WO2014060580A1

WO2014060580A1 - Lxvp-mediated calcineurin inhibition in macrophages

Info

Publication number: WO2014060580A1
Application number: PCT/EP2013/071842
Authority: WO
Inventors: Juan Miguel Redondo Moya; Amelia ESCOLANO ARTIGAS
Original assignee: Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii (Cnic)
Priority date: 2012-10-18
Filing date: 2013-10-18
Publication date: 2014-04-24
Also published as: WO2014060580A9

Abstract

The present invention refers to a composition (from hereinafter "composition of the invention") comprising a macrophage comprising an exogenous compound (from hereinafter "compound of the invention") capable of inhibiting the CN-NFAT signaling pathway in an immunophilin independent manner by binding to the CN A-CN B composite surface, wherein the CN A-CN B composite surface is composed of amino acids 341 to 363 of the calcineurin catalytic subunit A (CN A) as set forth in SEQ. ID No 4 and amino acids 113 to 127 of the calcineurin regulatory subunit B (CN B) as set forth in SEQ ID No 5.

Description

LxVP-MEDIATED CALCINEU IN INHIBITION IN MACROPHAGES

TECHNICAL FIELD

This invention relates to the biotechnological sector applied to the area of human health, and more specifically to a mechanism of LxVP-mediated calcineurin inhibition in macrophages in order to induce a switch of this type of cells towards an M2 anti-inflammatory phenotype.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention, and is not admitted to describe or constitute prior art to the present invention.

Inflammatory diseases, a leading cause of chronic illness and disability, are caused by inappropriate or excessive activation of the immune system. In autoimmune disorders and allergies the immune system reacts to self-antigens or other normally harmless substances, leading to chronic inflammation. In other situations, such as atherosclerosis or transplant rejection, the immune system is over-activated by injurious stimuli. Although both genetic and environmental factors contribute to the development of these disorders, their etiology is not known. Available treatments are therefore focused on relieving symptoms and slowing disease progression.

Expression of many cytokines during the inflammatory response is regulated by the calcium/calcineurin (CN) pathway. CN is a phosphatase that couples calcium-mobilizing signals to cell responses and is the target of the immunosuppressive (IS) drugs cyclosporin A (CsA) and FK506. Each of these drugs forms a complex with a specific immunophilin (IP) (cyclophilin and FK506 binding protein, respectively) and it is these IS/IP complexes that bind and inhibit CN. In addition to their use in preventing transplant rejection, IS drugs have been successfully used to treat atopic dermatitis, severe asthma and rheumatoid arthritis (RA). However, CN-independent actions of IS/IP complexes are linked to a number of side effects including hepatotoxicity, nephrotoxicity and high blood pressure.

Many CN-dependent processes described in mammals involve the regulation of the nuclear factor of activated T cells (NFAT) family of transcription factors. Structural and functional analyses of NFAT proteins have identified PxlxlT and LxVP motifs as docking sites involved in the interaction with CN. The inventors of the present invention have recently shown that a peptide based on LxVP interferes with the CN-NFAT interaction by binding to the same docking site on CN as the IS/IP complexes. Since it inhibits CN independently of IP, LxVP would lack the toxic side effects of IS/IP complexes.

A central role for the CN-NFAT pathway in adaptive immune responses has been documented through extensive studies in T cells; however, much less is known about its role in cells of the innate immune system such as macrophages. Not only does Macrophages constitute the first line of defense to an inflammatory insult, it also regulates the specific immune response by conditioning the cytokine milieu. Macrophages are reciprocally influenced by the surrounding environment, and can be classified into two main groups. Classically activated macrophages (Ml) support Thl/Thl7 responses and are characterized by the expression of pro-inflammatory cytokines and iNOS. In contrast, alternatively activated macrophages (M2) are defined as immunoregulators/immunosuppressors that produce low levels of pro-inflammatory cytokines but high levels of arginasel (Argl) and the anti-inflammatory cytokines IL10 or TGF$. M2 macrophages contribute to the suppressive microenvironment during tumorigenesis, maintain adipose tissue homeostasis and promote resolution of inflammation in atherosclerosis and myocardial infarction.

Here, we report that CN regulates M1/M2 macrophage polarization, and show that cell and gene therapy a pproaches based on suppression of CN signaling in macrophages promote the resolution of

inflammation.

BRIEF DESCRIPTION OF THE INVENTION

A first aspect of the present invention refers to a macrophage (from hereinafter "macrophage of the invention") comprising an exogenous compound (from hereinafter "compound of the invention") capable of inhibiting the CN-NFAT signaling pathway in an immunophilin independent manner by binding to the CN A-CN B composite surface or by allosterically modulating said surface, wherein the CN A-CN B composite surface is composed of amino acids 341 to 363 of the calcineurin catalytic subunit A (CN A) as set forth in SEQ ID No 4 and amino acids 113 to 127 of the calcineurin regulatory subunit B (CN B) as set forth in SEQ ID No 5, and wherein said exogenous compound induces a switch of the macrophage toward an anti-inflammatory phenotype.

