Tumor	Tumor Associated Target Antigens

Acute myelogenous leukemia	Wilms tumor 1 (WT1), preferentially
	expressed antigen of melanoma
	(PRAME), PR1,proteinase 3, elastase,
	cathepsin G
Chronic myelogenous leukemia	WT1, PRAME, PR1,proteinase 3,
	elastase, cathepsin G
Myelodysplastic syndrome	WT1, PRAME, PR1,proteinase 3,
	elastase, cathepsin G
Acute lymphoblastic leukemia	PRAME
Chronic lymphocytic leukemia	Survivin
Non-Hodgkin's lymphoma	Survivin
Multiple myeloma	NY-ESO-1
Malignant melanoma	MAGE, MART, Tyrosinase, PRAME
	GP100
Breast cancer	WT1, herceptin, epithelial tumor
	antigen (ETA)
Lung cancer	WT1
Ovarian cancer	CA-125
Prostate cancer	PSA
Pancreatic cancer	CA19-9, RCAS1
Colon cancer	CEA
Renal cell carcinoma (RCC)	Fibroblast growth factor 5
Germ cell tumors	AFP

A nucleic acid encoding the selected Th1-associated miR (such as a plasmid vector or viral vector encoding the miR) can be introduced into the isolated T cells, thus resulting in transformed T cells. Methods for transduction of such vectors into T cells are routine in the art.

In some embodiments, the nucleic acid molecule encoding the Th1-associated miR is a vector encoding the selected miR. A number of different plasmid and viral vectors have been described and are well known in the art. In some embodiments of the methods, the vector is a non-viral vector (for example, a plasmid). In other embodiments, the vector is a viral vector (for example, an adenoviral, adeno-associated viral, retroviral or lentiviral vector). Suitable vectors are well known in the art.

Retrovirus, including lentivirus, vectors can also be used with the methods described herein. Lentiviruses include, but are not limited to, human immunodeficiency virus (such as HIV-1 and HIV-2), feline immunodeficiency virus, equine infectious anemia virus and simian immunodeficiency virus. Other retroviruses include, but are not limited to, human T-lymphotropic virus, simian T-lymphotropic virus, murine leukemia virus, bovine leukemia virus and feline leukemia virus. Methods of generating retrovirus and lentivirus vectors and their uses have been well described in the art (see, for example, U.S. Pat. Nos. 7,211,247; 6,979,568; 7,198,784; 6,783,977; and 4,980,289, each of which is herein incorporated by reference).

In addition, adenovirus vectors can be first, second, third and/or fourth generation adenoviral vectors or gutless adenoviral vectors. Adenovirus vectors can be generated to very high titers of infectious particles; infect a great variety of cells; efficiently transfer genes to cells that are not dividing; and are seldom integrated in the host genome, which avoids the risk of cellular transformation by insertional mutagenesis (Douglas and Curiel,Science and Medicine,March/April 1997, pages 44-53; Zern and Kresinam,Hepatology25(2), 484-491, 1997). Representative adenoviral vectors are described by Stratford-Perricaudet et al. (J. Clin. Invest.90: 626-630, 1992); Graham and Prevec (In Methods in Molecular Biology: Gene Transfer and Expression Protocols 7: 109-128, 1991); and Barr et al. (Gene Therapy,2:151-155, 1995), which are herein incorporated by reference.

Adeno-associated virus (AAV) vectors can also be used. Methods of generating AAV vectors, administration of AAV vectors and their use are well known in the art (see, for example, U.S. Pat. No. 6,951,753; U.S. Patent Application Publication Nos. 2007-036757, 2006-205079, 2005-163756, 2005-002908; and PCT Publication Nos. WO 2005/116224 and WO 2006/119458).

Administration of a therapeutic amount of tumor antigen-specific T cells over-expressing a Th1-associated miR can be used to treat a primary tumor, to prevent recurrence of the tumor in the subject, or to treat a relapse of the tumor.

A therapeutically effective amount of TA-specific T cells engineered to express a Th1-associated miR is administered to the subject. Specific, non-limiting examples of a therapeutically effective amount of isolated T cells include cells administered at a dose of about 1×10⁵cells per kilogram of subject to about 1×10⁹cells per kilogram of subject, such as from about 1×10⁶cells per kilogram to about 1×10⁸cells per kilogram, such as from about 5×10⁶cells per kilogram to about 7.5×10⁷cells per kilogram, such as at about 2.5×10⁷cells per kilogram, or at about 5×10⁷cells per kilogram.

Isolated TA-specific T cells engineered to express a Th1-associated miR can be administered in single or multiple doses as determined by a clinician. For example, the cells can be administered at intervals of approximately one week, two weeks, four weeks, one month or two months depending on the response desired and the response obtained. In some examples, once the desired response is obtained, no further TA-specific T cells are administered. However, if the recipient displays one or more symptoms associated with the presence or growth of a tumor, a therapeutically effective amount of TA-specific T cells can be administered at that time. The administration can be local or systemic.

The purified antigen-specific T cells disclosed herein can be administered with a pharmaceutically acceptable carrier, such as saline. Other therapeutic agents can be administered before, during, or after administration of the TA-specific T cells engineered to express a Th1-associated miR, depending on the desired effect (as discussed in greater detail below).

