WO2013151490A1 - Methods and compounds for treating diseases - Google Patents

Abstract

The present invention provides a method for amplifying an immune response in an individual, comprising altering the composition of FoxP3 isoforms by inducing an expression of a FoxP3 isoform, such as FoxP3d2d7, that inhibits the function of FoxP3fl and/or FoxP3d2, and a method for treating a cancer or an infection or for inducing an increased vaccine response in an individual, comprising amplifying an immune response in said individual, compounds for use in such methods, pharmaceutical compositions, and a method for in vitro testing of a candidate medicinal product. The invention further relates to a method for diagnosing inflammatory bowel disease (IBD) in an individual, and use of FoxP3d2d7 as a diagnostic marker for IBD.

Description

METHODS AND COMPOUNDS FOR TREATING DISEASES

FIELD OF THE INVENTION

The present invention relates to a method for amplifying an immune response in an individual, a method for treating a cancer or an infection or for inducing an increased vaccine response in an individual, and further relates to compounds and pharmaceutical compositions for use in such methods. More particularly, it relates to a method comprising the alteration of the composition of FoxP3 isoforms in regulatory T cells. BACKGROUND

The immune system is the body's defense against infectious organisms. It is delicately regulated to allow responses against foreign antigens but not self-antigens. The process by which the immune system does not react against self-antigens is known as immunological tolerance. Several distinct mechanisms uphold immunological tolerance, including deletion of highly self-reactive lymphocytes during their development, lymphocyte hypo-responsiveness when antigen is encountered in the absence of co-stimulatory signals and suppression of immune responses by cells with regulatory capacity. Suppression of immune responses is beneficial in patients suffering from autoimmune diseases, asthma and allergies, or that have undergone transplantations. In contrast, it is beneficial to override tolerogenic mechanisms during vaccination, infections and cancers.

CD4+FoxP3+ regulatory T (TReg) cells are characterized by expression of the transcription factor FoxP3 [1-3] and in most cases by expression of CD25 [4], the IL-2 receptor a-subunit. The importance of FoxP3 and TReg cells is best illustrated by the development of fatal autoimmune disease in humans and mice with genetic deficiencies of FoxP3 [5-7]. FoxP3 belongs to the forkhead/winged-helix family of transcription factors. The human FoxP3 gene contains 11 coding exons and is located on the X chromosome. Human TReg cells express three different FoxP3 isoforms including full-length FoxP3 (FoxP3fl), FoxP3 that lacks exon 2 (FoxP3d2) and FoxP3 that lacks exon 2 and 7 (FoxP3d2d7). FoxP3fl and FoxP3d2 are the main forms expressed by human TReg cells and they confer an immunosuppressive phenotype to TReg cells whereas FoxP3d2d7 is counter-suppressive and inhibits the function of FoxP3fl and FoxP3d2. FoxP3d2d7 is usually expressed at very low levels in natural TReg cells, and up until now it has been suggested that it acts in tumor cells rather than in TReg cells [8] . Expression of FoxP3 in non-T cells is a very controversial subject. There have been a few studies suggesting FoxP3 expression in macrophages [9, withdrawn due to irreproducible results], in epithelial cells [10, which has been fiercely rebuffed by other research groups], [11], and in tumor cells [12]. The potential expression of FoxP3 in tumor cells has led to an interest in determining whether this could be exploited for diagnostic and therapeutic purposes. Induction of FoxP3 in tumor cells appears to result in growth inhibition and apoptosis, and has been suggested to be a viable anti-tumor treatment [13]. If this is to be used in the clinics, however, it will be crucial to only deliver or induce FoxP3 in tumor cells rather than in T cells, as it otherwise could result in increased TReg cell numbers and impaired antitumor responses. It has also been suggested that tumors can express novel isoforms of FoxP3 and that these potentially could be targeted by siRNA knockdown approaches or vaccine regimens without affecting the function of TReg cells [14, 15].

Deficiency in TReg cell function has also been suggested as an underlying cause for disease conditions ranging from autoimmune diseases to infectious diseases in several studies [8]. Many of these studies remain controversial; however, it is well established that manipulation of the number of TReg cells through cell transfers or deletion has dramatic effects on a variety of diseases. Addition of TReg cells through cell transfers can cure ongoing autoimmune disease [16-17] while deletion of TReg cells results in more severe disease [18]. In a similar manner addition of TReg cells promotes graft acceptance [19] and ameliorates allergic disease [20] while deletion results in improved vaccine responses [21-22], tumor responses [23] and increases atherosclerotic disease [24]. TReg cells also modulate immune responses to pathogens by limiting protective responses [25], which in some cases result in chronic infections necessary for maintenance of protective immunity [26]. TReg cells can also limit infection-associated immune-mediated pathology [27].

The observation that TReg cells inhibit protective anti-cancer and anti-pathogen responses has led to intense research in how to cancel out their immunosuppressive effect. Most strategies described up until today, include simply killing the TReg cells with antibodies or with antibodies coupled to toxins [reviewed in 28]. Global deletion of TReg cells is however inefficient as TReg cells have an enormous capability to self-renew and even very efficient depletion of the TReg is temporary. Repeated depletion is also inefficient as the immune system starts recognizing the depleting agents and neutralizes their function. Some attempts have also been made to directly target the transcription factor FoxP3 using anti-sense strategies [29]. Unfortunately, oligonucleotides used for gene-silencing technologies are normally not efficiently taken up by primary leukocytes in vivo. Thus this latter strategy will require improvements before it will be a viable approach to use in the clinics.