A preferred embodiment of the first aspect of the invention refers to the macrophage of the invention, wherein the compound of the invention is capable of activating p38 MAPK and inhibiting the CN-NFAT signaling pathway in an immunophilin independent manner by binding to the CN A-CN B composite surface or by allosterically modulating said surface, wherein the CN A-CN B composite surface is composed of amino acids 341 to 363 of the calcineurin catalytic subunit A (CN A) as set forth in SEQ ID No 4 and amino acids 113 to 127 of the calcineurin regulatory subunit B (CN B) as set forth in SEQ ID No 5, and wherein said compound of the invention induces a switch of the macrophage toward an anti- inflammatory phenotype.

In another preferred embodiment of the first aspect of the invention, the exogenous compound capable of inhibiting the CN-NFAT signaling pathway in an immunophilin independent manner binds directly to the CN A-CN B composite surface.

In a second aspect of the present invention, the macrophage of the invention is transformed or transduced with a nucleotide sequence coding for a peptide capable of inhibiting the CN-NFAT signaling pathway, wherein said peptide comprises the following sequence:

(I) R1-L-R2-V-P-R3

and wherein Rl is an aromatic amino acid of the Tyrosine or Phenylalanine type, R2 is an Alanine or Serine-type amino acid and R3 is not the amino acid Proline, in particular R3 is preferably a Glutamine, Serine or Alanine-type amino acid.

In a preferred embodiment of the second aspect of the invention, amino acid sequence (I) is selected from a peptide comprising any of the following sequences: a. SEQ ID No 1 (LxVPcl) or SEQ ID No 2 (LxVPc3) or SEQ ID No 3 (LxVPc4); or b. A variant of (a) which is at least 85%, preferentially 95% homologous to SEQ ID No 1 or to SEQ ID No 2 or to SEQ ID No 3 in terms of amino acid identity.

In another preferred embodiment of the second aspect of the invention, amino acid sequence (I) is selected from a peptide consisting of any of the following sequences:

a. SEQ ID No 1 (LxVPcl);

b. SEQ ID No 2 (LxVPc3); or

c. SEQ ID No 3 (LxVPc4).

In yet another preferred embodiment of the invention, the macrophage is at least positive for the phenotypic marker F4/80+, more preferably the macrophage is at least positive for the following phenotypic markers: F4/80+, CDllb+ and CDllc+.

A third aspect of the invention refers to a vector (from hereinafter "vector of the invention") comprising a compound of the invention comprising a polynucleotide sequence, preferably coding for amino acid sequence (I), which is capable of transporting or delivering said polynucleotide sequence into the macrophages of a subject.

In a preferred embodiment of the third aspect of the invention, the vector comprises a polynucleotide sequence coding for a peptide comprising any of the following sequences:

a. SEQ ID No 1 (LxVPcl) or SEQ ID No 2 (LxVPc3) or SEQ ID No 3 (LxVPc4); or

b. A variant of (a) which is at least 85%, preferably 95% homologous to SEQ ID No 1 or to SEQ ID No 2 or to SEQ ID No 3 in terms of amino acid identity.

In another preferred embodiment of the third aspect of the invention, the vector comprises a polynucleotide sequence coding for a peptide consisting of any of the following sequences:

a. SEQ ID No 1 (LxVPcl);

b. SEQ ID No 2 (LxVPc3); or

c. SEQ ID No 3 (LxVPc4).

In another preferred aspect of the invention, the vector is a viral vector encoding for an amino acid sequence as defined in any of the embodiments of the second aspect of the invention, wherein preferably the viral vector is a lentivirus. Preferably, said lentivirus are pseudotyped lentiviral vectors consisting of vector particles bearing glycoproteins (GPs) derived from other enveloped viruses. Among the first and still most widely used GPs for pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G), due to the very broad tropism and stability of the resulting pseudotypes. Lentiviruses particles bearing the vesicular stomatitis virus GP (VSV-G) have been found herein to predominantly infect macrophages. A further aspect of the invention refers to a non-viral vector (from hereinafter "non-viral vector of the invention") comprising, but not limited to, nanoparticles or lipids for the delivery of a compound of the invention into the macrophages of a subject. More preferably, said non-viral vector is used for the delivery of any of the following compounds:

a. A peptide comprising the following sequence 1-L-R2-V-P-R3, wherein said sequence is as defined above; or

b. An amino acid sequence selected from any of the following:

a. SEQ ID No 1 (LxVPcl) or SEQ ID No 2 (LxVPc3) or SEQ ID No 3 (LxVPc4); or

b. A variant of (a) which is at least 95% homologous to SEQ ID No 1 or to SEQ ID No 2 or to SEQ ID No 3 in terms of amino acid identity.