VI. Combination Treatment Methods

Th1-associated miR-transfected T cell immunotherapy can be used alone or can be accompanied by administration of other anti-cancer agents or therapeutic treatments (such as surgical resection of a tumor or radiation therapy). Any suitable anti-cancer agent can be administered to a patient as part of a treatment regimen that includes T cell immunotherapy. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy and antibodies that specifically target cancer cells.

Examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

Examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

Examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase).

Examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).

Examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone).

Examples of many of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.

Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

Another treatment for cancer is surgical treatment, for example surgical resection of the tumor or a portion of it. Another example of a treatment is radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.

When used in combination with T cell immunotherapy, the additional treatment methods described above can be administered or performed prior to, at the same time, or following T cell immunotherapy as appropriate for the particular patient, the cancer to be treated and the specific combination of therapies.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

ExamplesExample 1Materials and Methods

This example describes the experimental procedures used for the studies described in Example 2.

Reagents

RPMI 1640, FBS, L-glutamine, sodium pyruvate, 2-mercaptoethanol, nonessential amino acids, and penicillin/streptomycin were obtained from Invitrogen Life Technologies. Recombinant murine (rm) IL-12 was purchased from Cell Sciences Technologies. RmIL-4, recombinant human (rh) IL-4 and rhIL-2 were purchased from PeproTech. Purified mAbs against IL-12 (C15.6), IFN-γ (R4-6A2), IL-4 (11B11), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7) and CD49d (R1-2) were all purchased from BD Pharmingen. Purified mAbs against CD3 (UCHT1), CD28 (CD28.2) and IL-4 (MP4-25D2) were purchased from Biolegend. RT-PCR reagents and primers were purchased from Applied Biosystems and analyzed on a BioRad IQ5. WST-1 reagent was purchased from Roche. For isolation of T cells, immuno-magnetic isolation kits from Miltenyi Biotec were used. All reagents and vectors for lentiviral production were purchased from System Biosciences with the exception of LIPOFECTAMINE™ 2000, which was from Invitrogen.

Mice

C57BL/6 mice and C57BL/6 background STAT6 deficient mice (B6.129S2[C]-Stat6^tm1Gru/J), both 5-9 weeks of age, were purchased from The Jackson Laboratory. C57BL/6-background miR-17-92 transgenic (TG) mice (C57BL/6-Gt[ROSA]26Sor^{tm3(CAG-MIRN17-92,-EGFP)Rsky}/J; The Jackson Laboratory) were maintained as breeding colonies and bred to C57BL/6-background mice transgenic for Cre recombinase gene under the control of the Lck promoter (B6.Cg-Tg[Lck-cre]548J×m/J, the Jackson Laboratory) to obtain mice in which T cells expressed miR-17-92 at high levels (miR-17-92 TG/TG). For mouse tumor experiments, C57BL/6 mice and C57BL/6 background STAT6^−/− mice received subcutaneous injection of 1×10⁶B16 tumor cells resuspended in PBS into the right flank. Onday 15 following tumor inoculation, mice were sacrificed and splenic T cells were isolated.

T Cells from Healthy Donors and Patients with GBM

To determine the impact of IL-4, healthy donor-derived CD4⁺ T cells were isolated with immunomagnetic separation and stimulated with 100 IU/ml rhIL-2, anti-CD3 and anti-CD28 mAbs (1 μg/ml for each) in the presence or absence of rhIL-4 (10 ng/ml). RT-PCR analyses were performed with both healthy donor- and patient-derived T cells to determine the expression of miR-17-92 as described in the relevant section.

Th1 and Th2 Cell Culture

Th1 and Th2 cells were differentiated from immunomagnetically-separated CD4⁺ splenic T cells. Magnetic activated cell separation (MACS) was carried out using positive selection. Briefly, spleens were minced in complete media, resuspended in red blood cell lysis buffer and stained with immunomagnetically labeled anti-CD4 antibody. Cells were then washed and placed through the magnetic column in 500 μl of MACS buffer. The column was then washed 3 times with buffer and then removed from the magnet and labeled cells were extracted in 3 ml of MACS buffer.

For differentiation of T cells, purified CD4⁺ cells were stimulated in 48-well plates with anti-CD3 mAb (5 μg/ml) in the presence of irradiated C57BL/6 spleen cells (3000 Rad) as feeder cells. RmIL-12 (4 ng/ml), rmIFN-γ (4 ng/ml), anti-IL-4 mAb (10 μg/ml) and rhIL-2 (100 IU/ml) were added for Th1 development. Th2 cells were generated from the same CD4⁺ cell precursors stimulated with anti-CD3 mAb and feeder cells in the presence of rmIL-4 (50 ng/ml), two anti-IFN-γ mAbs (10 μg/ml), anti-IL-12 mAb (10 μg/ml) and rhIL-2 (100 IU/mL). After 10 days, cells were stained for IL-4 and IFN-γ to confirm differentiation. Neutral cell culture included anti-CD3, feeder cells and rhIL-2. For studies involving IL-4 blockade, 12.5 ng/ml anti-human IL-4 mAb (Biolegend) was used in human experiments and 2.5 μg/ml anti-mouse IL-4 mAb (11B11) in murine studies. IFN-γ and IL-4 in the culture supernatants were measured using specific ELISA kits (R&D Systems). For FACs analysis, cells were incubated with mAb at 4° C. for 30 minutes, washed twice in staining buffer, and fixed in 500 μl of 2% paraformaldehyde in PBS. Cells were stored in the dark at 4° C. until analysis. Flow cytometry was carried out on the Coulter XL four-color flow cytometer.