In summary, there exists a need to improve our ability to modulate TReg cell function, as it will result in increased immune responses that in turn could offer protection against cancer, infections and result in improved vaccine responses. SUMMARY OF THE INVENTION

To date, there have been no attempts to alter the isoform composition of FoxP3 in TReg cells in order to negate the function of the immune-suppressive FoxP3 isoforms (FoxP3fl and FoxP3d2). In the present study we provide several lines of evidence that we can increase the levels of the dominant negative FoxP3d2d7 isoform and thereby amplify immune responses. This could in turn offer protection against cancer, infections and result in improved vaccine responses. In addition, determining the levels of FoxP3d2d7 can serve as a diagnostic marker for inflammatory bowel disease and FoxP3d2d7 levels can be used to follow disease progression and monitor therapeutic efficacy. The invention provides methods to inhibit TReg cell function that result in amplified immune responses. This is done by increasing the levels of dominant negative FoxP3 isoforms using compounds such as IL-lbeta that cause preferential upregulation of non-suppressive isoforms of FoxP3 or by shifting the splicing pattern away from FoxP3fl and FoxP3d2 by using anti- sense oligonucleotides. This results in impaired immunosuppressive ability for the TReg cells.

The invention also provides methods for altering the phenotype of Treg cells and

consequently converts immunosuppressive Treg cells into immunostimulatory CD4+ effector T cells. This can also be done by increasing the levels of dominant negative FoxP3 isoforms using compounds that cause preferential upregulation of non-suppressive isoforms of FoxP3 or by shifting the splicing pattern away from FoxP3fl and FoxP3d2 by using anti-sense oligonucleotides.

The invention also provides methods for diagnosing the onset and monitoring disease progression of inflammatory bowel disease (IBD) by determining the levels of FoxP3d2d7 relative to the total expression of FoxP3. Increased relative levels of FoxP3d2d7 in peripheral blood or intestinal biopsies indicates onset of inflammatory bowel disease or ongoing inflammatory bowel disease. The invention also provides methods for determining treatment efficacy of inflammatory bowel disease by determining the levels of FoxP3d2d7 relative to the total expression of FoxP3. Decreased relative level of FoxP3d2d7 in peripheral blood or intestinal biopsies indicates a successful treatment regimen. According to a first aspect, the invention relates to a method for amplifying an immune response in an individual, comprising altering the composition of FoxP3 isoforms by inducing an expression of a FoxP3 isoform, such as FoxP3d2d7, that inhibits the function of FoxP3fl and/or FoxP3d2. In one embodiment, said method comprises administering to said individual at least one of the following:

(a) an oligonucleotide that has the capacity to bind the FoxP3 transcript and interfere with the splicing events required to generate FoxP3fl and/or FoxP3d2, and to promote formation of an alternatively spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner; or

(b) a pre-spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner; or

(c) a compound, such as IL-1 or IL-lbeta, that induces splicing of the FoxP3 transcript into a FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner.

Preferably, said oligonucleotide is administered in the form of a peptide nucleic acid (PNA), an alternating locked nucleic acid (LNA), a deoxynucleotide oligonucleotide, a fully modified (non-gapmer) 2 '-substituted oligonucleotide, or a phosphorodiamidate morpholino oligomer. In an embodiment of the method, the oligonucleotide is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of an intron-exon border or an exon-intron border in the FoxP3 transcript. Alternatively, the oligonucleotide is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of a branch site (required for RNA splicing) which is located near an intron-exon border or an exon-intron border in the FoxP3 transcript.

In another embodiment of the method, the FoxP3 isoform, which inhibits FoxP3fl and FoxP3d2 in a dominant negative manner, is FoxP3d2d7.

According to a preferred embodiment of the invention, the method comprises administering: - an oligonucleotide that is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of the intron-exon border of exon 2 of the FoxP3 transcript, i.e. has the capacity to bind with either of its ends to a nucleotide at any of the positions 7054-7073 of SEQ ID NO: 1; and

- an oligonucleotide that is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of the intron-exon border of exon 7 of the FoxP3 transcript, i.e. has the capacity to bind with either of its ends to a nucleotide at any of the positions 9309-9328 of SEQ ID NO: 1,

to promote formation of FoxP3d2d7. In a preferred embodiment, the oligonucleotide has a length of 5-50 nucleotides, more preferably 10-20 nucleotides.

According to another preferred embodiment of the invention, the alteration of the composition of FoxP3 isoforms takes place in regulatory T cells.

In an embodiment of the invention, the method further comprises administering an siRNA molecule to said individual to knock down an immunosuppressive isoform of FoxP3, such as FoxP3fl or FoxP3d2. According to a second aspect, the invention relates to a method for treating a cancer or an infection or for inducing an increased vaccine response in an individual, comprising amplifying an immune response in said individual according to the method as described above, including all embodiments. According to a third aspect, the invention relates to an oligonucleotide for use in the treatment of a cancer or an infection or for inducing an increased vaccine response in an individual, which oligonucleotide has the capacity to bind the FoxP3 transcript and interfere with the splicing events required to generate FoxP3fl and/or FoxP3d2, and to promote formation of an alternatively spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner.

In an embodiment, the oligonucleotide for use in the treatment of a cancer or an infection or for inducing an increased vaccine response in an individual is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of an intron-exon border or an exon-intron border in the FoxP3 transcript.

Alternatively, the oligonucleotide is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of a branch site (required for RNA splicing) which is located near an intron-exon border or an exon-intron border in the FoxP3 transcript.

