WO2013001372A2

WO2013001372A2 - Methods and compositions for inhibition of activation of regulatory t cells

Info

Publication number: WO2013001372A2
Application number: PCT/IB2012/001733
Authority: WO
Inventors: Kjetil Tasken; Maria Elisabeth KALLAND; Knut Martin TORGERSEN; Nikolaus G. OBERPRIELER; Torkel Vang
Original assignee: University Of Oslo
Priority date: 2011-06-30
Filing date: 2012-06-29
Publication date: 2013-01-03
Also published as: WO2013001372A3

Abstract

The present invention relates to human regulatory T cells (Tregs). In particular, the present invention relates to compositions and methods for inhibiting activation of Tregs and to methods of inhibiting excessive immunosuppressive activity in a cell, comprising contacting a cell with an inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof, wherein the contacting results in inhibition of activation of resting human regulatory T cells (rTregs) to active Tregs (aTregs). In other embodiments, the invention provides for the use of an inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof to treat a chronic infectious disease in a subject

Description

METHODS AND COMPOSITIONS FOR INHIBITION OF ACTIVATION OF

REGULATORY T CELLS FIELD OF THE INVENTION

The present invention relates to human regulatory T cells (Tregs). In particular, the present invention relates to compositions and methods for inhabiting activation of Tregs.

BACKGROUND OF THE INVENTION

Regulatory T cells (Tregs) constitute a sub-population of T lymphocytes with suppressive properties. Naturally occurring Tregs protect from autoimmune responses to self antigens and loss of Treg function is associated with autoimmune diseases. Peripherally induced or adaptive Tregs are normally induced at sites of antigenic activation and modulate immune responses with a time lag to avoid tissue damage and inflammation from exaggerated immune responses after an infection has cleared. In chronic infections and cancer, Treg-mediated immunosuppression is dysfunctional and inhibits clearance of infections or targeting of cancer cells by the immune system. Tregs are typically identified by the expression of the transcription factor FoxP3. In humans, CD25 and FoxP3 status does, however, not define Tregs equally well as in mice.

Furthermore, as FoxP3 is a nuclear factor, it can only be detected if cells are fixed and permeabilized and not in live cells. Staining to identify CD25^highCD 127^low or

CD25 FoxP3 CD127^low has been implemented to avoid contamination with activated effector T cells (Teffs) in humans, but does not fully discriminate between activated Teffs and Tregs.

Levels of Tregs have been shown to have clear correlation to outcome in an increasing number of cancers. Furthermore, it is clear that Tregs need to be activated in order to be fully suppressive and Treg activation status has strong interest in the field. So far, activated Tregs can be identified by their memory status (as CD45RA^" or CD45RO ), but markers better delineating various Treg compartments are clearly needed. Activated Tregs have been shown to be down- regulated in autoimmune diseases and upregulated in chronic inflammation indicating that the predictive value of activated Treg in clinical use could be even stronger than for total Tregs.

Basically, no or poor solutions exist for identification, diagnosis / prognostication, and/or blocking/stimulation or cell-based therapies aimed at Tregs exist. Thus, what is needed in the art are methods of activating and inhibiting Tregs.

SUMMARY OF THE INVENTION The present invention relates to human regulatory T cells (Tregs). In particular, the present invention relates to compositions and methods for inhabiting activation of Tregs.

For example, in some embodiments, the present invention provides a method of preventing excessive immunosuppressive activity in a cell, comprising: contacting said cell with an inhibitor of one of more genes selected from the group consisting of Mek, Erk and Aktl/2, wherein said contacting results in inhibition of activation of resting human regulatory T cells (rTregs) to active Tregs (aTregs). The present invention is not limited to a particular Mek, Erk or Aktl/2 inhibitor. Examples include, but are not limited to, an siR A, an antisense

oligonucleotide, an antibody and a small molecule drug. Exemplary inhibitors are known in the art or can be identified using the drug screening methods described herein. For example, known Mek inhibitors include, but are not limited to, AS703026, AZD6244 (Selumetinib),

AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI- 1040 (PD184352), PD0325901, PD318088, PD98059, U0126-EtOH, , GSK1120212, FR180204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, and GDC-0973 (XL518).. Known Aktl/2 inhibitors include, but are not limited to, A6730, B2311, 124018, GSK2141795, MK2206, GSK2110183, GSK690693, Perifosine (KRX-0401), GDC-0068, RX-0201, and VQD- 002. Known Erk inhibitors include, but are not limited to, PD98059, U0126, FR180204 , 3-(2- Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione, and pyrazolylpyrrole ERK

In some embodiments, the cell is in vitro, ex vivo or in vivo. In some embodiments, the cell is in an animal (e.g., a human or a non-human mammal). In some embodiments, the animal exhibits symptoms or is at risk of exhibiting symptoms of a disease characterized by excessive immunosuppressive activity (e.g., an autoimmune disease, a chronic infectious disease and cancer).

In some embodiments, the present invention provides for the use of an inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof to inhibit excessive immunosuppressive activity in a cell. In some embodiments, the MEK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of AS703026, AZD6244 (Selumetinib), AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD184352), PD0325901, PD318088, PD98059, and U0126-EtOH, GSK1120212, FR180204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, GDC-0973 (XL518). In some embodiments, the AKT1/2 inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of A6730, B2311, 124018, GSK2141795, MK2206, GSK2110183, GSK690693, Perifosine (KRX-0401), GDC-0068, RX-0201, VQD-002. In some embodiments, the ERK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of PD98059, U0126, FR180204, 3-(2-Aminoethyl)-5-((4- ethoxyphenyl)methylene)-2,4-thiazolidinedione, and pyrazolylpyrrole ERK Inhibitor. In some embodiments, the inhibition of excessive immunosuppressive activity is desired in a subject exhibiting symptoms of a disease characterized by excessive immunosuppressive activity. In some embodiments, the inhibition of excessive immunosuppressive activity is utilized for treatment of a disease selected from the group consisting of a chronic inflammatory disease, a chronic infectious disease and cancer. In some embodiments, the inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof is

coadministered with at least one additional therapeutic agent. In some embodiments, the least one additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an antiviral agent, an antibacterial agent, and a stem cell agent. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of a biologic therapeutic agent, a small molecule therapeutic agent, and an RNA-based therapeutic agent.

In some embodiments, the present invention provides for use of an inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof to treat a chronic infectious disease in a subject. In some embodiments, the chronic infectious disease is selected from the group consisting of Hepatitis B, Hepatitis C, tuberculosis, and HIV infection. In some embodiments, the MEK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of AS703026, AZD6244

(Selumetinib), AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD184352), PD0325901, PD318088, PD98059, and U0126-EtOH, GSK1120212, FR180204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, GDC-0973 (XL518). In some embodiments, the AKT1/2 inhibitor is selected from the group consisting of an siR A, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of A6730, B2311, 124018, GSK2141795, MK2206, GSK2110183, GSK690693, Perifosine (KRX-0401), GDC- 0068, RX-0201, VQD-002. In some embodiments, the ER inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of PD98059, U0126, FR180204, 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione, and pyrazolylpyrrole ERK Inhibitor. In some embodiments, the inhibition of excessive

immunosuppressive activity is desired in a subject exhibiting symptoms of a disease

characterized by excessive immunosuppressive activity. In some embodiments, the inhibition of excessive immunosuppressive activity is utilized for treatment of a disease selected from the group consisting of a chronic inflammatory disease, a chronic infectious disease and cancer. In some embodiments, the inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof is coadministered with at least one additional therapeutic agent. In some embodiments, the least one additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an antiviral agent, an antibacterial agent, and a stem cell agent. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of a biologic therapeutic agent, a small molecule therapeutic agent, and an RNA-based therapeutic agent.

In some embodiments, the present invention provides methods of treating a disease a patient suffering from a condition associated with inappropriate excessive immunosuppressive activity in a cell, comprising: coadministering to said patient a first therapeutic agent and a second therapeutic agent that is an inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof, wherein said administration of said second therapeutic agent results in inhibition of activation of resting human regulatory T cells (rTregs) to active Tregs (aTregs). In some embodiments, the first therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an antiviral agent, an antibacterial agent, and a stem cell agent. In some embodiments, the first therapeutic agent is selected from the group consisting of a biologic therapeutic agent, a small molecule therapeutic agent, and an RNA-based therapeutic agent. In some embodiments, the MEK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of AS703026, AZD6244 (Selumetinib), AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD184352), PD0325901, PD318088, PD98059, and U0126-EtOH, GSK1120212, FR180204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, GDC-0973 (XL518). In some embodiments, the AKT1/2 inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of A6730, B2311, 124018, GSK2141795, MK2206, GSK2110183, GSK690693, Perifosine (KRX-0401), GDC-0068, RX-0201, VQD-002. In some embodiments, the ERK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of PD98059, U0126, FR180204, 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4- thiazolidinedione, and pyrazolylpyrrole ERK Inhibitor. In some embodiments, the inhibition of excessive immunosuppressive activity is desired in a subject exhibiting symptoms of a disease characterized by excessive immunosuppressive activity. In some embodiments, the inhibition of excessive immunosuppressive activity is utilized for treatment of a disease selected from the group consisting of a chronic inflammatory disease, a chronic infectious disease and cancer.

In some embodiments, the present invention provides a pharmaceutical composition comprising a first therapeutic agent and a second therapeutic agent that is an inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof, wherein said administration of said second therapeutic agent results in inhibition of activation of resting human regulatory T cells (rTregs) to active Tregs (aTregs). In some embodiments, the first therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an antiviral agent, an antibacterial agent, and a stem cell agent. In some embodiments, the first therapeutic agent is selected from the group consisting of a biologic therapeutic agent, a small molecule therapeutic agent, and an RNA-based therapeutic agent. In some embodiments, the MEK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of AS703026, AZD6244 (Selumetinib), AZD8330(ARRY- 424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD 184352),

PD0325901, PD318088, PD98059, and U0126-EtOH, GSK1120212, FR180204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, GDC-0973 (XL518). In some embodiments, the AKT1/2 inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of A6730, B2311, 124018,

GSK2141795, MK2206, GSK2110183, GSK690693, Perifosine (KRX-0401), GDC-0068, RX- 0201, VQD-002. In some embodiments, the ERK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug. In some embodiments, the small molecule drug is selected from the group consisting of PD98059, U0126, FR180204 , 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione, and pyrazolylpyrrole ERK Inhibitor.

In some embodiments, the present invention provides for the use of the foregoing compositions for administration to a subject, preferably a human subject. In some embodiments, the present invention provides for the use of the foregoing compositions to treat a subject exhibiting symptoms of a disease characterized by excessive immunosuppressive activity. In some embodiments, the inhibition of excessive immunosuppressive activity is utilized for treatment of a disease selected from the group consisting of a chronic inflammatory disease, a chronic infectious disease and cancer.

Additional embodiments are described herein. DESCRIPTION OF THE FIGURES

Figure 1 provides results of high throughput analysis of T cell signaling profiles (A) Diagram showing workflow for phospho flow cytometry. (B) Anti-CD3 concentration-dependent increase in T cell signaling in both CD4⁺ and CD8⁺ T cells assessed by phospho flow cytometry. (C) Experiment as in (B), but data for T cells from three individuals are combined.