miR Microarray

Total RNA was isolated from Th1 and Th2 cells using the TRIZOL™ reagent and quality was confirmed with an A260/A280 ratio greater than 1.85. Two μg of total RNA was labeled with either Hy5 (red; Th1) or Hy3 (green; Th2) fluorescent dyes using miRCURY™ LNA microRNA labeling kit (Exiqon, Woburn, Mass.) according to the manufacturer's protocol. Labeled miR samples in duplicate were co-hybridized on miR array slides, a custom spotted miR array V4P4 containing duplicated 713 human, mammalian and viral mature antisense microRNA species (miRBase, version 9.1) plus 2 internal controls with 7 serial dilutions printed in house (Immunogenetics Laboratory, Department of Transfusion Medicine, Clinical Center, National Institutes of Health) (Ren et al.,Journal of Translational Medicine2009, 7:20). After washing, raw intensity data were obtained by scanning the chips with GENEPIX™ scanner Pro 4.0 and were normalized by median over entire array. Differentially expressed miRs were defined by mean (n=2) fold change (Th1/Th2 signal intensity)>2.

Quantitative RT-PCR

Total RNA was extracted using the Qiagen RNEASY™ kit and quality was confirmed with a A260/A280 ration greater than 1.85. RNA was subjected to RT-PCR analysis using the TAQMAN™ microRNA Reverse Transcription Kit, microRNA Assays (Applied Biosystems), and the Real-Time thermocycler iQ5 (Bio-Rad). The small nucleolar SNO202 was used as the housekeeping small RNA reference gene for all murine samples and RNU43 for human samples. All reactions were done in triplicate and relative expression of RNAs was calculated using the ΔΔC_Tmethod (Livak and Schmittgen,Methods2001, 25:402-408).

WST-1 Proliferation Assay

For WST-1 proliferation assays, 1×10⁴cells were cultured in a 96-well plate for 24-48 hours in 100 μl of complete media. Then, 10 μl of WST-1 reagent was added to each well. Cells were incubated at 37° C., 5% CO₂for 4 hours, and placed on a shaker for 1 minute. The plates were then read on a micro plate reader with a wavelength of 420 nm and a reference at 620 nm.

Assays Using Jurkat Lymphoma Cells Transduced with miR-17-92

Jurkat human T cell leukemia cells (American Type Culture Collection) were transduced by either one of the following pseudotyped lentiviral vectors: 1) control vector encoding GFP; 2) the 17-92-1 expression vector encoding miR-17, miR-18 and miR-19; or 3) the 17-92-2 expression vector encoding miR-20, miR-19b-1, and miR-92a-1. All vectors were purchased from SBI. Lentiviral particles were produced by co-transfecting confluent 293TN cells (SBI) with pPACK-H1 Lentivirus Packaging Kit (SBI) and the miR containing expression vectors (SBI) noted above using LIPOFECTAMINE™ 2000 reagent (Invitrogen). Supernatant was collected after 48 hour incubation at 37° C. with 5% CO₂and placed at 4° C. with PEG-it Virus Concentration Solution (SBI) for 24 hrs. Supernatants/PEG solutions were then centrifuged and the pellet was resuspended in a reduced volume of media as viral stock. Jurkat cells were further resuspended in the viral stock together with polybrene (8 μg/ml) for 24 hours. Fresh media was then added to the cells and transduction efficiency was evaluated by GFP expressing cells. For IL-2 production, transduced

Jurkat cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 10 ng/ml) and ionomycin (500 nM) overnight and supernatant was assayed for IL-2 by a human IL-2 ELISA kit. For AICD, cells were treated with 10 μg/ml purified anti-CD3 mAb (UCHT1) from Biolegend for 24 hours and then cell viability was measured using WST-1 reagent.

Statistical Methods

All statistical analyses were carried out on Graphpad Prism software. The statistical significance of differences between groups was determined using student t-test. Differences were considered significant when p<0.05. A post test for linear trend was used to determine linear trend and p<0.05 was considered to be significant.