According to a preferred embodiment, the invention provides an oligonucleotide that is at least 80%) complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of the intron-exon border of exon 2 of the

FoxP3 transcript, i.e. has the capacity to bind with either of its ends to a nucleotide at any of the positions 7054-7073 of SEQ ID NO: 1, and further provides an oligonucleotide that is at least 80%) complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of the intron-exon border of exon 7 of the FoxP3 transcript, i.e. has the capacity to bind with either of its ends to a nucleotide at any of the positions 9309-9328 of SEQ ID NO: 1.

In a preferred embodiment, the oligonucleotide has a length of 5-50 nucleotides, more preferably 10-20 nucleotides.

According to a fourth aspect, the invention relates to a pre-spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner, for use in the treatment of a cancer or an infection or for inducing an increased vaccine response in an individual. In a preferred embodiment, the pre-spliced FoxP3 isoform is FoxP3d2d7. According to a fifth aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one of the following:

(a) an oligonucleotide that has the capacity to bind the FoxP3 transcript and interfere with the splicing events required to generate FoxP3fl and/or FoxP3d2, and to promote formation of an alternatively spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner; or

(b) a pre-spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner; or

(c) a compound, such as IL-1 or IL-lbeta, that induces splicing of the FoxP3 transcript into a FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner.

In an embodiment of the pharmaceutical composition, the oligonucleotide is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of a branch site (required for RNA splicing) which is located near an intron-exon border or an exon-intron border in the FoxP3 transcript.

According to a preferred embodiment, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, an oligonucleotide that is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of the intron-exon border of exon 2 of the FoxP3 transcript, i.e. has the capacity to bind with either of its ends to a nucleotide at any of the positions 7054- 7073 of SEQ ID NO: 1, and an oligonucleotide that is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of the intron-exon border of exon 7 of the FoxP3 transcript, i.e. has the capacity to bind with either of its ends to a nucleotide at any of the positions 9309-9328 of SEQ ID NO: 1.

According to a sixth aspect, the invention relates to a method for in vitro testing of a candidate medicinal product, such as an oligonucleotide, for the treatment of cancer or infection of for increasing a vaccine response, comprising:

- transfecting a first portion of T cells (such as primary T cells or a T cell line) with a construct encoding pre-spliced FoxP3fl;

- transfecting a second portion of T cells with a construct encoding pre-spliced FoxP3fl and with a construct encoding the FoxP3 gene including introns;

- inducing an expression of said FoxP3 constructs;

- measuring the expression of FoxP3 target genes;

- contacting said T cells with a candidate medicinal product; and

- measuring the induction / expression of said FoxP3 target genes;

wherein a lower expression of said FoxP3 target genes in the second portion of T cells than in the first portion of T cells indicates that the candidate medicinal product has generated a FoxP3 isoform in said T cells or T cell line, which FoxP3 isoform inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner, and that said candidate medicinal product is suitable for the treatment of cancer or infection of for increasing a vaccine response.

In said method, the construct encoding a pre-spliced FoxP3fl and the construct encoding FoxP3 including introns are under the control of the same inducible promoter. Induction of the expression of said FoxP3 constructs can be made in the presence or absence of biotic or abiotic factors, according to methods known to persons skilled in the art. Examples of FoxP3 target genes are CD25 and CTLA-4, the expression of which is to be measured. Flow cytometry or transcriptional reporters, such as luciferase under the control of a CD25 or a CTLA-4 promoter, may be used to measure the expression of the different genes. The expression of FoxP3 target cells in the first portion of T cells serves as a base-line defining the CD25 and/or CTLA-4 expression level in the absence of inhibitory FoxP3 isoforms.

According to a further aspect, the invention provides a method for diagnosing IBD in an individual, comprising:

- providing a sample, such as a blood sample or a tissue sample, obtained from said individual;

- measuring the expression of FoxP3 isoforms in said sample;

- comparing said measured expression of FoxP3 isoforms to expression measured in healthy controls;

wherein an upregulated expression, such as a two-fold expression, of FoxP3d2d7 in said sample compared to expression measured in healthy controls is an indication of IBD in said individual.

Further, the invention relates to the use of FoxP3d2d7 as a diagnostic marker for

inflammatory bowel disease (IBD). Also contemplated is a method for determining treatment efficacy of inflammatory bowel disease by determining the levels of FoxP3d2d7 relative to the total expression of FoxP3.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 : FoxP3 isoforms in disease. FoxP3 isoform mRNA expression was measured by realtime PCR in intestinal biopsies from Crohn's disease patients and in peripheral blood mononuclear cells (PBMC) from Crohn's disease patients and healthy donors.

FIG. 2: FoxP3d2d7 isoform and IL-17 mRNA expression was measured by real-time PCR in intestinal biopsies from Crohn's disease patients and divided into two groups based upon the level of IL-17 production (50% with the lowest IL-17 production, 50% highest with the lowest IL-17 production).

FIG. 3 : FoxP3 expression in blood from IBD patients. Patients suffering from Crohn's disease were treated by removing gut-homing leukocytes by selective apheresis. FoxP3 isoform expression was measured using real time PCR.

FIG. 4: CD4+ T cells were stimulated with anti-CD3, anti-CD28 and TGF-beta in the presence or absence of IL-1. FoxP3 isoform expression was measured using real time PCR. FIG. 5: TReg cells that had been nucleofected with control morpholino oligonucleotides or morpholino oligonucleotides that promote removal of exon 2 were analysed using flow cytometry for expression of FoxP3 all isoforms (Y axis) and FoxP3 exon 2 (X axis).