Figure 2 provides results for a comparison of T cell signaling profiles for different naive and memory T cell subsets. (A) Primary human T cells were stimulated by cross-linking of the indicated combinations of antibodies with avidin and incubated for different time periods. (B) Experiment as in (A), but all values are relative to the phospho-signals obtained for the control sample of the naive CD4⁺ T cell population. Data are representative of experiments with T cells from three individual blood donors. (C) Experiment as in (B), but data for T cells from three individuals are combined. Data are presented as mean ± SEM (n = 3) of arcsinh median differences of the phospho-epitope specific fluorescence intensity signals.

Figure 3 provides results for an analysis of signaling in conventional and regulatory CD4⁺ T cell subsets reveals elevated Erk activation and lowered Akt activation in aTregs. (A) Gating strategy for distinguishing between CD4⁺ effector and regulatory T cell subsets using CD45RA and FoxP3 antibody staining. (B) Primary human T cells were stimulated by cross-linking of the indicated combinations of antibodies with avidin and incubated for different time periods. (D) Experiment as in the lower parts of the panels of (B) and (C), but data from three individuals are combined. Data are presented as mean ± SEM (n = 3) of arcsinh median differences of the phospho-epitope specific fluorescence intensity signals. (E) As in (D) but only unstimulated cells are included in the analysis.

Figure 4 provides results for costimulation with CD28 is essential for proper activation of Akt and NF-κΒ related signaling. (A) Primary human CD4⁺ T-cells were stimulated by cross- linking of the indicated combinations of antibodies with avidin and incubated for different time periods. (B) Amalgamated data with normalization to the CD4 CD45RA FoxP3^~ subset as in lower panels of A). Figure 5 provides results for Mek-dependent up regulation of FoxP3 in rTregs (A) Sorted rTregs were stimulated or not with aCD3/CD28/CD2-coated MicroBeads for the indicated time periods, followed by FoxP3 staining and FACS analysis. (B) Experiments as in (A) were performed with T cells from three individuals. (C) Experiment as in (A), but sorted rTregs were incubated with the indicated inhibitors of Mek (either PD0325901 or U0126), p38 (SB 203580) PI3K (PI-103 in combination with wortmannin), Akt (Aktl/2-inhibitor), mTOR (Rapamycin), or Calcineurin (CsA) for 20 min followed by stimulation with aCD3/CD28/CD2- coated MicroBeads for 36 hours. (D) Experiments as in (C) were performed with T cells from three individuals, and otherwise analyzed as outlined in (B).

Figure 6 provides results for induction of rTreg suppressive capacity is Mek-dependent. (A) Sorted rTregs were either directly added to CFSE-labeled CD4⁺ T cells, or first pre-treated with indicated inhibitor and then stimulated with aCD3/CD28/CD2-coated MicroBeads for 36 hours before being added to CFSE-labeled CD4⁺ T cells. (B) Data from three experiments as in (A) were combined. The suppressive capacity was calculated as Aproliferation = % proliferation (responder cells alone, 0: 1) - % proliferation (responder cells in presence of Tregs, 1 : 1).

Figure 7 provides results for purity of purified cells (A) CD3⁺ T cells purified with RosetteSep were stained for CD4 and CD3, and analyzed by FACS. Data are representative of four independent experiments. (B) CD3⁺ T cells purified with RosetteSep were stained for CD3, CD4 and CD8, and analyzed by FACS. Left panel shows CD3 and CD4 expression of all cells, while the CD3 CD4^" cells in this sample next were analyzed with regard to CD8 expression (right panel). Data are representative of nine individual blood donors.

Figure 8 provides results for effects of different anti-CD3 concentrations on signaling in CD4⁺ and CD8⁺ T cells. Data obtained with T cells from Donor I.

Figure 9 provides results for effects of different anti-CD3 concentrations on signaling in

CD4⁺ and CD8⁺ T cells. Data obtained with T cells from Donor II.

Figure 10 provides results for effects of different anti-CD3 concentrations on signaling in CD4⁺ and CD8⁺ T cells. Data obtained with T cells from Donor III. Figure 11 provides results for comparison of T cell signaling profiles for different naive and memory T cell subsets. Data obtained with T cells from Donor IV.

Figure 12 provides results for comparison of T cell signaling profiles for different naive and memory T cell subsets. Data obtained with T cells from Donor V.

Figure 13 provides results for comparison of T cell signaling profiles for different naive and memory T cell subsets. Data obtained with T cells from Donor VI.

Figure 14 provides results for comparison of T cell signaling profiles for different conventional and regulatory CD4⁺ T cell subsets. Data obtained with CD4⁺ T-cells from Donor VII.

Figure 15 provides results for comparison of T cell signaling profiles for different conventional and regulatory CD4⁺ T cell subsets. Data obtained with CD4⁺ T-cells from Donor VIII.

Figure 16 provides results for comparison of T cell signaling profiles for different conventional and regulatory CD4⁺ T cell subsets. Data obtained with CD4⁺ T-cells from Donor IX.

Figure 17 provides results for comparison of T cell signaling profiles for different conventional and regulatory CD4⁺ T cell subsets. Data obtained from Donor X.

Figure 18 provides results for comparison of T cell signaling profiles for different conventional and regulatory CD4⁺ T cell subsets. Data obtained from Donor XI.

Figure 19 provides results for comparison of T cell signaling profiles for different conventional and regulatory CD4⁺ T cell subsets. Data obtained from Donor XII.

Figure 20 provides results for purity of sorted cells (A) CD45RA and CD25 staining of purified CD4⁺ T cells before sorting was initiated. Populations to be sorted are indicated. (B) Immediately after sorting as described in (A) had been completed, sorted cells were analyzed by FACS to determine level of purity.

Figure 21 provides results for effect of different MEK inhibitors on FoxP3 upregulation upon stimulation of resting Tregs. Sorted resting Tregs were incubated with different

concentrations of the indicated MEK inhibitors, followed by stimulated with CD3/CD28/CD2- coated MicroBeads for 36h, followed by FoxP3 staining and FACS analysis.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below: As used herein, the term "inhibitor of MEK (MAPK/ER kinase)" refers to an agent that inhibits the expression or activity of MEK. The agent can, for example, be an siRNA, an antisense oligonucleotide, an antigen binding protein, or a small molecule drug.

As used herein, the term "inhibitor of ERK" refers to an agent that inhibits the expression or activity of ERK. The agent can, for example, be an siRNA, an antisense oligonucleotide, an antigen binding protein, or a small molecule drug.

As used herein, the term "inhibitor of AKT1/2 (RAC-alpha serine/threonine-protein kinase and RAC-beta serine/threonine-protein kinase, respectively)" refers to an agent that inhibits the expression or activity of AKT1/2. The agent can, for example, be an siRNA, an antisense oligonucleotide, an antigen binding protein, or a small molecule drug.

As used herein, the term "therapeutic agent" refers to a molecule or compound that is administered to an animal such as a human for therapeutic purposes.

As used herein, the term "chemotherapeutic agent" refers to an agent that is administered to a patient to treat a cancer.

As used herein, the term "antiviral agent" refers to an agent that is administered to a patient to treat a viral infection.

As used herein, the term "antibacterial agent" refers to an agent that is administered to a patient to treat a bacterial infection.

As used herein, the term "antifungal agent" refers to an agent that is administered to a patient to treat a fungal infection.

As used herein, the term "stem cell agent" refers to an agent comprising stem cells (adult or embryonic) that is administered to a patient to treat a disease.

As used herein, the term "biologic therapeutic agent" refers to a therapeutic agent that is prepared by a biological process such as cell culture. Biologic therapeutic agents include proteins such as growth factors, receptors and interferons as well as antigen binding proteins.

As used herein, the term "small molecule therapeutic agent" refers to a low molecular weight organic compound that is not a polymer.

As used herein, the term "RNA-based therapeutic agent" refers to an agent comprising an RNA moiety. Examples of RNA-based therapeutic agents include, but are not limited to, antisense RNA molecules and siRNA molecules.

As used herein, the terms "protein," "polypeptide," and "peptide" refer to a molecule comprising amino acids joined via peptide bonds. In general "peptide" is used to refer to a sequence of 20 or less amino acids and "polypeptide" is used to refer to a sequence of greater than 20 amino acids. As used herein, the term, "synthetic polypeptide," "synthetic peptide" and "synthetic protein" refer to peptides, polypeptides, and proteins that are produced by a recombinant process (i.e., expression of exogenous nucleic acid encoding the peptide, polypeptide or protein in an organism, host cell, or cell-free system) or by chemical synthesis.

As used herein, the term "native" (or wild type) when used in reference to a protein refers to proteins encoded by the genome of a cell, tissue, or organism, other than one manipulated to produce synthetic proteins.

As used herein, the term "antigen binding protein" refers to proteins that bind to a specific antigen. "Antigen binding proteins" include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, single chain, and humanized antibodies, Fab fragments,

F(ab')2 fragments, and Fab expression libraries. Various procedures known in the art are used for the production of polyclonal antibodies. For the production of antibody, various host animals can be immunized by injection with the peptide corresponding to the desired epitope including but not limited to rabbits, mice, rats, sheep, goats, etc. Various adjuvants are used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

A "subject" is an animal such as vertebrate, preferably a mammal such as a human, or a bird or a fish. Mammals are understood to include, but are not limited to, murines, simians, humans, bovines, cervids, equines, porcines, canines, felines etc.).

An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations.

As used herein, the term "purified" or "to purify" refers to the removal of components

(e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample. DETAILED DESCRIPTION OF THE INVENTION

Activation of CD4⁺ T cells is required for a majority of adaptive immune responses, and a balanced reaction is essential in order to avoid excessive tissue damage and autoimmunity.

However, these conventional CD4⁺ T cells are kept in check by another type of CD4⁺ T cells known as regulatory T cells (Tregs)²⁷. Tregs are characterized by the expression of the transcription factor FoxP3 and their ability to suppress conventional T cells^28"31. A recent paper indicated that CD4⁺ T cells in human blood can be divided into five different categories based on the expression of CD45RA, CD25 and FoxP3³². These subsets are: i) conventional naive CD4⁺ T cells (CD45RA⁺CD25^"FoxP3 ), ii) conventional effector/memory CD4⁺ T cells (CD45RA CD25^" FoxP3^"), iii) CD4⁺FoxP3⁺ effector T cells with cytokine-producing capabilities (CD45RA^" CD25⁺FoxP3⁺), iv) resting regulatory T cells (rTregs, CD45RA⁺CD25⁺FoxP3⁺), and v) activated regulatory T cells (aTregs, CD45RA^"CD25⁺⁺FoxP3⁺⁺). Given proper stimulatory conditions, naive conventional T cells can proliferate and differentiate to become effector/memory conventional T cells. In a similar manner, rTregs can be considered precursors of aTregs. The observation that rTregs in response to activation can proliferate and differentiate into Ki- 67 FoxP3 CD45RO cells with suppressive abilities comparable to aTregs supports this notion³².

Since the above-mentioned CD4⁺FoxP3⁺ T cell subsets combined constitute less than

10% of all CD4⁺ T cells in peripheral blood, traditional biochemical studies of signal

transduction in these cells have been challenging. Recent technical developments including fluorescent cell barcoding (FCB)³³ and a growing number of phosphoepitope-specific antibodies have, however, made it possible to use phospho flow cytometry-based methods to study signaling processes at single-cell resolution in several phenotypically defined T cell subsets

simultaneously^34"36. Furthermore, these developments have enabled increasing the resolution to a level where signaling differences can be linked to functional properties in small subsets of cells. Experiments conducted during the course of development of embodiments of the present invention used this technique to investigate signaling and the role of CD28 and CD2 co- stimulation in different human CD4⁺ T cell subsets, including the CD4⁺FoxP3⁺ subsets. We were able to reproducibly identify signaling characteristics and define distinct differences between CD28 and CD2 in activation of an Akt-NF-κΒ pathway and in activation of the S6-Ribosomal protein (S6-Rp) transcriptional program. In addition, differences in Akt versus Mekl/Erk signaling between rTregs and aTregs, combined with functional studies with signaling inhibitors, indicated that the activity level of Mekl/Erk and Akt pathways are involved in defining a functional switch between these two subsets.