Example 2miR-17-92 Expression in Differentiated T Cells

This example describes results demonstrating that (i) expression of the miR17-92 cluster is greater in Th1 cells compared to Th2 cells; (ii) the IL-4R/STAT6 signaling pathway regulates expression of miR-17-92; and (iii) over-expression of miR-17-92 in T cells promotes a Th1 phenotype and render cells resistant to AICD.

miR17-92 and its Paralogs are Overexpressed in Th1 Cells Compared with Th2 Cells

To identify differentially expressed miRs between Th1 and Th2 cells, miR microarray analysis was performed. From mouse splenic CD4⁺ T cells, Th1 and Th2 cells were generated as described in Example 1. These T cells exhibited expected cytokine profiles with Th1 cells dominantly producing IFN-γ, but not IL-4, while Th2 cells produced mostly IL-4 (FIG. 1A). Total RNA was extracted from these T cells, and analyzed for differential miR expression by miR microarray for 714 miRs (FIG. 1B). Hierarchical clustering of differentially expressed miRs revealed distinct miR expression profiles between the Th1 and Th2 cells. Eleven of the miRs from the miR-17-92 cluster and its paralogs were expressed at higher levels in Th1 cells than in Th2 cells. Next, the miRs preferentially expressed in Th1 cells were ranked according to the fold difference of expression when compared with Th2 cells (FIG. 1C). Interestingly, members of miRs in the miR-17-92 clusters were identified as the most differentially expressed of all miRs in Th1 cells compared to Th2 cells. Since miR-17-92 clusters appear to be transcribed as single polycistronic transcripts (FIG. 1D), it was expected that all the miRs from the miR-17-92 cluster would be consistently expressed at higher levels in Th1 cells than in Th2 cells, which was confirmed by RT-PCR analysis (FIG. 2A).

The miR-17-92 cluster has 2 paralog clusters: miR-106a-363 and miR-106b-25. These paralog clusters target similar mRNAs as the miR-17-92 cluster due to high sequence homology (Mendell,Cell2008, 133:217-222). To establish if these paralog miR clusters are also overexpressed in the Th1 vs. Th2 cells, RT-PCR was performed for miRs in each of these clusters. Representative for these paralog clusters are miR-106a and miR106b (FIG. 2B). These data demonstrate that the paralog clusters of miRs were also up-regulated in Th1 cells over Th2.

Neutralization of Endogenous IL-4 Up-Regulates miR-17-92 Cluster miRs in T-Cells

In order to identify factors that contribute to the differential expression of miR-17-92 cluster miRs between Th1 and Th2 cells, it was investigated whether a prototypical type-2 inducing cytokine, IL-4, would affect miR-17-92 expression in CD4⁺ T cells. Neutralization of endogenous IL-4 by specific mAb against IL-4 up-regulated miR-17-92 cluster miRs in CD4⁺ T cells stimulated with IL-2 without addition of Th1-inducing factors IL-12 or IFN-γ, by approximately 50% (FIG. 3A). The anti-IL-4 mAb also up-regulated miR-17-92 in Th2 culture conditions as well. To determine whether there is an IL-4 dose-dependent suppression of miR-17-92 cluster, CD4⁺ T cells were treated with increasing doses of IL-4 at 0, 10, 50 or 100 ng/ml and miR-17-5p expression was measured by RT-PCR (FIG. 3B). miR-17-92 suppression was a dose-dependent phenomenon.

Up-Regulated miR-17-92 Expression in STAT6-Deficient T Cells

To further elucidate the effect of IL-4 signaling on miR-17-92 cluster expression, CD4⁺ T cells were cultured under Th1 or Th2 skewing conditions from mice deficient of the critical IL-4 signaling molecule, STAT6 (Sasaki et al.,The Journal of Immunology2008, 181:104-108; Eguchi et al.,GeneTher2005, 12:733-741). Both Th1 and Th2 cultured cells induced from STAT6-deficient mice showed higher levels of miR-17-5p expression compared with corresponding WT Th cells, suggesting a novel critical role of IL-4R/STAT6-signaling in the down-regulation of miR-17 expression (FIG. 3C).

Suppression of miR-17-92 may Occur in Cancer-Bearing Hosts

These data led to the hypothesize that suppression of miR-17-92 would occur in cancer-bearing hosts where tumor-derived factors likely promote Th2-skewed immune responses and secretion of IL-4 (Roussel et al.,Clin Exp Immunol1996, 105:344-352). Indeed, CD4⁺ and CD8⁺ SPCs derived from wild type C57BL/6 mice bearing B16 subcutaneous tumors expressed lower levels of miR-17-5p when compared with those derived from non-tumor bearing mice (FIG. 4A). Interestingly, the tumor bearing condition did not suppress miR-17-5p expression by CD4⁺ T cells in STAT6^−/− mice. Furthermore, CD8⁺ T cells in STAT6^−/− mice demonstrated enhanced levels of miR-17-5p expression when these mice bore B16 tumors compared with non-tumor bearing mice. When wild type CD4⁺ T cells were stimulated with anti-CD3 mAb in vitro for 24 hours, the CD4⁺ T cells from tumor-bearing mice produced lower levels of IFN-γ when compared with ones from non-tumor bearing wild type mice (FIG. 4B). These data suggest that tumor-associated immunosuppression may involve the down-regulation of miR-17-92 through a STAT6 dependant pathway.