FIG. 6: Suppressive ability of TReg cells. TReg cells that had been nucleofected with control morpholino oligonucleotides or morpholino oligonucleotides that promote removal of exons 2 and 7 were tested for immunosuppressive ability in a suppression assay.

FIG. 7: TReg cells that had been nucleofected with control morpholino oligonucleotides or morpholino oligonucleotides that promote removal of exons 2 and 7 were cultured for 10 days in IL-1, IL-2, anti-CD3, anti-CD28 micro beads. IL-17A was measured using cytokine bead arrays.

FIG. 8: Mouse FoxP3 isoforms in TReg cells. Real time PCR of mouse leukocytes cells using probes that recognize splicing events that couple exons 1 and 3 and 6 and 8. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to alteration of different isoforms of the forkhead box P3 (FoxP3) gene (Genbank accession no. NG 007392, in accordance with SEQ ID NO: 1), e.g. by splice-shifting. Splicing of FoxP3 occurs via two sequential transesterification reactions. First, the 2ΌΗ of a specific branch-point nucleotide within the intron that is defined during spliceosome assembly performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site forming the lariat intermediate. Second, the 3ΌΗ of the released 5' exon then performs a nucleophilic attack at the last nucleotide of the intron at the 3' splice site thus joining the exons and releasing the intron lariat.

Technology for exon-skipping, and thereby for splice-shifting, is currently geared towards the use of antisense oligonucleotides. The skipping of an exon is preferably induced by the binding of antisense oligonucleotides targeting either one or both of the splice sites, or exon- internal sequences known as exon inclusion signals (EISs). It is thought that an EIS is a particular structure of an exon that allows splice acceptor and donor to assume a particular spatial conformation. In addition it could be possible induce exon skipping by targeting cis- acting RNA sequences that are required for the correct splicing of exons in a transcript. In particular, supplementary elements such as intronic or exonic splicing enhancers or silencers are identified to regulate specific and efficient splicing of constitutive and alternative exons [30].

To be effective in modification of splicing, oligonucleotides must bind to pre-mRNA without inducing degradation of the RNA duplexed with the oligonucleotide by RNase H, a ubiquitous enzyme. Different types of nucleic acid monomers may be used to generate a suitable oligonucleotide. A nucleic acid may have a backbone, a sugar and/or a base modification compared to an RNA-based oligonucleotide. The chemistries that have been shown to work in animal models include peptide nucleic acids (PNAs), alternating locked nucleic acids (LNAs) and deoxynucleotide oligonucleotides, fully modified (non-gapmer) 2'- substituted oligonucleotides and phosphorodiamidate morpholino oligomers-based oligomers. The latter two chemistries have been used in clinical trials that tested splicing modulation as a treatment for Duchenne muscular dystrophy [30].

MATERIALS AND METHODS

Patient material

The patients in this study were recruited from the Gastroenterology ward at Sodersjukhuset in Stockholm and were assessed by Harvey Bradshaw Index (HBI). Biopsies were taken and immediately frozen and analyzed at a later time.

Peripheral blood mononuclear cells from healthy individuals Peripheral blood mononuclear cells (PBMC) from buffy coats of healthy donors were prepared by Ficoll-Paque density gradient centrifugation.

Real time-PCR

mRNA was isolated with Trizol (Invitrogen), cDNA from human and murine T cells was generated by using Vilo cDNA Synthesis Kit (Invitrogen). RT-PCR was carried out with exon overlapping primer pairs: human FOXP3 :

5'-CAGCTGCAGCTGCCCACACTG-3' (sense) (SEQ ID NO: 2) and

5 ' -GCCTTGAGGGAGAAGACC-3 ' (antisense) (SEQ ID NO: 3) for FOXP3fl

5'-CAGCTGCAGCTCTCAACGGTG-3' (sense) (SEQ ID NO: 4) and

5 '-GCCTTGAGGGAGAAGACC-3' (antisense) (SEQ ID NO: 5) for FOXP3D2 and 5'-GAGCAGCAGGCATCATCCG-3' (sense) (SEQ ID NO: 6) and

5'-CTGGGAATGTGCTGTTTCC-3' (antisense) (SEQ ID NO: 7) for FOXP3D2D7 mouse FoxP3 isoforms:

5'-CAGCTGCAGCTGCCTACA-3' (sense) (SEQ ID NO: 8) and

5 ' -GATCCC AGGTGGC AGGC-3 ' (antisense) (SEQ ID NO: 9) for FoxP3fl

5'-CAGCTGCAGCTCTCCACT-3' (sense) (SEQ ID NO: 10) and

5' -GATCCC AGGTGGC AGGC-3' (antisense) (SEQ ID NO: 11) for FoxP3D2

5'-GAGCAGCAGGCCTCAATG-3' (sense) (SEQ ID NO: 12) and

5 ' -CC ATGTTGTGGAAGAACTCT-3 ' (antisense) (SEQ ID NO: 13) for FoxP3D2D7

Cell culture

Human PBMCs were separated by Ficoll (GE Healthcare) centrifugation. Isolated T cells were incubated in serum free X-VIVO 15 Medium (Lonza) at 37°C and 6% C02.

T cell stimulation T cells were stimulated with micro beads with anti-CD3 and a-CD28 antibodies (Miltenyi). In Thl7 skewing conditions T cells were treated with lOng/ml TGF-b with or without lOng/ml IL-1 (all Peprotech). Cell isolation

Treg cells were isolated by using a-CD25 PE antibody (Invitrogen) in combination with a-PE beads (Miltenyi) using magnetic cell sorting via AutoMacs (Miltenyi). CD4+CD25- T cells were obtained by labelling the cells with anti-CD4 beads (Miltenyi) and subsequent magnetic cell sorting via AutoMacs (Miltenyi).