Experiments conducted during the course of development of embodiments of the present invention investigated how T cell signaling networks elicited by triggering of the T cell receptor and different co-stimuli (CD28, CD2) differ in T cell subsets such as CD4 and CD8 naive and effector/memory T cells and in resting and activated regulatory T cells (rTreg, aTreg).

Phosphoflow cytometry of fluorescent cell bar-coded (FCB) samples was utilized to

concomitantly analyze the effects of TCR triggering and CD28 and/or CD2 co-stimuli in up to 5 different subsets of human primary T cells at 6 different time points on phosphorylation levels of 18 different signaling proteins detected by a panel of phospho-specific antibodies, carefully composed, balanced and titrated for this purpose. This advanced technology developed specifically for the purpose allowed acquisition and composition of high-resolution maps of T cell signaling networks.

From the global overview of T cell signaling networks that these analyses provided, it was possible to extract information of differences in signaling patterns in T cell subsets revealing that while rTregs have low basal, but inducible levels of Erk and strong stimulation-dependent Akt activation, aTregs displayed a constitutively active Erk and little Akt activation. This information was used to perturb the Erk pathway upstream by Mek inhibitors. It was shown that this inhibits up-regulation of Foxp3 upon activation of rTregs, as well as the stimulation-induced increase in Treg suppressive activity which was a surprising finding that could not be inferred from the available literature or from the state-of -the-art at the time of invention.

Accordingly, in some embodiments, the present invention provides compositions and methods for using Mek inhibitors to block the transition of rTregs to aTregs upon activation. The compositions and methods of the present invention find use in research, screening (e.g., drug screening) and clinical (e.g., prevention of excess Treg-mediated immunosuppression in relevant clinical conditions) applications.

In some embodiments, the compositions and methods described herein find use in the treatment and prevention of a variety of clinical conditions that result from excess

immunosuppressive activity. Examples include, but are not limited to immunomodulation in autoimmune diseases, chronic inflammatory diseases, chronic infectious diseases and/or cancer.

In some embodiments, the present invention provides compositions and methods for inhibiting activation of rTreg to prevent excess immunosuppressive activity by inhibiting Mek and/or of Akt 1/2. I. Therapeutic Applications

In some embodiments, the present invention provides therapies for cancer, autoimmune and chronic inflammatory disorders. In some embodiments, therapies directly or indirectly target the expression or activity MEK, ERK and/or AKTI/2. Accordingly, the therapies comprise administration of agents that inhibit expression of the MEK, ERK and/or AKTI/2 genes as well as agents which inhibit the activity of the MEK, ERK and/or AKTI/2 proteins.

A. RNA Interference and Antisense Therapies

In some embodiments, the present invention targets the expression of MEK, ERK and/or AKTI/2 genes. For example, in some embodiments, the present invention employs compositions comprising oligomeric antisense or RNAi compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding MEK, ERK and/or AKTI/2 genes, ultimately modulating the amount of MEK, ERK and/or AKTI/2 expressed. 1. RNA Interference (RNAi)

In some embodiments, RNAi is utilized to inhibit or modulate MEK, ERK and/or AKTI/2 expression. RNAi represents an evolutionary conserved cellular defense for controlling the expression of foreign genes in most eukaryotes, including humans. RNAi is typically triggered by double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation of single- stranded target RNAs homologous in response to dsRNA. The mediators of mRNA degradation are small interfering RNA duplexes (siRNAs), which are normally produced from long dsRNA by enzymatic cleavage in the cell. siRNAs are generally approximately twenty-one nucleotides in length (e.g. 21-23 nucleotides in length), and have a base-paired structure characterized by two nucleotide 3 '-overhangs. Following the introduction of a small RNA, or RNAi, into the cell, it is believed the sequence is delivered to an enzyme complex called RISC (RNA-induced silencing complex). RISC recognizes the target and cleaves it with an endonuclease. It is noted that if larger RNA sequences are delivered to a cell, RNase III enzyme (Dicer) converts longer dsRNA into 21-23 nt ds siRNA fragments. Chemically synthesized siRNAs have become powerful reagents for genome-wide analysis of mammalian gene function in cultured somatic cells.

Beyond their value for validation of gene function, siRNAs also hold great potential as gene- specific therapeutic agents (Tuschl and Borkhardt, Molecular Intervent. 2002; 2(3): 158-67, herein incorporated by reference).

The transfection of siRNAs into animal cells results in the potent, long-lasting post- transcriptional silencing of specific genes (Caplen et al, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al, Nature. 2001 ; 41 1 :494-8; Elbashir et al, Genes Dev. 2001 ; 15 : 188-200; and Elbashir et al., EMBO J. 2001 ; 20: 6877-88, all of which are herein incorporated by reference). Methods and compositions for performing RNAi with siRNAs are described, for example, in U.S. Pat. 6,506,559, herein incorporated by reference.

siRNAs are extraordinarily effective at lowering the amounts of targeted RNA, and by extension proteins, frequently to undetectable levels. The silencing effect can last several months, and is extraordinarily specific, because one nucleotide mismatch between the target RNA and the central region of the siRNA is frequently sufficient to prevent silencing

(Brummelkamp et al, Science 2002; 296:550-3; and Holen et al, Nucleic Acids Res. 2002;

30: 1757-66, both of which are herein incorporated by reference).

An important factor in the design of siRNAs is the presence of accessible sites for siRNA binding. Bahoia et al, (J. Biol. Chem., 2003; 278: 15991-15997; herein incorporated by reference) describe the use of a type of DNA array called a scanning array to find accessible sites in mRNAs for designing effective siRNAs. These arrays comprise oligonucleotides ranging in size from monomers to a certain maximum, usually Comers, synthesized using a physical barrier (mask) by stepwise addition of each base in the sequence. Thus the arrays represent a full oligonucleotide complement of a region of the target gene. Hybridization of the target mRNA to these arrays provides an exhaustive accessibility profile of this region of the target mRNA. Such data are useful in the design of antisense oligonucleotides (ranging from 7mers to 25mers), where it is important to achieve a compromise between oligonucleotide length and binding affinity, to retain efficacy and target specificity (Sohail et al, Nucleic Acids Res., 2001 ; 29(10): 2041- 2045). Additional methods and concerns for selecting siRNAs are described for example, in WO

05054270, WO05038054A1 , WO03070966A2, J Mol Biol. 2005 May 13;348(4):883-93, J Mol Biol. 2005 May 13;348(4):871-81 , and Nucleic Acids Res. 2003 Aug l ;31(15):4417-24, each of which is herein incorporated by reference in its entirety. In addition, software (e.g. , the MWG online siMAX siRNA design tool) is commercially or publicly available for use in the selection of siRNAs.

In some embodiments, the present invention utilizes siRNA including blunt ends (See e.g., US20080200420, herein incorporated by reference in its entirety), overhangs (See e.g., US20080269147A1 , herein incorporated by reference in its entirety), locked nucleic acids (See e.g., WO2008/006369, WO2008/043753, and WO2008/051306, each of which is herein incorporated by reference in its entirety). In some embodiments, siRNAs are delivered via gene expression or using bacteria (See e.g., Xiang et al, Nature 24: 6 (2006) and WO06066048, each of which is herein incorporated by reference in its entirety). In other embodiments, shR A techniques (See e.g., 20080025958, herein incorporated by reference in its entirety) are utilized. A small hairpin R A or short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it. shRNA is transcribed by RNA polymerase III.

The present invention also includes pharmaceutical compositions and formulations that include the RNAi compounds of the present invention as described below.

2. Antisense

In other embodiments, protein expression is modulated using antisense compounds that specifically hybridize with one or more nucleic acids encoding MEK, ERK and/or AKTl/2. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds that specifically hybridize to it is generally referred to as "antisense." The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity that may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of Mek. In the context of the present invention, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.

It is preferred to target specific nucleic acids for antisense. "Targeting" an antisense compound to a particular nucleic acid, in the context of the present invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding a Mek of the present invention. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g. , detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in

prokaryotes). Eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the present invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene.

Translation termination codon (or "stop codon") of a gene may have one of three sequences {i.e., 5'-UAA, 5'-UAG and 5'-UGA; the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction {i.e., 5 ' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction {i.e., 5 ' or 3') from a translation termination codon.

The open reading frame (ORF) or "coding region," which refers to the region between the translation initiation codon and the translation termination codon, is also a region that may be targeted effectively. Other target regions include the 5' untranslated region (5' UTR), referring to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3' UTR), referring to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene. The 5' cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap. The cap region may also be a preferred target region. Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," that are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. mRNA splice sites (i.e., intron-exon junctions) may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

In some embodiments, target sites for antisense inhibition are identified using

commercially available software programs (e.g., Biognostik, Gottingen, Germany; SysArris Software, Bangalore, India; Antisense Research Group, University of Liverpool, Liverpool, England; GeneTrove, Carlsbad, CA). In other embodiments, target sites for antisense inhibition are identified using the accessible site method described in PCT Publ. No. WO0198537A2, herein incorporated by reference.

Once one or more target sites have been identified, oligonucleotides are chosen that are sufficiently complementary to the target (i.e., hybridize sufficiently well and with sufficient specificity) to give the desired effect. For example, in preferred embodiments of the present invention, antisense oligonucleotides are targeted to or near the start codon.

In the context of this invention, "hybridization," with respect to antisense compositions and methods, means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. It is understood that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired (i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed).

Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with specificity, can be used to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. The specificity and sensitivity of antisense is also applied for therapeutic uses. For example, antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides are useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues, and animals, especially humans.

While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 30 nucleobases (i.e., from about 8 to about 30 linked bases), although both longer and shorter sequences may find use with the present invention. Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 25 nucleobases.

Specific examples of preferred antisense compounds useful with the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be

oligonucleosides.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral

phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,

thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e., the backbone) of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an amino ethylgly cine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science 254:1497 (1991).

Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular— CH2,

-NH-O-CH2-, ~CH2~N(CH3)--0~CH2~ [known as a methylene (methylimino) or MMI backbone], -CH2~0-N(CH₃)-CH2~, ~CH2~N(CH3)-N(CH₃)--CH2~, and

— O— N(CH3)— CH2— CH2— [wherein the native phosphodiester backbone is represented as

— O— P— O--CH2— ] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above -referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugar moieties.

Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C \ to C \ Q alkyl or C2 to C \ Q alkenyl and alkynyl. Particularly preferred are 0[(CH₂)_nO]_mCH3, 0(CH₂)_nOCH₃, 0(CH₂)_nNH₂,

0(CH₂)_nCH₃, 0(CH₂)_nONH₂, and 0(CH2)_nON[(CH2)_nCH₃)]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C\ to C \ 0 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF₃, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes

2'-methoxyethoxy (2'-0~CH2CH20CH3, also known as 2'-0-(2-methoxy ethyl) or 2'-MOE)

(Martin et al., Helv. Chim. Acta 78:486 [1995]) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy {i.e., a 0(CH2)20N(CH3)2 group), also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as

2^*-0-dimethylaminoethoxyethyl or 2^*-DMAEOE), i.e., 2^*-0~CH2~0-CH2~N(CH2)2- Other preferred modifications include 2'-methoxy (2'-0— CH3), 2'-aminopropoxy

(2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.

Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and

2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,

8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo,

5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and

7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and

3- deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines,

6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2. °C and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar

modifications.

Another modification of the oligonucleotides of the present invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, (e.g.,

hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain, (e.g., dodecandiol or undecyl residues), a phospholipid, (e.g. , di-hexadecyl-rac-glycerol or triethylammonium

l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a polyethylene glycol chain or adamantane acetic acid, a palmityl moiety, or an octadecylamine or

hexylamino-carbonyl-oxycholesterol moiety.

One skilled in the relevant art knows well how to generate oligonucleotides containing the above-described modifications. The present invention is not limited to the antisense oligonucleotides described above. Any suitable modification or substitution may be utilized.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds that are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of the present invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving R A:DNA or R A:RNA hybrids. By way of example, R aseH is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter

oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. Chimeric antisense compounds of the present invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above.

The present invention also includes pharmaceutical compositions and formulations that include the antisense compounds of the present invention as described below.

B. Genetic Therapy

The present invention contemplates the use of any genetic manipulation for use in modulating the expression of the MEK, ERK and/or AKTI/2 genes. Examples of genetic manipulation include, but are not limited to, gene knockout (e.g., removing the MEK, ERK and/or AKTI/2 genes from the chromosome using, for example, recombination), expression of antisense constructs with or without inducible promoters, and the like. Delivery of nucleic acid construct to cells in vitro or in vivo may be conducted using any suitable method. A suitable method is one that introduces the nucleic acid construct into the cell such that the desired event occurs (e.g. , expression of an antisense construct). Genetic therapy may also be used to deliver siR A or other interfering molecules that are expressed in vivo (e.g., upon stimulation by an inducible promoter (e.g., an androgen-responsive promoter)).

Introduction of molecules carrying genetic information into cells is achieved by any of various methods including, but not limited to, directed injection of naked DNA constructs, bombardment with gold particles loaded with said constructs, and macromolecule mediated gene transfer using, for example, liposomes, biopolymers, and the like. Preferred methods use gene delivery vehicles derived from viruses, including, but not limited to, adenoviruses, retroviruses, vaccinia viruses, and adeno-associated viruses. Because of the higher efficiency as compared to retroviruses, vectors derived from adenoviruses are the preferred gene delivery vehicles for transferring nucleic acid molecules into host cells in vivo. Adenoviral vectors have been shown to provide very efficient in vivo gene transfer into a variety of solid tumors in animal models and into human solid tumor xenografts in immune-deficient mice. Examples of adenoviral vectors and methods for gene transfer are described in PCT publications WO 00/12738 and WO

00/09675 and U.S. Pat. Appl. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128, 5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544, each of which is herein incorporated by reference in its entirety.

Vectors may be administered to subjects in a variety of ways. For example, in some embodiments of the present invention, vectors are administered into tissue using direct injection. In other embodiments, administration is via the blood or lymphatic circulation (See e.g., PCT publication 99/02685 herein incorporated by reference in its entirety). Exemplary dose levels of adenoviral vector are preferably 10⁸ to 10¹¹ vector particles added to the perfusate.

C. Antibody Therapy

In some embodiments, the present invention provides antibodies that target cells that express the MEK, ERK and/or AKT1/2 genes or MEK, ERK and/or AKT1/2 gene products themselves. Any suitable antibody (e.g., monoclonal, polyclonal, or synthetic) may be utilized in the therapeutic methods disclosed herein. In preferred embodiments, the antibodies used for clinical application are humanized antibodies. Methods for humanizing antibodies are well known in the art (See e.g., U.S. Pat. Nos. 6,180,370, 5,585,089, 6,054,297, and 5,565,332; each of which is herein incorporated by reference).

In some embodiments, the therapeutic antibodies comprise an antibody generated against a MEK, ERK and/or AKT1/2, wherein the antibody is conjugated to a cytotoxic agent. In such embodiments, a therapeutic agent is generated that does not target normal cells, thus reducing many of the detrimental side effects of traditional chemotherapy. For certain applications, it is envisioned that the therapeutic agents will be pharmacologic agents that will serve as useful agents for attachment to antibodies, particularly cytotoxic or otherwise anticellular agents having the ability to kill or suppress the growth or cell division of endothelial cells. The present invention contemplates the use of any pharmacologic agent that can be conjugated to an antibody, and delivered in active form. Exemplary anticellular agents include chemo therapeutic agents, radioisotopes, and cytotoxins. The therapeutic antibodies of the present invention may include a variety of cytotoxic moieties, including but not limited to, radioactive isotopes (e.g., iodine-131, iodine-123, technicium-99m, indium-I l l, rhenium-188, rhenium-186, gallium-67, copper-67, yttrium-90, iodine- 125 or astatine -211), hormones such as a steroid, antimetabolites such as cytosines (e.g. , arabinoside, fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycin C), vinca alkaloids (e.g., demecolcine; etoposide; mithramycin), and antitumor alkylating agent such as chlorambucil or melphalan. Other embodiments include agents such as a coagulant, a cytokine, growth factor, bacterial endotoxin or the lipid A moiety of bacterial endotoxin. For example, in some embodiments, therapeutic agents include plant-, fungus- or bacteria-derived toxin, such as an A chain toxins, a ribosome inactivating protein, a-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin, to mention just a few examples. In some preferred embodiments, deglycosylated ricin A chain is utilized.

In any event, it is proposed that agents such as these may, if desired, be successfully conjugated to an antibody, in a manner that will allow their targeting, internalization, release or presentation to blood components at the site of the targeted cells as required using known conjugation technology {See, e.g., Ghose et al., Methods EnzymoL, 93:280 [1983]).

For example, in some embodiments the present invention provides immunotoxins targeting MEK, ERK and/or AKTl/2. Immunotoxins are conjugates of a specific targeting agent typically a cell or tumor-directed antibody or fragment, with a cytotoxic agent, such as a toxin moiety. The targeting agent directs the toxin to, and thereby selectively kills, cells carrying the targeted antigen. In some embodiments, therapeutic antibodies employ crosslinkers that provide high in vivo stability (Thorpe et ah, Cancer Res., 48:6396 [1988]).

In other embodiments, particularly those involving treatment of solid tumors, antibodies are designed to have a cytotoxic or otherwise anticellular effect against the tumor vasculature, by suppressing the growth or cell division of the vascular endothelial cells. This attack is intended to lead to a tumor-localized vascular collapse, depriving the tumor cells, particularly those tumor cells distal of the vasculature, of oxygen and nutrients, ultimately leading to cell death and tumor necrosis.

In preferred embodiments, antibody based therapeutics are formulated as pharmaceutical compositions as described below. In preferred embodiments, administration of an antibody composition of the present invention results in a measurable decrease in disease.

The present invention also includes pharmaceutical compositions and formulations that include the antibody compounds of the present invention as described below.

D. Small Molecules

In some embodiments, small molecule inhibitors are used to inhibit or modulate MEK, Erk and/or Aktl/2 activity. Exemplary MEK inhibitors include, but are not limited to, commercially available inhibitors (e.g., including but not limited to, AS703026, AZD6244 (Selumetinib), AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD184352), PD0325901, PD318088, PD98059, and U0126-EtOH) and inhibitors identified using the drug screening methods described herein.

Commercially available AKTl/2 inhibitors include, but are not limited to, 1,3-Dihydro-l- (l-((4-(6-phenyl-lH-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H- benzimidazol-2-one trifluoroacetate salt hydrate, Akt Inhibitor VIII trifluoroacetate salt hydrate, Akti-1/2 trifluoroacetate salt hydrate (A6730); 5-(2-Benzothiazolyl)-3-ethyl-2-[2-

(methylphenylamino)ethenyl]- 1 -phenyl- lH-benzimidazo Hum iodide (B2311); and 1,3-Dihydro- l-(l-((4-(6-phenyl-lH-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H- benzimidazol-2-one, Akti-1/2 (124018; Akt Inhibitor VIII, Isozyme-Selective, Akti-1/2). Commercially available ER inhibitors include, but are not limited to, PD98059, U0126,

E. Cell Based Therapeutics

In some embodiments, cell based therapies such as stem cells and ex vivo manipulation of cells and autologous transfer back into the same human or leukoferesis techniques removing or modulating cells, etc are utilized. For example, in some embodiments, cells are removed from a subject, altered (e.g., the expression or activity of MEK, ERK and/or AKT1/2 is altered) and the Cells are re-introduced into the autologous subject.

Cells may be purified from blood using known methods such as leukoferesis, by identification of the markers described herein, or a combination of such methods. Such isolation methods find use in the research and clinical applications described herein. F. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions (e.g. , comprising pharmaceutical agents that modulate the expression or activity of MEK, ERK and/or AKT1/2). The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated and the pharmaceutical agent that is selected. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular

administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention also include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active pharmaceutical agent with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The pharmaceutical compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium

carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual agents, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the agent is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

G. Combination therapy

In some embodiments, the present invention provides therapeutic methods comprising one or more MEK, ERK and/or AKT1/2 inhibitors in combination with an additional agent (e.g., a chemotherapeutic agent or agent useful in treating autoimmune or chronic inflammatory diseases). The present invention is not limited to a particular additional agents. Additional agents include, but are not limited to, chemotherapeutic agents, antiviral agents, antibacterial agents, antifungal agents and stem cell agents as well as biologic therapeutic agents, small molecule therapeutic agents, and oligonucleotide therapeutic agents such as RNA-based therapeutic agents.

Certain embodiments of the invention provide pharmaceutical compositions containing (a) an inhibitor of MEK, ERK and/or AKTl/2and (b) one or more other chemotherapeutic agents that function by an antisense ornon-antisense mechanism. Examples of such chemotherapeutic agents include, but are not limited to, anticancer drugs such as blocking antibodies, non-steroidal anti-inflammatory agents, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine,

6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

Various classes of antineoplastic (e.g., anticancer) agents are contemplated for use in certain embodiments of the present invention. Anticancer agents suitable for use with embodiments of the present invention include, but are not limited to, agents that induce apoptosis, agents that inhibit adenosine deaminase function, inhibit pyrimidine biosynthesis, inhibit purine ring biosynthesis, inhibit nucleotide interconversions, inhibit ribonucleotide reductase, inhibit thymidine monophosphate (TMP) synthesis, inhibit dihydrofolate reduction, inhibit DNA synthesis, form adducts with DNA, damage DNA, inhibit DNA repair, intercalate with DNA, deaminate asparagines, inhibit RNA synthesis, inhibit protein synthesis or stability, inhibit microtubule synthesis or function, and the like.