It was next evaluated whether the observed IL-4-mediated and tumor-induced suppression of miR-17-92 are relevant in human T cells. When healthy donor-derived CD4⁺ T cells were stimulated with rhIL-2, anti-CD3 and anti-CD28 mAbs, consistent with the mouse data, addition of rhIL-4 in the cultures suppressed expression of miR-17-5p (FIG. 4C). Moreover, CD4⁺ T cells obtained from patients with GBM exhibited significantly decreased levels of miR-17-5p when compared with ones from healthy donors (FIG. 4D). Thus, both IL-4 and GBM-bearing conditions suppress miR-17-5p expression in CD4⁺ T cells. Although not statistically significant, CD8⁺ T cells demonstrated a trend towards decreased levels of miR-17-5p expression in GBM patients when compared with healthy donors (FIG. 4D).

T Cells Derived from miR-17-92 Transgenic Mice Display Enhanced Type-1 Phenotype

The data discussed above strongly suggest GBM-associated factors and a type-2 promoting cytokine (IL-4) down-regulate miR-17-92 in T cells. miR-17-92 is expected to play pivotal roles in T cell functions. Experiments were therefore carried out to determine whether ectopic expression of miR-17-92 would promote the type-1 phenotype of T cells. As detailed in Example 1, mice that overexpress miR-17-92 specifically in T cells (miR-17-92 TG/TG) were generated. CD4⁺ splenocytes were isolated from these mice and the expression of miR-17-5p was evaluated (FIG. 5A). CD4⁺ cells from TG/TG mice displayed a greater than 15-fold increase in miR-17-p5 expression as compared with controls. These cells also expressed elevated levels of CD49d, which is a subunit of a type-1 T cell marker VLA-4 (FIG. 5B). Although CD49d (also known as α4-integrin) can form heterodimers with both β1 (CD29) and β7 integrins, α4β7 complexes were not expressed by either Th1 cells or Th2 cells, suggesting that CD49d is a suitable surrogate for VLA-4 expression levels (Sasaki et al.,The Journal of Immunology2008, 181:104-108; Sasaki et al.,Eur J Immunol2008, 38:2865-2873; Zhu et al.,J Transl Med2007, 5:10; Sasaki et al.,Cancer Res2007, 67:6451-6458). miR-17-92-TG/TG CD4⁺ cells also demonstrated enhanced ability to produce IFN-γ upon stimulation (FIG. 5C). Similar data were obtained with CD8⁺ T cells isolated from these TG/TG mice. These findings suggest that miR-17-92 promotes the type-1 phenotype in differentiating T cells.

Ectopic Expression of miR-17-92 Promotes IL-2 Production and Resistance Against Activation-Induced Cell Death (AICD) in Jurkat Cells

miR-17-92 is expected to play pivotal roles in T cell survival as well as functions. To evaluate these aspects, Jurkat cells were transduced with lentiviral vectors encoding GFP and either the miR-17-92-1 expression vector encoding miR-17, miR-18 and miR-19a, or the 17-92-2 expression vector encoding miR-20, miR-19b, and miR-92. The control vector encodes GFP, but not miRs. Transduced Jurkat cells were stimulated with PMA and ionomycin overnight before the supernatants were assayed for IL-2 production by ELISA (FIG. 6A). Transduction of either miR-vector promoted IL-2 production in Jurkat cells.

AICD and chemotherapy-induced suppression of T cells represent major obstacles for efficient T cell-based cancer immunotherapy (Kennedy and Celis,Immunological Reviews2008, 222:129-144; Brenner et al.,Critical Reviews in Oncology/Hematology2008, 66:52-64). It was next examined whether transfection of Jurkat cells with miR-17-92 renders T cells resistant to AICD. AICD was induced by cultivation of Jurkat cells in the presence of 10 μg/ml anti-CD3 mAb, which is hyper-stimulatory and used as a standard method to induce AICD (Jiang et al.,Clin Exp Med2009). As demonstrated in (FIG. 6B), the growth of control Jurkat cells was significantly suppressed by nearly 25% in the AICD inducing condition compared with the same cells with the regular (growth-promoting) dose of anti-CD3 mAb (1 μg/ml). In contrast, the growth of Jurkat cells transduced with either miR-17-92-1 or miR-17-92-2 was not significantly altered by the high dose (10 μg/ml) of anti-CD3 mAb, suggesting that the miR-17-92 transfection confers T cells with substantial resistance against AICD. These findings point to a potential utility for miR-17-92 transfected T cells in cancer immunotherapy.

Summary of Results

Attaining effective tumor immunity is a major goal of modern biologic therapy, limited by the tumor microenvironment and profound regulatory mechanisms limiting T cell and NK cell effectors. It is demonstrated herein that the type-2-skewing tumor microenvironment induces down-regulation of miR-17-92 expression in T cells, thereby hampering anti-tumor T cell responses. It also suggests that development of immunotherapy using miR-17-92-transduced T cells is warranted based on these findings demonstrating that ectopic expression of miR-17-92 in T cells leads to improved type-1 functions, including increased VLA-4 expression and IFN-γ production.