Splice-shifting

Enhanced splicing was achieved using Morpholino Antisense Oligonucleotides (MAO) (GeneTools) spanning the intron-exon boundaries of human FOXP3. T cells were transfected with 15μΜ MAO by using a Nucleotransfector device (Lonza) according to the protocol for human primary cells (P3 Primary Cell Nucleofector Kit).

MAO Sequences were as followed:

5'-TGCCCATTCACCGTCCATACCTGGT-3' (SEQ ID NO: 14) Fluorescein for FOXP3D2 5 ' - AGCTGTGAAATGGC AC AAAC ATGAG-3 ' (SEQ ID NO: 15) Fluorescein for

FOXP3D2D7

Flow cytometry

Cytokine expression and FOXP3 isoforms pattern were analyzed by monoclonal fluorophore- labelled antibodies. T cells were fixed and permeabilized according to the protocol using FOXP3 staining kit (ebioscience). FOXP3fl was stained with an exon 2 specific clone: FJK 16s (ebioscience) and FOXP3D2 was detected with a clone recognizing all isoforms:

236A/E7 (ebioscience). IL-17A expression was measured with a-IL17A antibody (biolegend) in cells that were stimulated 24h before flow cytometry with 50ng/ml PMA (Sigma) and 1 μg/ml Ionomycin (Sigma) in the presence of GolgiBlocker (BD Biosciences). Cytometric Bead array

Supernatant of cells were taken and cytokines were quantified using BD Cytometric Bead Array (CBA) for mouse Thl/Th2/Thl7 Cytokine Kit (BD Biosciences) following the user instructions. Western blot

SDS gel electrophoresis (BioRad) was carried out on T cell lysates to separate FoxP3 isoforms. Proteins were blotted on Immobilon P membranes (Millipore) using a Transblot (BioRad). Murine FoxP3 isoforms were detected with a-FoxP3 antibody clone eBio7979 (ebioscience), recognizing full length as well as exon 2 and exon 7 depleted isoforms.

Incubation of the membrane with HRP-coupled secondary antibody goat a-mouse IgG (Santa Cruz) and ECL Western Blot detection reagent (Amersham) resulted in FOXP3 bands emitting light that was detected by a light sensitive film (SigmaAldrich). Additional experiments

We are comparing the potency of a large pool of siRNA molecules with the potency of several splice-shifting oligonucleotides with respect to inhibition of TReg cells'

immunosuppressive ability, ability to induce effector cell cytokines and ability to convert TReg cells to effector T cells.

We are determining whether combinations of siRNA molecules with splice-shifting oligonucleotides are more potent than siRNA molecules only.

We are determining the transcriptomic profile of single cells to determine the phenotype of cells expressing FoxP3d2d7.

We are currently generating antibodies that will be able to distinguish between the three different FoxP3 isoforms, FoxP3fl, FoxP3d2 and FoxP3d2d7. We are determining the function of FoxP3d2d7 in vivo. According to the literature, mice only express a single form of FoxP3. The support for this idea is weak as it is based upon a long chain of citations to unpublished data. We have found that mice express all 3 isoforms of FoxP3 using both real-time PCR and western blot. This finding allows us to generate a transgenic mouse where we can express mouse FoxP3d2d7 at will. In brief, FoxP3d2d7 is expressed under the control of tetracycline response elements (TRE). These transgenic mice are then crossed to mice expressing tetracycline-controlled transactivator (tTA) or reverse tetracycline-controlled transactivator (rtTA) under tissue specific promoters. Transcription can then be reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline). We will then determine how turning on FoxP3d2d7 expression would affect immune homeostasis and if they are more or less susceptible to cancer and infections.

The novel observation that mice express different FoxP3 isoforms allows us to administer splice-shifting oligonucleotides that induce FoxP3d2d7 and determine their effect in experimental disease systems. We are currently addressing the curative potential of such oligonucleotides in models of established tumors and infections.

We are currently determining if inhibiting TReg cell function through splice-shifting, from FoxP3fl and FoxP3d2 to FoxP3d2d7, inhibits tumor metastasis. The rationale is that functional TReg cells are a major source of TGF-beta, and TGF-beta promotes epithelial to mesenchymal transition, which is a key step in cancer metastasis.

We are currently also determining if splice-shifting, from FoxP3fl and FoxP3d2 to

FoxP3d2d7, affects the plasticity of TReg cells i.e. if a portion of the treated cells starts losing FoxP3 expression and becomes effector T cells.

RESULTS AND DISCUSSION

We hypothesized that FoxP3d2d7 is upregulated by proinflammatory mediators, facilitates immune responses and amplifies immune responses. In order to test this hypothesis we initially decided to study patients suffering from inflammatory bowel disease (IBD). IBD comprise a group of inflammatory conditions of the colon and small intestine. The major types of IBD are Crohn's disease and ulcerative colitis. Experimental studies suggest that TReg cells play an essential role in limiting the inflammatory response in IBD, based upon that: 1) homeostatic proliferation of T cells in the absence of TReg cells results in colitis [16]; and 2) adoptive transfer of TReg cells can cure established colitis [16]. Thus we believe that IBD is suitable for starting to study the effects of FoxP3d2d7. The following results support our hypothesis: First, we found that patients suffering from IBD displayed an increase in FoxP3d2d7 mRNA levels relative to the levels of the FoxP3fl and FoxP3d2 mRNA when compared to the FoxP3 isoform levels in peripheral blood mononuclear cells from healthy donors. This was true in both peripheral blood mononuclear cells and intestinal biopsies from these IBD patients (fig. 1). Second, we measured the levels of the pro-inflammatory cytokine IL-17 and divided the samples into two groups based upon the 50% highest and 50% lowest IL-17 mRNA expression. We found that the group that produced the most IL-17 mRNA also had the highest amounts of FoxP3d2d7 (fig. 2).