In some embodiments, exemplary anticancer agents suitable for use in compositions and methods of embodiments of the present invention include, but are not limited to: 1) alkaloids, including microtubule inhibitors (e.g., vincristine, vinblastine, and vindesine, etc.), microtubule stabilizers (e.g., paclitaxel (TAXOL), and docetaxel, etc.), and chromatin function inhibitors, including topoisomerase inhibitors, such as epipodophyllotoxins (e.g., etoposide (VP- 16), and teniposide (VM-26), etc.), and agents that target topoisomerase I (e.g., camptothecin and isirinotecan (CPT-11), etc.); 2) covalent DNA-binding agents (alkylating agents), including nitrogen mustards (e.g. , mechlorethamine, chlorambucil, cyclophosphamide, ifosphamide, and busulfan (MYLERAN), etc.), nitrosoureas (e.g., carmustine, lomustine, and semustine, etc.), and other alkylating agents (e.g. , dacarbazine, hydroxymethylmelamine, thiotepa, and mitomycin, etc.); 3) noncovalent DNA-binding agents (antitumor antibiotics), including nucleic acid inhibitors (e.g., dactinomycin (actinomycin D), etc.), anthracyclines (e.g., daunorubicin

(daunomycin, and cerubidine), doxorubicin (adriamycin), and idarubicin (idamycin), etc.), anthracenediones (e.g., anthracycline analogues, such as mitoxantrone, etc.), bleomycins (BLENOXANE), etc., and plicamycin (mithramycin), etc.; 4) antimetabolites, including antifolates (e.g., methotrexate, FOLEX, and MEXATE, etc.), purine antimetabolites (e.g., 6- mercaptopurine (6-MP, PURINETHOL), 6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir, chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and 2'-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (e.g., fluoropyrimidines (e.g., 5-fluorouracil

(ADRUCIL), 5-fluorodeoxyuridine (FdUrd) (floxuridine)) etc.), and cytosine arabinosides (e.g., CYTOSAR (ara-C) and fludarabine, etc.); 5) enzymes, including L-asparaginase, and hydroxyurea, etc.; 6) hormones, including glucocorticoids, antiestrogens {e.g., tamoxifen, etc.), nonsteroidal antiandrogens {e.g., flutamide, etc.), and aromatase inhibitors {e.g., anastrozole (ARIMIDEX), etc.); 7) platinum compounds {e.g., cisplatin and carboplatin, etc.); 8) monoclonal antibodies conjugated with anticancer drugs, toxins, and/or radionuclides, etc.; 9) biological response modifiers {e.g., interferons {e.g., IFN-a, etc.) and interleukins {e.g., IL-2, etc.), etc.); 10) adoptive immunotherapy; 11) hematopoietic growth factors; 12) agents that induce tumor cell differentiation {e.g., all-trans-retinoic acid, etc.); 13) gene therapy techniques; 14) antisense therapy techniques; 15) tumor vaccines; 16) therapies directed against tumor metastases {e.g., batimastat, etc.); 17) angiogenesis inhibitors; 18) proteosome inhibitors {e.g., VELCADE); 19) inhibitors of acetylation and/or methylation {e.g., HDAC inhibitors); 20) modulators of NF kappa B; 21) inhibitors of cell cycle regulation {e.g., CDK inhibitors); 22) modulators of p53 protein function; and 23) radiation.

Any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of embodiments of the present invention. For example, the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S.F.D.A. maintain similar formularies.

In some embodiments, agents for use in combination therapy comprise antigen binding proteins (e.g., anti-cancer blocking antibodies) or other biological agents. Preferred biological agents include, but are not limited to Abatacept, Adalimumab, Alefacept, Erythropoietin,

Etanercept, Infliximab, Trastuzumab, Ustekinumab, Denileukin diftitox, Rituximab, Anakinra, Tocilizumab, Aldesleukin, Advate®, Alferon N®, Aldurazyme®, Amevive®, Aranesp®, Avastin®, Benefix®, Botox®, Campath®, Elitek®, Enbrel®, Erbitux®, Fabrazyme®,

Flebogamma®, Gamine®, Genotropin®, Humate P®, Infergen®, Kogenate®, Kineret®, Leukine®, Myobloc®, Mylotarg®, Neumega®, Neulasta®, NovoSeven®, Oncospar®,

Orthoclone OKT3®, Pegasys®, Pulmozyme®, Raptiva® (efalizumab), Refacto®, Reppro® (abciximab), Rebif® (interferon beta- la), Retavase® (reteplase), Roferon-A® (interferon A), Santyl® (collagenase), Simulect® (basiliximab), TNKase® (tenecteplase), Xolair®

(omalizumab), Zemaira® (alpha- 1 proteinase inhibitor), Zenapax® (daclizumab), and Zevalin® (ibritumomab tiuxetan).

Other classes of useful agents for combination therapy include, for example, non-steroidal anti-inflammatory drugs (NSAIDS)(for example, selected from the following categories: (e.g., propionic acid derivatives, acetic acid derivatives, fenamic acid derivatives, biphenylcarboxylic acid derivatives and oxicams); steroidal anti-inflammatory drugs including hydrocortisone and the like; antihistaminic drugs (e.g., chlorpheniramine, triprolidine); antitussive drugs (e.g., dextromethorphan, codeine, carmiphen and carbetapentane); antipruritic drugs (e.g.,

methidilizine and trimeprizine); anticholinergic drugs (e.g., scopolamine, atropine, homatropine, levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine, meclizine, chlorpromazine, buclizine); anorexic drugs (e.g., benzphetamine, phentermine, chlorphentermine, fenfluramine); central stimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamine and methylphenidate); antiarrhythmic drugs (e.g., propanolol, procainamide, disopyraminde, quinidine, encainide); P-adrenergic blocker drugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol); cardiotonic drugs (e.g., milrinone, amrinone and dobutamine);

antihypertensive drugs (e.g., enalapril, clonidine, hydralazine, minoxidil, guanadrel,

guanethidine);diuretic drugs (e.g., amiloride and hydrochlorothiazide); vasodilator drugs (e.g., diltazem, amiodarone, isosuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs (e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs (e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine, bupivacaine, chlorprocaine, dibucaine);

antidepressant drugs (e.g., imipramine, desipramine, amitryptiline, nortryptiline); tranquilizer and sedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide, flurazapam, hydroxyzine, loxapine and promazine); antipsychotic drugs (e.g., chlorprothixene, fluphenazine, haloperidol, molindone, thioridazine and trifluoperazine); antimicrobial drugs (antibacterial, antifungal, antiprotozoal and antiviral drugs).

Antimicrobial drugs which are preferred for incorporation into the present combination therapies include, for example, pharmaceutically acceptable salts of β-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin, triclosan, doxycycline, capreomycin, chlorhexidine, chlortetracycline, oxytetracycline, clindamycin, ethambutol, hexamidine isothionate, metronidazole; pentamidine, gentamycin, kanamycin, lineomycin, methacycline, methenamine, minocycline, neomycin, netilmycin, paromomycin, streptomycin, tobramycin, miconazole, and amanfadine.

Antiviral drugs which are preferred for incorporation into the present combination therapies include, for example, Abacavir, Aciclovir, Acyclovir, Adefovir, Amantadine,

Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Boceprevir, Cidofovir, Combivir, Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine,

Enfuvirtide, Entecavir, Famciclovir, Fomivirsen, Fosamprenavir, Foscamet, Fosfonet,

Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Interferon type III, Interferon type II, Interferon type I, Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazone, Nelfmavir, Nevirapine, Nexavir, Oseltamivir (Tamiflu), Peginterferon alfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Raltegravir,

Ribavirin, Rimantadine, Ritonavir, Pyramidine, Saquinavir, Stavudine, Tea tree oil, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir

(Relenza) and Zidovudine.

Antifungal drugs which are preferred for incorporation into the present combination therapies include, for example, Nystatin, Amphotericin B, Griseofulvin, Miconazole,

Ketoconazole, Terbinafme, Itraconazole, Fluconazole, Posaconazole, and Voriconazole.

The combination therapy can also comprise use with hormones (e.g.,

medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide or somatostatin); muscle relaxant drugs (e.g., cinnamedrine, cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine, idaverine, ritodrine, dephenoxylate, dantrolene and azumolen); antispasmodic drugs; bone-active drugs (e.g., diphosphonate and phosphonoalkylphosphinate drug compounds);

endocrine modulating drugs (e.g., contraceptives (e.g., ethinodiol, ethinyl estradiol,

norethindrone, mestranol, desogestrel, medroxyprogesterone), modulators of diabetes (e.g., glyburide or chlorpropamide), anabolics, such as testolactone or stanozolol, androgens (e.g., methyltestosterone, testosterone or fluoxymesterone), antidiuretics (e.g., desmopressin) and calcitonins); estrogens (e.g., diethylstilbesterol), glucocorticoids (e.g., triamcinolone,

betamethasone, etc.) and progenstogens, such as norethindrone, ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g., liothyronine or levothyroxine) or anti-thyroid agents (e.g., methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormone suppressors (e.g., danazol or goserelin), oxytocics (e.g., methylergonovine or oxytocin) and prostaglandins, such as mioprostol, alprostadil or dinoprostone; immunomodulating drugs (e.g., antihistamines, mast cell stabilizers, such as lodoxamide and/or cromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone, dexamethasone, prednisolone, methylprednisolone, beclomethasone, or clobetasol), histamine H₂ antagonists (e.g., famotidine, cimetidine, ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc. Other drugs of use in conjunction with the present invention will be apparent to those of skill in the art. III. Drug Screening Applications

In some embodiments, the present invention provides drug screening assays {e.g., to screen for anticancer drugs). The screening methods of the present invention utilize MEK, ERK and/or AKT1/2 genes or proteins. For example, in some embodiments, the present invention provides methods of screening for compounds that alter {e.g., decrease) the expression of Mek, Erk and/or Aktl/2. The compounds or agents may interfere with transcription, by interacting, for example, with the promoter region. The compounds or agents may interfere with m NA produced from the Mek, Erk and/or Aktl/2 (e.g., by R A interference, antisense technologies, etc.). The compounds or agents may interfere with pathways that are upstream or downstream of the biological activity of the Mek, Erk and/or Aktl/2. In some embodiments, candidate compounds are antisense or interfering RNA agents (e.g. , oligonucleotides) directed against MEK, ERK and/or AKTI/2 gene expression. In other embodiments, candidate compounds are antibodies or small molecules that specifically bind to a MEK, ERK and/or AKTI/2 regulator or expression products of the present invention and inhibit its biological function.

In one screening method, candidate compounds are evaluated for their ability to alter

MEK gene expression by contacting a compound with a cell expressing the MEK, ERK and/or AKTI/2 genes and then assaying for the effect of the candidate compounds on expression. In some embodiments, the effect of candidate compounds on expression of MEK, ERK and/or AKTI/2 genes is assayed for by detecting the level of MEK, ERK and/or AKTI/2 mRNA expressed by the cell. mRNA expression can be detected by any suitable method.

In other embodiments, the effect of candidate compounds on expression of MEK, ERK and/or AKTI/2 gene expression is assayed by measuring the level of polypeptide encoded by the respective MEK, ERK and/or AKTI/2 gene. The level of polypeptide expressed can be measured using any suitable method, including but not limited to, those disclosed herein.

Specifically, the present invention provides screening methods for identifying

modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to MEK, ERK and/or AKTI/2, have an inhibitory (or stimulatory) effect on, for example, MEK, ERK and/or AKTI/2 gene expression or MEK, ERK and/or AKTI/2 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a MEK, ERK and/or AKTI/2 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., MEK, ERK and/or AKTI/2) either directly or indirectly in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

Compounds that inhibit the activity or expression of MEK, ERK and/or AKTI/2 genes or gene products are useful in the treatment of disorders, e.g., cancer, and immune disorders.

In one embodiment, the invention provides assays for screening candidate or test compounds that are substrates of a MEK, ERK and/or AKTI/2 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of a MEK, ER and/or AKT1/2 protein or polypeptide or a biologically active portion thereof.

