said siRNA or shRNA; or (ii) (1) a gene editing system targeting one or more regions in a gene encoding Tet2 or in its regulatory elements; (2) a nucleic acid encoding one or more components of said gene editing system; or (3) combinations of them. 25. The method of claim 24 wherein said contacting takes place ex vivo . 26. The method of claim 26 wherein said contacting occurs in vivo . 27. The method of claim 26, wherein the contacting occurs in vivo prior to delivery of the CAR-encoding nucleic acid into the cell. 28. The method of claim 26, wherein the contacting occurs in vivo after the cells are administered to an individual in need thereof. 29. The use of immune effector cells according to any one of paragraphs. 1-23 for treating a disease associated with the expression of a tumor antigen in an individual. 30. Use according to claim 29, wherein the method further comprises administering to said individual a Tet2 inhibitor. 31. Use according to claim 29 or 30, wherein the tumor antigen expression associated disease is selected from proliferative disease, premalignant condition, cancer, and non-cancer associated disease associated with tumor antigen expression. 32. The use of a CAR-expressing immune effector cell in combination with a Tet2 inhibitor to treat a disease associated with tumor antigen expression in an individual, wherein the CAR contains an antigen-binding domain that binds to a tumor antigen, a transmembrane domain, and an intracellular signaling domain, wherein said Tet2 inhibitor is selected from the group consisting of (i) an siRNA or shRNA specific for Tet2 or a nucleic acid encoding said siRNA or shRNA; or (ii) (1) a gene editing system targeting one or more regions in a gene encoding Tet2 or in their regulatory elements; (2) a nucleic acid encoding one or more components of said gene editing system; or (3) combinations thereof; thus carrying out the treatment of the specified subject. 33. The use of claim 32, wherein the subject is pre-treated with a Tet2 inhibitor prior to initiation of administration of the CAR-expressing immune effector cell. 34. Use according to claim 32, wherein said individual is treated concurrently with a Tet2 inhibitor and treated with CAR expressing cells. 35. The use of claim 32 wherein the subject is treated with a Tet2 inhibitor following therapy with CAR expressing cells. 36. Application according to any one of paragraphs. 31-35, wherein the subject has a disease associated with the expression of a tumor antigen, or a proliferative disease, a precancerous condition, a cancer, and a non-cancer associated disease associated with the expression of a tumor antigen. 37. The use of claim 36 wherein the cancer is a hematologic cancer selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL ), T-cell acute lymphoblastic leukemia (T-ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, m acroglobulinemia Waldenström or preleukemia. 38. Use according to claim 36, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung carcinoma, small intestine cancer, esophageal cancer, melanoma, bone cancer, cancer pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, endometrial cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, endometrial cancer, fallopian tube carcinoma, endometrial carcinoma, carcinoma cervix, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine malignancy, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, urinary cancer bladder, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), first CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, malignant tumors induced by environmental factors, combinations of these malignant tumors and metastatic foci of these malignant tumors . 39. A method for the production of CAR-expressing cells, comprising: (a) introducing a nucleic acid encoding a CAR into a cell such that said nucleic acid is integrated into the cell's genome in the Tet2 gene, or said integration occurs in an intron or exon of the Tet2 gene such that expression and/or function of Tet2 is reduced or abolished; or (b) providing a cell derived from an individual and contacting the cell ex vivo with a Tet2 inhibitor, wherein the contacting occurs before, simultaneously with, or after said cell is modified to express CAR, where said CAR contains an antigen-binding domain that binds to a tumor antigen, a transmembrane domain, and an intracellular signaling domain. 40. A method for producing a CAR-expressing cell, comprising contacting said CAR-expressing cell ex vivo with a Tet2 inhibitor, where said CAR contains an antigen-binding domain that binds to a tumor antigen, a transmembrane domain, and an intracellular signaling domain, where said Tet2 inhibitor is: (i) an siRNA or shRNA specific for Tet2 or a nucleic acid encoding said siRNA or shRNA; or (ii) (1) a gene editing system targeting one or more regions in a gene encoding Tet2 or in their regulatory elements; (2) a nucleic acid encoding one or more components of said gene editing system; or (3) combinations of them. 41. A vector for the production of CAR containing a sequence encoding a CAR and a sequence encoding a Tet2 inhibitor, where the specified CAR contains an antigen-binding domain that binds to a tumor antigen, a transmembrane domain and an intracellular signaling domain, where the Tet2 inhibitor is: (i) siRNA or shRNA specific for Tet2; or (ii) a gene editing system targeting one or more regions in a gene encoding Tet2 or in its respective regulatory elements. 42. The vector of claim 41, wherein the CAR coding sequence and the Tet2 inhibitor coding sequence are separated by region 2A. 43. Composition for the production of a CAR-expressing cell ex vivo containing a Tet2 inhibitor, where said Tet2 inhibitor is: (i) an siRNA or shRNA specific for Tet2 or a nucleic acid encoding said siRNA or shRNA; (ii) (1) a gene editing system targeting one or more regions in a gene encoding Tet2 or in its respective regulatory elements; (2) a nucleic acid encoding one or more components of said gene editing system; or (3) combinations of them. 44. The population of immune effector cells containing one or more cells according to any one of paragraphs. 1-23, for the treatment of a disease associated with the expression of a tumor antigen, where the cell population contains a higher percentage of Tscm cells or CD45RA+CD62L+CCR7+CD27+CD95+ T cells than the cell population that does not contain one or more cells, in which the expression and/or function of Tet2 is reduced or eliminated.

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