Blockade of endogenous IL-4 by inhibitory mAb or disruption of STAT6 signaling was sufficient to up-regulate miR-17-92 in T cells (FIG. 3). These findings suggest that STAT6 may negatively regulate miR-17-92 expression in T cells. Several transcription factors have been identified that regulate expression of this miR cluster, including the E2 transcription factor (E2F) family members (Woods et al.,J Biol Chem2007, 282:2130-2134; Sylvestre et al.,J Biol Chem2007, 282:2135-2143), c-Myc (O'Donnell et al.,Nature2005, 435:839-843), STAT3 (Brock et al.,Circ Res2009, 104:1184-1191), as well as the sonic hedgehog pathway (Northcott et al.,Cancer Res2009, 69:3249-3255; Uziel et al.,Proc Natl Acad Sci USA2009, 106:2812-2817). With regard to the effects of IL-4/STAT6 signaling on Th1 vs. Th2 functions, the inventors have recently demonstrated that STAT6^−/− Th2 cells exhibit Th1 phenotype with increased surface expression of VLA-4 (Sasaki et al.,Journal of Immunotherapy2009, 32:793-802). These observations have led to the hypothesis that STAT6-regulated miR-17-92 may contribute to the promotion of type-1 T cell functions.

These findings indicate that the tumor-bearing host down-regulates miR-17-92 in T cells (FIGS. 3 and 4). Interestingly, not only are STAT6^−/− T cells resistant to tumor-induced inhibition of miR-17-5p, but CD8⁺ T cells in tumor bearing STAT6^−/− mice exhibited higher levels of miR-17-5p when compared with CD8⁺ T cells obtained from non-tumor bearing STAT6^−/− mice. In addition to IL-4, other tumor-derived factors are likely to be involved in these events.

While tumor bearing mice demonstrated decreased levels of miR-17-92 in both CD4⁺ and CD8⁺ cells, human GBM patients exhibited a statistically significant decrease of miR-17-92 in CD4⁺ cells but not in CD8⁺ cells (FIG. 4). However, there is a trend towards lower miR-17-92 expression in GBM patient-derived CD8⁺ cells compared with those obtained from healthy donors. The type-1 vs. type-2 differentiation appears to be more distinct for CD4⁺ T cells than for CD8⁺ cells (Ehi et al.,Oncol Rep2008, 19:601-607; Tatsumi et al.,Cancer Res2003, 63:4481-4489), and this may also be the case for miR-17-92.

Messages encoding proteins that are targeted by miR-17-92 cluster miRs include: E2F1, E2F2, E2F3 (O'Donnell et al.,Nature2005, 435:839-843; Brock et al.,Circ Res2009, 104:1184-1191), P21 (Inomata et al.,Blood2009, 113:396-402), anti-angiogenic thrombospondin-1 and connective tissue growth factor (Dews et al.,Nat Genet2006, 38:1060-1065), proapoptotic Bim, and phosphatase and tensin homolog (PTEN) (Xiao et al.,Nat Immunol2008, 9:405-414). These proteins are all involved in cell cycle regulation or apoptotic cell death, further supporting the importance of miR-17-92 cluster in T cell biology. In fact, Bim and PTEN are down-regulated in T cells overexpressing miR-17-92 (Xiao et al.,Nat Immunol2008, 9:405-414). Furthermore, TGF-β receptor II (TGFBRII) is one of the established targets of miR-17-92 (Volinia et al.,Proc Natl Acad Sci USA2006, 103:2257-2261).

The findings demonstrating increased IFN-y production from miR-17-92 TG/TG T cells compared with control cells suggest that miR-17-92 promotes the type-1 skewing of T cells (FIGS. 5 and 6C). As miR-17-92 targets hypoxia-inducible factor (HIF)-1α in lung cancer cells (Taguchi et al.,Cancer Res2008, 68:5540-5545), enhanced miR-17-92 expression in activated T cells may promote the type-1 function of T cells at least partially through down-regulation of HIF-1α. Although HIF-1 expression provides an important adaptation mechanism of cells to low oxygen tension (Sitkovsky and Lukashev,Nat Rev Immunol2005, 5:712-721; Semenza,Current Opinion in Genetics&Development1998, 8:588-594), it does not appear to be critical for survival of T cells, unlike its apparent role in macrophages (Cramer et al.,Cell2003, 112:645-657). T cells do not depend on HIF-1α for survival to the same degree as macrophages since activated T cells produce ATP by both glycolysis and oxidative phosphorylation (Brand and Hermfisse,FASEB J1997, 11:388-395). Rather, HIF-1α in T cells appears to play an anti-inflammatory and tissue-protecting role by negatively regulating T cell functions (Sitkovsky and Lukashev,Nat Rev Immunol2005, 5:712-721; Neumann et al.,Proc Natl Acad Sci USA2005, 102:17071-17076; Eltzschig et al.,Blood2004, 104:3986-3992). Indeed, T cell-targeted disruption of HIF-1α leads to increased IFN-γ secretion and/or improved effector functions (Kojima et al.,Proc Natl Acad Sci USA2002, 99:2170-2174; Lukashev et al.,J Immunol2006, 177:4962-4965; Guo et al.,Int Arch Allergy Immunol2009, 149:98-102; Thiel et al.,PLoS One2007, 2:e853). Although available data on gene expression profiles in Th1 and Th2 cells do not suggest differential expression of HIF-1α mRNA between these cell populations (Nagai et al.,Int Immunol2001, 13:367-376), as is often the case in miR-mediated gene expression regulation, miR-17-92 may still regulate HIF-1α protein expression at post-transcriptional levels. These data collectively suggest that miR-17-92 expression in activated T cells promotes the type-1 function of T cells at least partially through down-regulation of HIF-1α.