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al, J. Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are preferred for use with peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90:6909 [1993]; Erb et al, Proc. Nad. Acad. Sci. USA 91 : 11422 [1994]; Zuckermann et al, J. Med. Chem. 37:2678 [1994]; Cho et al, Science 261 : 1303 [1993]; Carrell et al, Angew. Chem. Int. Ed. Engl. 33.2059 [1994]; Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061 [1994]; and Gallop et al, J. Med. Chem. 37: 1233

[1994].

Libraries of compounds may be presented in solution {e.g. , Houghten, Biotechniques

13:412-421 [1992]), or on beads (Lam, Nature 354:82-84 [1991]), chips (Fodor, Nature 364:555- 556 [1993]), bacteria or spores (U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids (Cull et al, Proc. Nad. Acad. Sci. USA 89: 18651869 [1992]) or on phage (Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406 [1990]; Cwirla et al, Proc. Natl. Acad. Sci. 87:6378-6382 [1990]; Felici, J. Mol. Biol. 222:301 [1991]).

EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example 1

Materials and Methods

Reagents Cyclosporin A (cat. no. 239835), PI-103 (cat. no. 528100), Akt Inhibitor VIII (cat. no 124017), Rapamycin (cat. no. 553211), and SB 203580 (cat. no. 559395) were purchased from Calbiochem, U0126 (cat. no. 9903) and wortmannin (cat. no. 9951) from Cell Signaling, PD0325901 (cat. no. 1408) from Axon Medchem. Antibodies used for T cell stimulation were: anti-CD3 (clone OKT3) custom produced from the hybridoma by Diatec, anti-CD28

(eBioscience, cat. no. 13-0289), and anti-CD2 (eBioscience, cat. no. 13-0029). Avidin (cat. no. 43-4401) was from Invitrogen, 7-amino-actinomycin D (7-AAD) from Becton Dickinson (BD, cat. no. 559925). Antibodies used to detect the phosphorylation of CD3ζ(Y142) (cat. no.

558489), LAT(Y171) (cat. no. 558518), MEK1(S298) (cat. no. 560043), NF-κΒ p65(S529) (cat. no. 558422), SLP76(Y128) (cat. no. 558438), STAT3(Y705) (cat. no. 557815), Rb(S807/S811) (cat. no. 558590), ZAP70/Syk(Y319/Y352) (cat. no. 557817), and the isotype control IgGl Kappa (cat. no. 557783) were from BD. Antibodies used to detect the phosphorylation of Akt/PKB(S473) (cat. no. 4075), histone H3(S10) (cat. no. 9716), NF-κΒ p65(S536) (cat. no. 4887), MAPKAPK-2(T334) (cat. no. 4320), S6-Ribosomal protein(S235/236) (cat. no. 4851), tyrosine (T100) (cat. no. 9415), 44/42 MAPK(T202/Y204) (cat. no. 4375), and p38

MAPK(T180/Y182) (cat. no. 4552) were from Cell Signaling Technology. Antibody used to detect the phosphorylation of ATF-2(T71) (cat. no. sc-8398) was from Santa Cruz. Furthermore, antibodies used to detect the expression of CD3 (cat. no. 345766), CD4 (cat. no. 348809 and 557922), CD25 (cat. no. 557741), CD45RA (cat. no. 555489), CD45RO (cat. no. 555493), and FoxP3 (cat. no. 560047 and 560045) were from BD, while the antibody used to detect the expression of CD8 (cat. no. 9536-09) was from Southern Biotech.

T cell purification, stimulation and fixation

Buffy coats were obtained from healthy blood donors (Oslo University Hospital Blood Centre, Oslo, Norway; studies approved by the Regional Ethics Review Board). T cells, either CD3⁺ or CD4⁺, were purified by negative selection from buffy coats using RosetteSep™

Enrichment kits (StemCell Technologies) according to the manufacturer's instructions. Unless otherwise stated, purified T cells were resuspended in RPMI 1640 GlutaMAX™ (Gibco®) with 1% fetal calf serum (FCS). Prior to stimulation, cells were pre-equlibrated at 37°C for 5 min. Thereafter, biotinylated antibodies were added [different concentrations of anti-CD3 alone (range 1 ng/ml - 10 μg/ml), or a fixed concentration of anti-CD3 (1 μg/ml) alone or together with either anti-CD28 (5 μg/ml), anti-CD2 (5 μg/ml) or anti-CD28 and anti-CD2 combined (both 5 μg/ml)]. Two min later, avidin (50 μg/ml) was added to allow cross-linking, and incubations were continued for different time periods. In some experiments (specifically stated in the figure legends), cells were stimulated using a different protocol that included incubation on ice with biotinylated antibodies for 30 min, one wash, then addition of pre-warmed avidin and incubation at 37°C for different time periods. All harvested samples were fixed immediately using pre- warmed BD Phosflow Fix Buffer I (BD Biosciences) for 10 min at 37°C, followed by

centrifugation (830 x g, 4°C, 5 min), one wash with flow washing solution (PBS containing 1% FCS and 0.09% sodium azide) and one wash with PBS.

Fluorescent cell barcoding (FCB)

Three-dimensional FCB was carried out as previously described¹'². In brief, fixed cells were incubated with varying concentrations of esters conjugated to Pacific Blue™ (100, 25, 6.3, 0.7 pg/μΐ; Molecular Probes, Invitrogen), Pacific Orange™ (1000, 250, 41.7, 4.2 pg/μΐ;

Molecular Probes, Invitrogen), and Alexa Fluor® 488 (50, 12.5, 3.1, 0.3 pg/μΐ; Molecular Probes, Invitrogen) for 20 min at room temperature. Each sample was incubated with a unique combination of dye concentrations, allowing identification of each sample based on its color coding combination. Following FCB, all samples were centrifuged and separately washed once in PBS with 3% FCS. Later, all samples were combined and washed once with flow washing solution, permeabilized (-20°C cold BD Phosflow Perm Buffer III, BD Biosciences) and stored at -80°C (henceforth called FCB cell stock). Staining of samples and analysis by phospho flow cytometry

FCB cell stocks were rehydrated with PBS and washed once in flow washing solution. Aliquots of FCB cells were then incubated with different combinations of Alexa Fluor 647 (Ax647) conjugated phospho-epitope specific antibodies and fluorescently labeled cell surface- marker antibodies (30 min at room temperature), washed twice with flow washing solution, and made ready for flow cytometric analysis by resuspension in flow washing solution. FoxP3 staining, when included, was performed on FCB cell stocks prior to permabilization and storage at -80°C, using a FoxP3 staining kit from BD Pharmingen (cat. no. 560098). Finally, all samples were analyzed using a BD FACSCanto™ II (4-2-2) cytometer equipped with 405, 488, and 633 nm lasers. For each sample, at least 200,000 events were recorded (corresponding to >3000 events per square in the heatmaps for prevalent cell populations and >300 events for less prevalent populations). For fluorochrome compensation, PerCP-conjugated CD3 antibody, PE- Cy⁷-conjugated CD4 antibody, and PE-conjugated CD8, CD45RA or CD45RO antibody staining was used on unstimulated, non-FCB cells. In order to compensate for the phospho-epitope specific antibodies conjugated to Ax647, Ax647-conjugated CD3 antibody was used. The data analysis program Cytobank and FlowJo 8.8.2 (TreeStar, Ashland, OR) were used for further analysis and visualization of data.

Sorting of CD4⁺ T cell subsets

Purified CD4⁺ T cells in PBS with 2% FCS were incubated with fluorescently labeled antibodies (anti-CD45RA-PE and anti-CD25-PE-Cy⁷, anti-CD4-Ax700 was also used unless subsequent suppression assays were to be performed) for 30 min on ice. Thereafter, cells were washed once in PBS with 2% FCS, resuspended in PBS with 2% FCS, and sorted on a BD FACSAria™ IIu cytometer (5-2) equipped with 488 and 633 nm lasers and set up with a sheath pressure of 70 psi and a 70-μιη nozzle.

In vitro stimulation of resting regulatory T cells

Resting regulatory T cells (rTregs) defined as CD4⁺CD45RA⁺CD25 T cells were sorted as described. Sorted cells were resuspended at 1 ^χ 10⁶ cells/ml in complete medium (RPMI 1640 containing 10% FCS, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 1 mM sodium pyruvate, and non-essential amino acids), incubated with or without specific inhibitors of PI3K (0.5 μΜ of PI-103 in combination with 0.1 μΜ of wortmannin), Akt (either 1 μΜ of Aktl/2-inhibitor or 10 nM of the mTOR inhibitor Rapamycin), Mek (either 1 μΜ of PD0325901 or 10 μΜ of U0126), p38 (5 μΜ of SB 203580) or NFAT (0.1 μΜ of the Calcineurin-inhibitor Cyclosporin A, CsA) pathways for 20 min at 37°C, followed by stimulation with aCD3/CD28/CD2-coated

MicroBeads (Miltenyi Biotec; bead-to-cell ratio of 1 :5, this beadxell ratio was used in all bead- based experiments throughout the paper) for different time periods. One sample was left unstimulated as a reference. Unstimulated, sorted activated Tregs (aTregs, defined as

CD4⁺CD45RA^"CD25⁺⁺ T cells) and naive conventional T cells (defined as CD4⁺CD45RA⁺CD25^" T cells) were included as controls. After stimulation for the intended time periods, samples were washed once in flow washing solution before staining with 7-AAD. Later, cells were fixed with Buffer A from a FoxP3 staining kit (BD Pharmingen™) according to the manufacturer's protocol and stored at -80°C. Subsequently, all samples were stained for FoxP3 and surface markers, and analyzed as described above.

Suppression assay

Sorted rTregs were either added directly into a suppression assay, or pre-treated (30 min at 37°C) or not with specific inhibitors of PI3K, Akt, Mek, p38 and NFAT pathways, followed by 36 hours of culture in complete medium alone or in presence of aCD3/CD28/CD2-coated MicroBeads. After two rounds of washing, cells were mixed with CFSE-stained, purified CD4 T cells (called responder cells) at a 1 : 1 ratio and stimulated with aCD3/CD28/CD2-coated

MicroBeads for 84 hours. Cells were then stained with 7-AAD and subjected to flow cytometric analysis using a BD FACSCanto™ II. The suppressive capacity of different rTreg populations was determined by level of CFSE dilution in responder cells using the Flow Jo™ 8.8.2 software.

Processing of data and statistical analysis

Changes in phosphorylation of signaling proteins following activation of T cells were calculated using the inverse hyperbolic sine (arcsinh) of the median fluorescence intensity (MFI) of stimulated versus unstimulated cell populations. The reason for choosing arcsinh for calculating changes are explained by Irish et al.³. Comparison of the effects of pre-incubation of rTregs with different specific inhibitors on FoxP3 expression was analyzed using one-way analysis of variance (ANOVA) specifying unstimulated rTregs as control group. Comparisons of the effects of specific inhibitors on the suppressive capacity of rTregs were also analyzed using one-way ANOVA, where stimulated rTregs pre -incubated in the absence of an inhibitor was used as the control group. Differences in mean values were considered statistically significant when p < 0.05. The statistical analyses were conducted in SigmaPlot^® 11.2 (Systat Software).

Results

High-throughput analysis of T cell signaling profiles. Our experimental setup is outlined in Figure 1 A. After subjecting cells to different stimulatory conditions and fixation with formaldehyde, cells from each stimulatory condition are stained with a unique combination of FCB reagents, and can therefore be tracked from all other sample populations in subsequent assays. Thus, FCB allows for combining all cell samples prior to staining with fluorescently labeled antibodies against intracellular phospho-epitopes and cell surface markers, thereby analyzing all samples with the same baseline, minimizing intra-assay variability and allowing for high-throughput analysis³³.