Third, we studied patients in a phase I clinical trial in which gut-homing leukocytes were removed from peripheral blood by selective apheresis. The treated patients (n=14) did better than the placebo treated group (n=10) (data not shown). The treated patients displayed decreased levels of FoxP3d2d7 mRNA but no differences in FoxP3fl or FoxP3d2 mRNA expression (fig. 3).

These results suggest that FoxP3d2d7 is found at greater levels during chronic inflammatory conditions than in healthy individuals. In addition, it appears that FoxP3d2d7 levels correlate with the intensity of the pro-inflammatory response as suggested by the correlation between high IL-17 mRNA levels and high FoxP3d2d7 mRNA levels. Finally these results suggest that FoxP3d2d7 could be useful as a marker both for diagnosing IBD and for monitoring IBD disease progression. Alternative splicing is a normal phenomenon in eukaryotes, where it greatly increases the diversity of proteins that can be encoded by the genome. It allows the same gene to generate different protein isoforms that can perform different functions. The different isoforms of a protein can also be differentially regulated which allows further diversification of function from the products of a single gene. In our initial hypothesis we stated that we expected FoxP3d2d7 to be upregulated by proinflammatory mediators. This appears to indeed be the case, as activation of naive T cells in the presence of TGF-beta results in FoxP3 induction, and addition of IL-1 to these cultures resulted in preferential induction of FoxP3d2d7 (Fig. 4). This is perfectly in line with the initial hypothesis, as a proinflammatory mediator such as IL- 1 is meant to induce immune responses and induction of FoxP3d2d7 could be one means of doing so.

The findings described above supported our belief that FoxP3d2d7 could amplify immune responses. This opens the very intriguing possibility of altering the composition of FoxP3 isoforms in TReg cells, thereby resulting in an increased immune response. It is known that antisense oligonucleotides that are stable against nucleolytic degradation and that do not induce RNase H catalyzed hydrolysis of complementary mRNA are powerful modulators of pre-mRNA splicing in vitro and in vivo. Such oligonucleotides have successfully been used in the past to shift pre-mRNA splicing of several genes including: CFTR, IL-5R, c-myc, tau, SMN-2, bcl-x, β-globin and dystrophin. Amongst the oligonucleotide analogues most intensively investigated for splice shifting are the anionic analogues 2'-OMe-RNA and 2'- methoxyethyl (MOE)-RNA, as well as the charge neutral analogues morpholino-DNA and PNA. Thus to directly test if FoxP3d2d7 promote proinflammatory responses we decided to alter the composition of FoxP3 isoforms in TReg cells. The following results demonstrate that we indeed can induce a shift of FoxP3 isoforms from FoxP3fl and FoxP3d2 to FoxP3d2d7 and that such a shift indeed promotes immune responses.

First, we isolated TReg cells and delivered a morpholino oligonucleotide by nucleofection, which interfered with the splice events at exon 2 by steric hindrance. We could indeed see that this caused the splice-pattern of FoxP3 to change, as antibodies that bound all FoxP3 isoforms still gave an equally strong signal as control treated cells whereas antibodies that recognized exon 2 gave a much weaker signal than control treated cells (Fig 5). In this experiment a scrambled morpholino oligonucleotide was used as control. Second, we targeted both exon 2 and exon 7 in TReg cells to be removed by alternative splicing using nucleofection with morpholino oligonucleotides. We confirmed successful deletion of exon 2 and exon 7 in these TReg cells using real-time PCR (data not shown). The TReg cells expressing increased levels of FoxP3d2d7 were less immunosuppressive when compared to control treated TReg cells when assayed in an in vitro suppression assay (Fig. 6).

Third, TReg cells, which have an altered FoxP3 isoform composition towards the FoxP3d2d7 isoform as described above, also produced more of the proinflammatory cytokine IL-17 in response to TCR stimulation in the presence of IL-1 (Fig.7). We have found that mice express all 3 isoforms of FoxP3 using both real-time PCR (Fig. 8) and western blot (data not shown). This observation will allow us to determine the role of FoxP3d2d7 in vivo and allow us to test the therapeutic potency of splice-altering

oligonucleotides in different disease models. It is to our understanding the first time anyone has altered the balance of the pre-existing isoform composition of FoxP3. The data presented above support that altering the FoxP3 isoform pattern from the FoxP3fl and FoxP3d2, which confers a suppressive phenotype to TReg cells, to FoxP3d2d7 results in amplified immune responses. The fact that FoxP3d2d7 counteracts the function of FoxP3fl and FoxP3d2 will likely make splice-shifting a more potent strategy than siRNA silencing as it will always be difficult to target every single mRNA molecule with siRNA silencing. In the case of splice-shifting, all mRNA molecules will still be translated and the resulting FoxP3d2d7 will inhibit any FoxP3 molecule that remains unaltered. Further, combinations of oligonucleotides that induce changes in FoxP3 isoform composition may possibly be successfully combined with conventional siRNA- mediated targeting of FoxP3, and it may be possible to specifically degrade FoxP3d2d7 mRNA and restore TReg cell function during inflammatory conditions. It may also be possible to restore TReg cell function during inflammatory conditions by enforcing exon 7 to be retained in the processed mRNA by blocking exonic splicing silencers with

oligonucleotides.