The human Jurkat T cell line with ectopic expression of miR-17-92 cluster members demonstrate increased IL-2 production and improved viability following treatment with the AICD condition (FIG. 6). The Jurkat cell line was established from the peripheral blood of a T cell leukemia patient in the 1970s. This cell line is often used to recapitulate what would happen in humans T cells as the line retains many T cell properties, such as CD4, a T cell receptor, and ability to produce IL-2 (Abraham and Weiss,Nat Rev Immunol2004, 4:301-308). For these reasons, Jurkat cells were for the experiments disclosed herein.

miRs in the miR-17-92 clusters are amplified in various tumor types including B cell lymphoma and lung cancer, and promote proliferation and confer anti-apoptotic function in tumors, thereby promoting tumor-progression and functioning as oncogenes (He et al.,Nature2005, 435:828-833; Hayashita et al.,Cancer Res2005, 65:9628-9632; Matsubara et al.,Oncogene2007, 26:6099-6105; Lawrie,Expert Opin Biol Ther2007, 7:1363-1374; Rinaldi et al.,Leuk Lymphoma2007, 48:410-412). However, miR-17-92 by itself may not be responsible for oncogenesis as transgenic mice with miR-17-92 overexpressed in lymphocytes develop lymphoproliferative disorder and autoimmunity but not cancer (Xiao et al.,Nat Immunol2008, 9:405-414). miR-17-92 may cooperate with other oncogenes to promote the oncogenic process. Transgenic mice overexpressing both miR-17-92 and c-Myc in lymphocytes develop early onset lymphomagenesis disorders (He et al.,Nature2005, 435:828-833). On the other hand, knockout studies of the miR-17-92 cluster in mice have demonstrated the importance of this cluster in mammalian biology. While knockout of the miR-17-92 cluster results in immediate post-natal death of all progeny, knockout of either or both the miR-106a or miR-106b clusters are viable without an apparent phenotype (Ventura et al.,Cell2008, 132:875-886). However knock out of the miR-17-92 cluster together with miR-106a or 106b cluster results in embryonic lethality (Xiao and Rajewsky,Cell2009, 136:26-36).

During lymphocyte development, miR-17-92 miRs are highly expressed in progenitor cells, with the expression level decreasing 2- to 3-fold following maturation (Xiao et al.,Nat Immunol2008, 9:405-414). In addition, the inventors have evaluated relative expression of miR-17-92 in a variety of Th cells as well as naïve CD4⁺ cells. Naïve CD4⁺ cells express miR-17-92 at the highest level among the cell populations examined. Albeit lower than that in naïve CD4⁺ cells, Th1 cells express miR-17-92 at higher levels than T neutral (anti-CD3, feeder cells and IL-2) and Th17 cells, and Th2 cells consistently exhibit the lowest levels of miR-17-92 among the populations tested.

Example 3Inhibition of Tumor Growth in miR-17-92 TG Mice

This example supports the methods of engineering tumor antigen (TA)-specific CTLs to express the miR-17-92 cluster that provide potent and durable antitumor activity by potentiating TA-specific type-1 CTL functions. This example describes studies in which C57BL/6-background mice that overexpress miR-17-92 in T cells (miR-17-92-TG mice) or control Lck-Cre mice were challenged with two different tumor systems (FIGS. 7A-7B).

First, mice received s.c. challenge with B16 melanoma cells (1×10⁵/mouse) and were sacrificed onday 15 following tumor challenge because the size of most tumors exceeded 2 cm²in control mice. As shown inFIG. 7A, tumors in miR-17-92-TG mice were significantly smaller than those that developed in control mice (p=0.0034 by student-t test, n=9 and 10 for control and miR-17-92-TG mice, respectively).

As a second model, mice received i.c. inoculation of GL261 glioma cells (1×10⁵/mouse) and were followed for symptom-free survival. As shown inFIG. 7B, all control mice died byday 25 after the tumor inoculation (median 23 days), whereas the survival of miR-17-92-TG mice was significantly longer than that of control mice (median 31 days; p=0.0426 by Logrank test), and 2 of 7 mice were still alive 50 days following tumor inoculation.

The results of these in vivo studies demonstrate that overexpression of miR-17-92 in T cells significantly inhibits tumor growth and/or tumor induced death.

Example 4miR-17-92-TG T Cells Infiltrate Gliomas More Intensively Than Control T Cells

The glioma experiment described in Example 3 and depicted inFIG. 7B was repeated by challenging miR-17-92-TG or control Lck-Cre mice (n=4/group) with i.c. inoculation of syngeneic GL261 glioma cells, with the exception that these mice were sacrificed on day 23 to evaluate the immunological microenvironment of the glioma sites by flow-cytometric evaluation of brain-infiltrating leukocytes (BILs). As shown inFIG. 8, BILs were stained for T cell marker CD3 as well as VLA-4 as a critical homing receptor to the brain tumor sites (Sasaki et al.,J Immunol181:104-108, 2008; Sasaki et al.,Eur J Immunol38:2865-2873, 2008; Sasaki et al.,Cancer Res67:6451-6458, 2007; Zhu et al.,J Transl Med5:10, 2007).