In order to define the sensitivity of the experimental system, initial assays involved incubation of purified CD3⁺ human T cells (95 ± 3% purity, see Figure 7A) with different concentrations of anti-CD3 antibody (range from 1 ng/ml to 10 μg/ml) on ice, followed by one round of washing, and then cross-linking with avidin at 37°C for up to 60 min. Figure IB shows the subsequent analysis of these cells with regard to the phosphorylation status of five selected signaling intermediates downstream of the TCR (for 13 additional signaling parameters and data from more individuals, see Figures 8-10). As evident, TCR proximal signaling molecules (such as ζ- chain, Zap-70, LAT and Slp-76) were considerably activated/phosphorylated only at anti-CD3 concentrations of 1 μ /ηι1 or higher, and then peaked after 1-3 min (Figure IB). In accordance with the principle of signal cascade amplification, signaling mediators located more downstream (e.g. Erk, p38, NF-κΒ and S6-Rp) were activated at lower levels of stimulation and with delayed kinetics compared to TCR proximal signaling molecules (Figure IB and Figures 8-10). These observations were apparent for both CD4⁺ and CD8⁺ T cells. Moreover, combination of data from three independent donors revealed a consistent pattern, testifying to the robustness of the method (Figure 1C).

Alternatively, purified CD3⁺ T cells were pre-equilibrated at 37°C prior to addition of anti-CD3 antibody, followed 2 min later by cross-linking with avidin and continued incubation for up to 60 min (Figures 11-13). When analyzing the whole data set on signaling responses and comparing data obtained from cells incubated on ice to cells only treated at 37°C, distinct differences were observed (Figures 11-13). Phosphorylation of TCR-proximal molecules (such as ζ-chain and Zap-70) appeared stronger in cells that had been incubated on ice compared to cells only treated at 37°C. Phosphorylation of TCR-distal molecules such as Mekl, p38, and NF-KB were elevated at time zero following incubation on ice, indicating a direct effect of the temperature changes on several signaling processes. As a result of this, and to optimize signal-to- noise across the full panel of signal markers, all further experiments were conducted on cells that had been pre-equilibrated at 37°C.

Comparison of T cell signaling profiles for different naive and effector/memory T cell subsets. Having established a well-working phospho-epitope specific flow cytometry protocol for a set of markers across relevant signal pathways in T cell activation, we next wanted to dissect signaling profiles in different T cell subsets. Three different stimulatory conditions were used: i) cross-linking of anti-CD3 alone, ii) cross-linking of anti-CD3/anti-CD28, and iii) cross-linking of anti-CD3/anti-CD28/anti-CD2. For all these experiments, a sub-optimal concentration of anti-CD3 (1 μg/ml) was used in order to capture effects of the different co- stimulatory conditions. In addition to the panel of 18 phospho-specific antibodies described above, staining for CD45RO was also included so that na^'ive T cells could be discerned from effector/memory T cells (CD45RO^" and CD45RO⁺, respectively). Due to the limited number of channels in the FACS analysis, CD 8 staining had to be omitted in this setup. Still, more than 90% of CD3 CD4^" peripheral T cells were CD3 CD8 T cells, indicating that CD4 negativity within the population of peripheral T cells was a good surrogate marker for CD8 positivity (Figure 7B). In the following, CD3⁺CD4^~CD45RO^~ and CD3⁺CD4^~CD45RO⁺ T cells are referred to as naive and effector/memory CD8 T cells, respectively, while CD3 CD4 CD45RO^" and CD3 CD4 CD45RO T cells denote naive and effector/memory CD4 T cells. The signaling in each subset was analyzed initially relative to the control sample for the same subset (Figure 2A). Compared to analysis of the entire populations of CD4⁺ and CD8⁺ peripheral T cells, the addition of CD45RO-based sub-gating of CD4⁺ and CD8⁺ T cells revealed differences in signaling responses. When co-stimulation was not present, the amplitude of the signaling responses seemed to be stronger in naive cells compared to effector/memory cells. This was particularly evident when looking at Mekl and Erk, both of which demonstrated a strong signaling response in naive CD4⁺ T cells, but less so in memory cells. The addition of CD28 co-stimulation only had a modest effect on the signaling amplitudes in all four subsets, and then preferentially on downstream mediators such as S6-Rp. In contrast, combined co-stimulation of CD28 and CD2 consistently gave high signaling amplitudes for all signaling molecules tested in all the subsets (Figure 2A).

Next, signaling responses in all subsets where analyzed using unstimulated na^'ive CD4⁺ T cells as a reference (Figure 2B and C). With such an approach, several features were observed. First, compared to CD3 stimulation alone, the addition of CD28 co-stimulation resulted in higher signaling responses in na^'ive CD4⁺ T cells, even at the level of TCR-proximal molecules such as ζ-chain and Zap-70. Second, all four subsets displayed higher signaling responses with two over one co-stimulatory factor present, and with an especially strong effect of CD2 co-stimulation. Third, while phosphorylation of TCR-proximal signaling molecules (such as ζ-chain, Zap-70 and Slp-76) generally was stronger in na^'ive than effector/memory T cells (both CD4⁺ and CD8⁺ cells), the opposite was the case for more downstream mediators (e.g. Erk and S6-Rp). Fourth, in na^'ive T cells (both CD4⁺ and CD 8 ) phosphorylation of Mekl peaked after 1 min of stimulation and subsequently displayed a second wave of activation in the presence of CD2 co-stimulation. This indicated involvement of a positive feedback loop as a result of co-receptor signaling. The signaling patterns of Erk did not relate directly to their upstream activator Mekl . Finally, the relatively high basal phosphorylation of S6-Rp in effector/memory subsets indicated an activated transcription and translation program, as expected for effector cell populations (Figure 2C). It was also observed that addition of CD2 co-stimulation produced a reproducible increase in S6- Rp phosphorylation in all the subsets irrespective of basal status, supporting a role for CD2 induced signals in the regulation of translation at the level of S6-Rp³⁷.

Analysis of signaling in conventional and regulatory CD4⁺ T cell subsets revealed elevated Erk activation and lowered Akt activation in aTregs. As previously described for humans³²'³⁸, CD4 Tregs can be divided into two functionally distinct subsets based on CD45RA and FoxP3 expression: CD4⁺CD45RA⁺FoxP3⁺ rTregs and CD4⁺CD45RA^~FoxP3⁺⁺ aTregs (Figure 3A). It was contemplated that this functional delineation would be reflected in signal transduction processes and used the established phospho-specific flow cytometry protocol to investigate signaling in these subsets. For global overview purposes, the analyses also included na^'ive

(CD45RA⁺FoxP3^~) and effector/memory (CD45RA^~FoxP3^~) CD4⁺ conventional T cells as well as the CD4 CD45RA^~FoxP3⁺ effector T cell subset with cytokine secreting ability³².

As shown in Figure 3B and D, phosphorylation levels of ζ-chain (as well as other TCR- proximal signaling molecules, Figures 14-16) were comparable between aTregs, rTregs and na^'ive CD4⁺ conventional T cells, both in response to CD3 stimulation and when different types of co-stimulation were added. The same observation was made for rTregs and na^'ive CD4⁺ conventional T cells with regard to Mekl phosphorylation, while the signals for aTregs were weaker and comparable to the ones seen for effector/memory CD4⁺ conventional T cells. Levels of Erk activation in aTregs were very high, both in unstimulated cells and after CD3 stimulation with or without co-stimulation. For all other CD4⁺ T cell subsets tested, robust Erk activation was observed in response to stimulation, but the signals were always significantly weaker than those in aTregs. aTregs also differed from most other subsets with regard to Akt signaling (Figure 3C and D), which was almost absent in aTregs, even in presence of the strongest stimulus tested (combined cross-linking of CD3/CD28/CD2). In contrast, CD4⁺ conventional T cells as well as rTregs displayed potent Akt activation, especially in the presence of co-stimulation. Again, all subsets significantly increased the levels of S6-Rp phosphorylation when CD2 co- stimulation was added, even when the basal activity was elevated, as seen for CD4⁺

effector/memory T cells. The basal activities and patterns of responses were reproducible in all three donors tested (Figure 3D and E, and Figures 14-16).

CD28 and CD2 co-stimulation trigger overlapping but distinct signaling pathways. In order to delineate differences between CD28 and CD2 in co-stimulation, we examined more carefully the relative contribution of CD2 and CD28 by cross-linking with CD3 either alone or combined. Co-stimulation with CD28 or CD2 separately increased the amplitude of proximal TCR signaling events at the level of ζ-chain- and Slp-76 phosphorylation compared to CD3 stimulation alone (Figures 4A and B). Similar responses were seen for phosphorylation of Zap-70, Mekl and histone 3 (Figures 17-19). Additive effects of CD28 and CD2 were generally observed (as also seen in Figures 3B, C and D). At the level of Erk activation, however, the addition of CD28 and/or CD2, compared to CD3 alone, had only modest effect on the signaling amplitudes in the different subsets tested, indicating that Erk phosphorylation depends mainly on the TCR signal (Figures 4 A and B). The phosphorylation responses of Akt and NF-κΒ depended mainly on CD28, indicating that the CD28 signal is involved in activation of an Akt-NF-κΒ pathway (Figure 4B and Figures 17-19). In contrast, CD2 appeared to give stronger activation of S6-Rp compared to CD28, especially in conventional memory cells and aTregs. A clear additive effect of the activation of S6-Rp was observed when both co-stimulators were present.

Mek-Erk dependent up regulation of FoxP3 in rTregs. rTregs most likely represent a thymus- derived population that upon stimulation can expand and mature both in vitro and in vivo to become aTregs, which are characterized by enhanced suppressive capabilities ³². Key events in this maturational process are increased FoxP3 expression and proliferation. Given the significant differences between rTregs and aTregs with respect to activation of Akt and Erk (as described in Figure 3), we aimed to address the importance of different signaling pathways in the transition of rTregs to aTregs. In order to first assess the induction of FoxP3 protein in rTregs upon activation, CD4⁺CD25⁺CD45RA⁺ T cells were sorted and stimulated with aCD3/CD28/CD2-coated Micro- beads in vitro for up to 92 hours, followed by flow cytometry analysis of FoxP3 levels (denoted stimulated rTregs). As seen in Figure 5A and B, FoxP3 expression in rTregs increased markedly in a time-dependent manner in response to stimulation. Peak levels, which were reached after 36- 44 hours, even exceeded the FoxP3 levels observed for aTregs isolated directly from blood (Figure 5A). At later time points, FoxP3 expression in stimulated rTregs dropped, indicating that transiently high expression of FoxP3 was necessary to drive the transcriptional program necessary for the maturation of these cells.