REFERENCES

1. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol. 2003 Apr;4(4):337-42.

2. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003 Apr;4(4):330-6.

3. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003 Feb 14;299(5609): 1057-61.

4. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995 Aug l; 155(3): 1151-64.

5. Brunkow ME, Jeffery EW, Hjerrild KA, Paeper B, Clark LB, Yasayko SA, Wilkinson JE, Galas D, Ziegler SF, Ramsdell F. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001 Jan;27(l):68-73.

6. Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, Kelly TE, Saulsbury FT, Chance PF, Ochs HD. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (TPEX) is caused by mutations of FOXP3. Nat Genet. 2001 Jan;27(l):20-1.

7. Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, Buist N, Levy-Lahad E, Mazzella M, Goulet O, Perroni L, Bricarelli FD, Byrne G, McEuen M, Proll S, Appleby M, Brunkow ME. X-linked neonatal diabetes mellitus, enteropathy and endocnnopathy syndrome is the human equivalent of mouse scurfy. Nat Genet. 2001 Jan;27(l): 18-20.

8. Mailer RK, Falk K, Rotzschke O. Absence of leucine zipper in the natural

FOXP3Delta2Delta7 isoform does not affect dimerization but abrogates suppressive capacity. PLoS One. 2009 Jul l;4(7):e6104. 9. Manrique SZ, Correa MA, Hoelzinger DB, Dominguez AL, Mirza N, Lin HH, Stein- Streilein J, Gordon S, Lustgarten J. Foxp3 -positive macrophages display immunosuppressive properties and promote tumor growth. J Exp Med. 2011 Jul 4;208(7): 1485-99.

10. Chen GY, Chen C, Wang L, Chang X, Zheng P, Liu Y. Cutting edge: Broad expression of the FoxP3 locus in epithelial cells: a caution against early interpretation of fatal inflammatory diseases following in vivo depletion of FoxP3 -expressing cells. J Immunol. 2008 Apr 15; 180(8):5163-6.

11. Kim J, Lahl K, Hori S, Loddenkemper C, Chaudhry A, deRoos P, Rudensky A,

Sparwasser T. Cutting edge: depletion of Foxp3+ cells leads to induction of autoimmunity by specific ablation of regulatory T cells in genetically targeted mice. J Immunol. 2009 Dec 15; 183(12):7631-4.

12. Zuo T, Wang L, Morrison C, Chang X, Zhang H, Li W, Liu Y, Wang Y, Liu X, Chan MW, Liu JQ, Love R, Liu CG, Godfrey V, Shen R, Huang TH, Yang T, Park BK, Wang CY, Zheng P, Liu Y. FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene. Cell. 2007 Jun 29; 129(7): 1275-86.

13. Patent publication US20090325868

14. Patent publication US20100143359 15. Ebert LM, Tan BS, Browning J, Svobodova S, Russell SE, Kirkpatrick N, Gedye C, Moss D, Ng SP, MacGregor D, Davis ID, Cebon J, Chen W. The regulatory T cell-associated transcription factor FoxP3 is expressed by tumor cells. Cancer Res. 2008 Apr 15;68(8):3001- 9.

16. Mottet C, Uhlig HH, Powrie F. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J Immunol. 2003 Apr 15;170(8):3939-43. 17. Tarbell KV, Petit L, Zuo X, Toy P, Luo X, Mqadmi A, Yang H, Suthanthiran M, Mojsov S, Steinman RM. Dendritic cell-expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice. J Exp Med. 2007 Jan 22;204(1): 191-201. 18. McGeachy MJ, Stephens LA, Anderton SM. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system. J Immunol. 2005 Sep l; 175(5):3025-32.

19. Hara M, Kingsley CI, Niimi M, Read S, Turvey SE, Bushell AR, Morris PJ, Powrie F, Wood KJ. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol. 2001 Mar 15; 166(6):3789-96.

20. Lin W, Truong N, Grossman WJ, Haribhai D, Williams CB, Wang J, Martin MG, Chatila TA. Allergic dysregulation and hyperimmunoglobulinemia E in Foxp3 mutant mice. J Allergy Clin Immunol. 2005 Nov; 116(5): 1106-15.

21. Suvas S, Kumaraguru U, Pack CD, Lee S, Rouse BT. CD4+CD25+ T cells regulate virus- specific primary and memory CD8+ T cell responses. J Exp Med. 2003 Sep 15; 198(6):889- 901.

22. Toka FN, Suvas S, Rouse BT. CD4+ CD25+ T cells regulate vaccine-generated primary and memory CD8+ T-cell responses against herpes simplex virus type 1. J Virol. 2004 Dec;78(23): 13082-9. 23. Onizuka S, Tawara I, Shimizu J, Sakaguchi S, Fujita T, Nakayama E. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res. 1999 Jul 1;59(13):3128-33.

24. Ait-Oufella H, Salomon BL, Potteaux S, Robertson AK, Gourdy P, Zoll J, Merval R, Esposito B, Cohen JL, Fisson S, Flavell RA, Hansson GK, Klatzmann D, Tedgui A, Mallat Z. Natural regulatory T cells control the development of atherosclerosis in mice. Nat Med. 2006 Feb; 12(2): 178-80. 25. Hisaeda H, Maekawa Y, Iwakawa D, Okada H, Himeno K, Kishihara K, Tsukumo S, Yasutomo K. Escape of malaria parasites from host immunity requires CD4+ CD25+ regulatory T cells. Nat Med. 2004 Jan;10(l):29-30. 26. Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature. 2002 Dec

5;420(6915):502-7.