Interestingly, tumors in miR-17-92-TG mice were infiltrated more heavily by CD3⁺VLA-4⁺ cells compared with control mice. Enumeration of CD3⁺VLA-4⁺ cells by multiplying the total number of leukocyte-gated BILs and the percentage of this population revealed 1.3×10⁴/mouse in the control mice compared with 6.4×10⁴/mouse in miR-17-92-TG mice. As transgenic expression of miR-17-92 in miR-17-92-TG mice is restricted in their T cells owing to Lck-Cre system, these results suggest that transgenic expression of miR-17-92 in T cells promotes trafficking of T cells to glioma sites, thereby promoting anti-tumor immunity.

Example 5miR-17-92 Expression Confers T Cell Resistance to AICD of Primary Mouse T Cells

AICD represents major obstacles for efficient T cell-based cancer immunotherapy. Example 2 describes results in a T cell line (Jurkat) demonstrating that miR-17-92 overexpression confers resistance to AICD. It was next examined whether transgenic miR-17-92 expression confers primary T cells resistant to AICD. AICD was induced by cultivation of miR-17-92 TG Tc1 or control TG Tc1 in the presence of 10 μg/ml anti-CD3 mAb. The number of control Tc1 cells was significantly reduced by nearly 45% in the AICD inducing condition compared with the same cells with the regular (growth-promoting) dose of anti-CD3 mAb (1 μg/ml). In contrast, the number of T cells transgenic with miR-17-92 was not significantly altered by the high dose (10 μg/ml) of anti-CD3 mAb, suggesting that the miR-17-92 transfection confers T cells with substantial resistance against AICD.

Example 6T Cells Over-Expressing miR17-92 for Cancer Immunotherapy

This example illustrates methods to further demonstrate miR-17-19b over-expression can promote proliferation of adoptively-transferred tumor-antigen specific T cells in situ and exert potent anti-tumor response in vivo. This method also provides an example of how to perform cancer immunotherapy.

The efficiency of T cell-adoptive transfer therapy largely depends upon the persistence and proliferation of transferred T cells in vivo, and this has been the rate-limiting step for development of successful immunotherapy. Tumor-induced type-2 deviation and other immuno-suppression mechanisms appear to induce inactivation and death of anti-tumor T cells. In order to sustain effective proliferation of adoptively-transferred tumor-specific Tc1 cells, genetically engineered miR-17-19b over-expressing Tc1 and Tc2 are generated and adoptively transferred into intracranial GL261-bearing mice.

Using a Hamilton syringe, 1×10⁵GL261 glioma cells are stereotactically injected through an entry site at the bregma, 3 mm to the right of sagittal suture and 4 mm below the surface of the skull of anesthetized mice using a stereotactic frame (Kopf). The miR-17 or control backbone vector are infected into Tc1 and Tc2 cells. Onday 10, five mice per group receive an i.v. injection with 2×10⁷miR-17 or control vector-transfected Tc1 cells that have been cultured for 9 days with 100 U/ml of hIL-2. Mice are closely monitored for any neurological signs, or any signs of weakness or malaise, which are considered to be endpoints. When endpoints are observed, the mice are sacrificed. The survival of mice is analyzed using the Kaplan Mayer method. As a means of immunological monitoring, blood is drawn from mice treated with engineered Tc1 cells, then serum IFN-γ and IL-4 levels are assessed by ELISA.

In another set of experiments, mice are given BrdU in the drinking water for 5 days after i.v. transfer of engineered Tc1 cells. Atday 6 after adoptive-transfer, mice are sacrificed and BILs are isolated as described previously (Nishimura et al.,Cancer Res.66:4478-4487, 2006). BILs are stained extracellularly with PE-gp100_25-33⁻H-2D^b-tetramer permeabilized using the CYTOFIX/CYTOPERM™ kit (BD Biosciences), and stained with FITC-anti-BrdU mAb. It is anticipated that miR-17-19b-transfected Tc1 cells will demonstrate higher proliferation levels than control-transfected Tc1 cells after encountering the tumor-antigens in situ.

To eliminate concerns for development of lymphoma by the stable gene-transfer of miR-17, normal tumor-free C57BL/6 mice (5 mice/group) receive i.v. transfer of 2×10⁷miR-17- or control-transfected Tc1 cells per mouse. Mice have blood (50 μl) drawn from the tail vein, and are then monitored for the presence of leukemia by blood smear analysis at 5, 10, 20 and 40 weeks after the adoptive-transfer. Mice are also observed for any changes in physical appearance and demeanor including weakness, hunch back, weight-loss and general malaise. Mice demonstrating any of the signs above, or those surviving longer than 120 days, are sacrificed. Lymphoid (cervical, axillary, and inguinal, mesenteric lymph-nodes, spleen) and non-lymphoid (brain, kidney, liver, bone-marrow, intestine) organs are harvested. To this end, hematoxylin and eosin (H&E) staining is employed to examine the presence of malignancies.

The treatment methods described herein can easily be adapted for other species or subjects, such as humans.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.