It was next tested to what extent inhibition of the Mek-Erk or Akt signaling pathways affected the stimulation-induced up-regulation of FoxP3 in rTregs. To do so, sorted rTregs were incubated with a panel of inhibitors prior to stimulation for 36 hours as shown in Figure 5C. Mek inhibitors (PD 0325901 and U0126) that would prevent activation of Erk potently inhibited the stimulation-induced up-regulation of FoxP3 (>90%), while pre-treatment with inhibitors against PI3K (PI- 103 in combination with wortmannin), mTOR (Rapamycin), Akt (Aktl/2-inhibitor) or Calcineurin (CsA) reduced the FoxP3 induction by approximately 50%. These effects were consistent between several donors (Figure 5D), indicating a role for Mek-related signaling in activation-induced up-regulation of FoxP3 in rTregs. In comparison, inhibition of p38 (SB 203580) had no significant effect. Additional inhibitors are shown in Table 1 below. Induction of rTreg suppressive capacity is Mek-dependent. We next tested the functional consequence of blocking the activation-induced up-regulation of FoxP3 in rTregs. In these assays, Treg function was defined as ability to suppress the proliferation of CFSE-labeled purified CD4⁺ T cells. Notably, no suppressive function could be demonstrated for sorted rTregs in these assays (Figure 6 A and B). However, when rTregs were stimulated for 36 hours (to assure proper FoxP3 up-regulation) before initiation of the CFSE assay, significant suppression was observed. Finally, incubation of rTregs with a Mek inhibitor prior to stimulation and subsequent initiation of the CFSE assay completely blocked the ability of these cells to become suppressive. In comparison, inhibition of Akt resulted in 50% reduction in suppressive capacity. These results were consistent among all donors tested (Figure 6B) and indicate that the degree to which these inhibitors prevent FoxP3 up-regulation defines their potency to restrain induction of suppressive function in rTregs.

Table 1

PD325901 Pfizer allosteric MEK inhibitor, analog of CI- 1040

GSK2118436 GlaxoSmithKline MEK inhibitor

ARRY-300 Novartis /Array Biopharma MEK inhibitor

MK-2206 Merck Akt inhibitor

GSK2110183 GlaxoSmithKline Akt inhibitor

GSK690693 GlaxoSmithKline Akt inhibitor

GSK2141795 GlaxoSmithKline Akt inhibitor

GDC-0068 Genentech Akt inhibitor

VQD-002 VioQuest Akt inhibitor

PBI-05204 Phoenix Biotechnology Akt inhibitor

Perifosine Aeterna Zentaris Akt inhibitor

EXAMPLE 2

This example describes the effect of different MEK inhibitors on FoxP3 upregulation upon stimulation of rTregs. Sorted resting Tregs were incubated with different concentrations of the indicated MEK inhibitors, followed by stimulation with CD3/CD28/CD2-coated MicroBeads for 36h, followed by FoxP3 staining and FACS analysis as described above in Example 1. The results are presented in Figure 21.

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Claims

CLAIMS We claim:

1. A method of inhibiting excessive immunosuppressive activity in a cell, comprising:

contacting said cell with an inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof,

wherein said contacting results in inhibition of activation of resting human regulatory T cells (rTregs) to active Tregs (aTregs).

2. The method of claim 1, wherein said MEK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

3. The method of claim 2, wherein said small molecule drug is selected from the group consisting of AS703026, AZD6244 (Selumetinib), AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD184352), PD0325901, PD318088, PD98059, and U0126-EtOH, GSKl 120212, FR180204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, GDC-0973 (XL518).

4. The method of claim 1, wherein said AKT1/2 inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

5. The method of claim 4, wherein said small molecule drug is selected from the group consisting of A6730, B2311 and 124018, GSK2141795, MK2206, GSK2110183,

GSK690693, Perifosine (KRX-0401), GDC-0068, RX-0201, VQD-002.

6. The method of claim 1, wherein said ERK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

7. The method of claim 6, wherein said small molecule drug is selected from the group consisting of PD98059, U0126, FR180204 , 3-(2-Aminoethyl)-5-((4- ethoxyphenyl)methylene)-2,4-thiazolidinedione, and pyrazolylpyrrole ER Inhibitor.

8. The method of claim 1, wherein said cell is in vitro or ex vivo.

9. The method of claim 1, wherein said cell is in an animal.

10. The method of claim 9, wherein said animal is a human or a non-human mammal.

11. The method of claim 9, wherein said animal exhibits symptoms of a disease characterized by inappropriate or excessive immunosuppressive activity.

12. The method of claim 9, wherein said disease is selected from the group consisting of a chronic inflammatory disease, a chronic infectious disease and cancer.

13. The method of claim 1, wherein said inhibitor is coadministered with at least one additional therapeutic agent.

14. The method of claim 13, wherein said at least one additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an antiviral agent, an

antibacterial agent, and a stem cell agent.

15. The method of claim 13, wherein said at least one additional therapeutic agent is selected from the group consisting of a biologic therapeutic agent, a small molecule therapeutic agent, an R A-based therapeutic agent.

16. Use of an inhibitor selected from the group consisting of MEK, ERK, and AKTl/2 inhibitors and combinations thereof to inhibit excessive immunosuppressive activity in a cell.

17. Use of claim 16, wherein MEK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

18. Use of claim 17, wherein said small molecule drug is selected from the group consisting of AS703026, AZD6244 (Selumetinib), AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD184352), PD0325901, PD318088, PD98059, and U0126-EtOH, GSKl 120212, FRl 80204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, GDC-0973 (XL518).

19. Use of claim 16, wherein said AKTl/2 inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

20. Use of claim 19, wherein said small molecule drug is selected from the group consisting of A6730, B2311, 124018, GSK2141795, MK2206, GSK2110183, GSK690693, Perifosine (KRX-0401), GDC-0068, RX-0201, VQD-002 .

21. Use of Claim 26, wherein said ERK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

22. Use of Claim 21, wherein said small molecule drug is selected from the group consisting of PD98059, U0126, FR180204 , 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)- 2,4-thiazolidinedione, and pyrazolylpyrrole ERK Inhibitor.

23. Use of claims 16 to 22, wherein said inhibition of excessive immunosuppressive activity is desired in a subject exhibiting symptoms of a disease characterized by excessive immunosuppressive activity.

24. Use of claims 16 to 22, wherein said inhibition of excessive immunosuppressive activity is utilized for treatment of a disease selected from the group consisting of a chronic inflammatory disease, a chronic infectious disease and cancer.

25. Use of claims 16 to 24, wherein said inhibitor selected from the group consisting of MEK, ERK, and AKTl/2 inhibitors and combinations thereof is coadministered with at least one additional therapeutic agent.

26. Use of claim 25, wherein said at least one additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an antiviral agent, an antibacterial agent, and a stem cell agent.

27. Use of claim 25, wherein said at least one additional therapeutic agent is selected from the group consisting of a biologic therapeutic agent, a small molecule therapeutic agent, and an RNA-based therapeutic agent.

28. Use of an inhibitor selected from the group consisting of MEK, ER , and

AKT1/2 inhibitors and combinations thereof to treat a chronic infectious disease in a subject.

29. Use of claim 28, wherein said chronic infectious disease is selected from the group consisting of Hepatitis B, Hepatitis C, tuberculosis, and HIV infection.

30. Use of claims 28 or 29, wherein said MEK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

31. Use of claim 30, wherein said small molecule drug is selected from the group consisting of AS703026, AZD6244 (Selumetinib), AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD184352), PD0325901, PD318088, PD98059, and U0126-EtOH, GSKl 120212, FRl 80204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, GDC-0973 (XL518).

32. Use of claim 28 or 29, wherein said AKT1/2 inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

33. Use of claim 32, wherein said small molecule drug is selected from the group consisting of A6730, B2311 and 124018, GSK2141795, MK2206, GSK2110183, GSK690693, Perifosine (KRX-0401), GDC-0068, RX-0201, VQD-002.

34. Use of claim 28 or 29, wherein said ERK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

35. Use of Claim 34, wherein said small molecule drug is selected from the group consisting of PD98059, U0126, FR180204 , 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)- 2,4-thiazolidinedione, and pyrazolylpyrrole ERK Inhibitor.

36. Use of claims 28 to 35 wherein said inhibitor selected from the group consisting of MEK, ERK, and AKTl/2 inhibitors and combinations thereof is coadministered with at least one additional therapeutic agent.

37. Use of claim 36, wherein said at least one additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an antiviral agent, an antibacterial agent, and a stem cell agent.

38. Use of claim 36, wherein said at least one additional therapeutic agent is selected from the group consisting of a biologic therapeutic agent, a small molecule therapeutic agent, and an RNA-based therapeutic agent.

39. A method of treating a disease a patient suffering from a condition associated with inappropriate excessive immunosuppressive activity in a cell, comprising:

coadministering to said patient a first therapeutic agent and a second therapeutic agent that is an inhibitor selected from the group consisting of MEK, ERK, and AKTl/2 inhibitors and combinations thereof, wherein said administration of said second therapeutic agent results in inhibition of activation of resting human regulatory T cells (rTregs) to active Tregs (aTregs).

40. The method of claim 39, wherein said first therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an antiviral agent, an antibacterial agent, and a stem cell agent.

41. The method of claim 39, wherein said first therapeutic agent is selected from the group consisting of a biologic therapeutic agent, a small molecule therapeutic agent, and an RNA-based therapeutic agent.

42. The method of claim 39, wherein said MEK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

43. The method of claim 42, wherein said small molecule drug is selected from the group consisting of AS703026, AZD6244 (Selumetinib), AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD184352), PD0325901, PD318088, PD98059, and U0126-EtOH, GSK1120212, FR180204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, GDC-0973 (XL518).

44. The method of claim 39, wherein said AKT1/2 inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

45. The method of claim 44, wherein said small molecule drug is selected from the group consisting of A6730, B2311 and 124018, GSK2141795, MK2206, GSK2110183,

GSK690693, Perifosine (KRX-0401), GDC-0068, RX-0201, VQD-002.

46. The method of claim 39, wherein said ER inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

47. The method of claim 46, wherein said small molecule drug is selected from the group consisting of PD98059, U0126, FR180204 , 3-(2-Aminoethyl)-5-((4- ethoxyphenyl)methylene)-2,4-thiazolidinedione, and pyrazolylpyrrole ERK Inhibitor.

48. A pharmaceutical composition comprising a first therapeutic agent and a second therapeutic agent that is an inhibitor selected from the group consisting of MEK, ERK, and AKT1/2 inhibitors and combinations thereof, wherein said administration of said second therapeutic agent results in inhibition of activation of resting human regulatory T cells (rTregs) to active Tregs (aTregs).

49. The composition of claim 48, wherein said first therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an antiviral agent, an antibacterial agent, and a stem cell agent.

50. The composition of claim 48, wherein said first therapeutic agent is selected from the group consisting of a biologic therapeutic agent, a small molecule therapeutic agent, and an RNA-based therapeutic agent.

51. The composition of claim 48, wherein said MEK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

52. The composition of claim 51, wherein said small molecule drug is selected from the group consisting of AS703026, AZD6244 (Selumetinib), AZD8330(ARRY-424704), BIX 02188, BIX 02189, BMS 777607, BMS 777607, CI-1040 (PD184352), PD0325901, PD318088, PD98059, and U0126-EtOH, GSKl 120212, FR180204, MEK162, BAY86-9766 (RDEA 119), R05126766, R04987655, GDC-0973 (XL518).

53. The composition of claim 48, wherein said AKT1/2 inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

54. The composition of claim 53, wherein said small molecule drug is selected from the group consisting of A6730, B2311 and 124018, GSK2141795, MK2206, GSK2110183, GSK690693, Perifosine (KRX-0401), GDC-0068, RX-0201, VQD-002.

55. The composition of claim 48, wherein said ERK inhibitor is selected from the group consisting of an siRNA, an antisense oligonucleotide, an antibody and a small molecule drug.

56. The composition of claim 55, wherein said small molecule drug is selected from the group consisting of PD98059, U0126, FR180204 , 3-(2-Aminoethyl)-5-((4- ethoxyphenyl)methylene)-2,4-thiazolidinedione, and pyrazolylpyrrole ERK Inhibitor.