27. Suvas S, Azkur AK, Kim BS, Kumaraguru U, Rouse BT. CD4+CD25+ regulatory T cells control the severity of viral immunoinflammatory lesions. J Immunol. 2004 Apr

l; 172(7):4123-32.

28. Byrne WL, Mills KH, Lederer JA, O'Sullivan GC. Targeting regulatory T cells in cancer. Cancer Res. 2011 Nov 15;71(22):6915-20.

29. Morse MA, Hobeika A, Serra D, Aird K, McKinney M, Aldrich A, Clay T, Mourich D, Lyerly HK, Iversen PL, Devi GR. Depleting regulatory T cells with arginine-rich, cell- penetrating, peptide-conjugated morpholino oligomer targeting FOXP3 inhibits regulatory T- cell function. Cancer Gene Ther. 2012 Jan; 19(l):30-7.

30. Kole R, Krainer AR and Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nature reviews drug discovery (2012) 11 : 125-140.

Claims

1. A method for amplifying an immune response in an individual, comprising altering the composition of FoxP3 isoforms by inducing an expression of a FoxP3 isoform, such as

FoxP3d2d7, that inhibits the function of FoxP3fl and/or FoxP3d2.

2. The method of claim 1, comprising administering to said individual at least one of the following:

(a) an oligonucleotide that has the capacity to bind the FoxP3 transcript and interfere with the splicing events required to generate FoxP3fl and/or FoxP3d2, and to promote formation of an alternatively spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner; or

(b) a pre-spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner; or

(c) a compound, such as IL-1 or IL-lbeta, that induces splicing of the FoxP3 transcript into a FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner.

3. The method of claim 2, wherein said oligonucleotide is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within 10 nucleotides of an intron-exon border or an exon-intron border in the FoxP3 transcript.

4. The method of claim 2, wherein said FoxP3 isoform, which inhibits FoxP3fl and FoxP3d2 in a dominant negative manner, is FoxP3d2d7.

5. The method of any preceding claim, comprising administering:

- an oligonucleotide that is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide at any of the positions 7054-7073 of SEQ ID NO: 1; and

- an oligonucleotide that is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide at any of the positions 9309-9328 of SEQ ID NO: 1,

to promote formation of FoxP3d2d7.

6. The method of any preceding claim, wherein the alteration of the composition of FoxP3 isoforms takes place in regulatory T cells.

7. A method for treating a cancer or an infection or for inducing an increased vaccine response in an individual, comprising amplifying an immune response in said individual according to the method of any one of claims 1-6.

8. An oligonucleotide for use in the treatment of a cancer or an infection or for inducing an increased vaccine response in an individual, which oligonucleotide has the capacity to bind the FoxP3 transcript and interfere with the splicing events required to generate FoxP3fl and/or FoxP3d2, and to promote formation of an alternatively spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner.

9. The oligonucleotide for use according to claim 8, which is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide within

10 nucleotides of an intron-exon border or an exon-intron border in the FoxP3 transcript.

10. A pre-spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner, for use in the treatment of a cancer or an infection or for inducing an increased vaccine response in an individual.

11. The pre-spliced FoxP3 isoform for use according to claim 10, which is FoxP3d2d7.

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one of the following:

(a) an oligonucleotide that has the capacity to bind the FoxP3 transcript and interfere with the splicing events required to generate FoxP3fl and/or FoxP3d2, and to promote formation of an alternatively spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner; or

(b) a pre-spliced FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner; or

(c) a compound, such as IL-1 or IL-lbeta, that induces splicing of the FoxP3 transcript into a FoxP3 isoform, which inhibits FoxP3fl and/or FoxP3d2 in a dominant negative manner.

13. The pharmaceutical composition of claim 12, comprising:

- an oligonucleotide that is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide at any of the positions 7054-7073 of SEQ ID NO: 1; and

- an oligonucleotide that is at least 80% complementary to the FoxP3 transcript and has the capacity to bind with either of its ends to a nucleotide at any of the positions 9309-9328 of SEQ ID NO: 1.

14. A method for in vitro testing of a candidate medicinal product, such as an oligonucleotide, for the treatment of cancer or infection of for increasing a vaccine response, comprising:

- transfecting a first portion of T cells with a construct encoding pre-spliced FoxP3fl;

- transfecting a second portion of T cells with a construct encoding pre-spliced FoxP3fl and with a construct encoding the FoxP3 gene including introns;

- inducing an expression of said FoxP3 constructs;

- measuring the expression of FoxP3 target genes;

- contacting said T cells with a candidate medicinal product; and

- measuring the induction / expression of said FoxP3 target genes;

wherein a lower expression of said FoxP3 target genes in the second portion of T cells than in the first portion of T cells indicates that the candidate medicinal product has generated a FoxP3 isoform in said T cells or T cell line, which FoxP3 isoform inhibits FoxP3fl and/or

FpxP3d2 in a dominant negative manner, and that said candidate medicinal product is suitable for the treatment of cancer or infection of for increasing a vaccine response.

15. A method for diagnosing inflammatory bowel disease (IBD) in an individual, comprising: - providing a sample, such as a blood sample or a tissue sample, obtained from said individual;

- measuring the expression of FoxP3 isoforms in said sample;

- comparing said measured expression of FoxP3 isoforms to expression measured in healthy controls;

wherein an upregulated expression, such as a two-fold expression, of FoxP3d2d7 in said sample compared to expression measured in healthy controls is an indication of IBD in said individual.

16. Use of FoxP3d2d7 as a diagnostic marker for inflammatory bowel disease (IBD).