In addition, a further embodiment of the invention refers to the use of the non-viral or viral vectors of the invention for the ex vivo transformation or transduction of macrophages.

Also provided herein is a method of increasing or enhancing the clinical status and perception of the well-being of a subject with an inflammatory disease, preferably with arthritis, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a vector of the invention or any of the compositions of the invention and, optionally, a pharmaceutically acceptable carrier.

A further aspect of the invention refers to a composition (from hereinafter composition of the invention), preferably a pharmaceutical composition (from hereinafter "pharmaceutical composition of the invention"), which comprises the vector and/or the macrophage of the invention and optionally a pharmaceutically acceptable carrier.

A fourth aspect of the invention refers to the pharmaceutical composition of the invention, the composition of the invention, the macrophage of the invention, the vector of the invention or the non- viral vector of the invention, for its use in therapy.

A fifth aspect of the invention refers to the pharmaceutical composition of the invention, the composition of the invention, the macrophage of the invention, the vector of the invention or the non- viral vector of the invention, for its use in the treatment or prevention of an inflammatory disease.

In a preferred embodiment of the fifth aspect of the invention, the inflammatory disease can be selected from the list consisting of rheumatoid arthritis, encephalomyelitis, lupus erythematosus, psoriasis, atopic dermatitis, allergies , contact hypersensitivity, hepatitis, atherosclerosis, obesity, diabetes and Crohn^'s disease.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Locally-delivered LxVP cl lentivirus prophylactically protects against collagen-induced arthritis by selectively transducing macrophages, (a) The scheme shows the CIA protocol, indicating the time of lentivirus injection. Charts show time profiles of arthritic score (left) and incidence (right) in mice transduced in the right hind footpad on day -5 with LxVP or mutLxVP lentivirus. Data are means ± s.e.m from a representative experiment of two; n=10 mice (10 paws) per group. * P< 0.05; **P< 0.01; ***p< 0.001. (b) Masson's trichrome staining in joints of paws locally transduced on day -5 with mutLxVP (top) or LxVP (bottom), (c) Footpad-injected lentivirus targets popliteal lymph-nodes (LN). The chart shows luciferase activity in different LN-cell homogenates from mice inoculated in the right hind footpad with luciferase-encoding lentivirus. (d) Flow cytometry analysis of F4/80, CDllb and CDllc expression in sorted GFP+ cells obtained from popliteal LN after inoculation of footpads with GFP-encoding lentivirus. (e) Confocal immunofluorescence showing colocalization of virus-encoded GFP and F4/80 in sections of inflamed paws locally-injected with lentiviral vector.

Figure 2. Systemically-delivered LxVP lentivirus therapeutically protects against CIA by selectively transducing macrophages, (a) The scheme shows the CIA protocol, indicating the time of lentivirus injection. The chart shows the time profile of arthritic score in mice inoculated i.p. with LxVP or mutLxVP lentivirus at disease onset (day 28). Data are means ± s.e.m of three independent experiments; n = 20 mice per group. * P< 0.05. (b) Masson's trichrome staining in paw joints of animals inoculated i.p. at disease onset with mutLxVP (top) or LxVP (bottom), (c) Top, GFP expression in gated B220, CD4 and CD8 cells from peritoneal cell exudate. Bottom, Flow cytometry analysis of F4/80, CDllb and CDllc expression in gated GFP+ cells from peritoneal exudate obtained after i.p. injection of GFP-encoding lentivirus. Data are from a representative experiment of at least three, (d) i.p.-injected lentivirus targets parathymic lymph-nodes (LN). The chart shows luciferase activity in different LN-cell homogenates from mice inoculated i.p. with luciferase-encoding lentivirus.

Figure 3. Macrophages migrate from the peritoneal cavity to inflammation sites and mediate antiinflammatory effects, (a) In vivo tracking of i.p.-injected DIR-labeled non-transduced macrophages in mice with zymosan-induced acute inflammation in the right hindpaw. In vivo images (top) and quantification (bottom). Zym, zymosan. ***P <0.001 (means ± s.d.; n=3). (b) Time profile of DIR signal in inflamed paws after i.p. injection of DIR-labeled macrophages. *P< 0.05 (means ± s.d.; n>3). (c) In vivo image analysis of the migratory capacity of DIR-labeled LxVP- or mutLxVP-transduced macrophages to inflamed paws, (d) GFP immunostaining in paw sections from animals inoculated i.p. with LxVP or mutLxVP lentivirus. (e) CIA score in arthritic mice inoculated in the footpads with LxVP- or mutLxVP- transduced macrophages. Macrophages were injected on day 20 to ensure exposure to the booster collagen treatment. Data are means ± s.e.m. of three independent experiments; n= 30 mice per group. *P< 0.05; **P< 0.01. (f) In vivo tracking of i.p.-injected DIR-labeled non-transduced macrophages in mice with oxazolone-induced contact hypersensitivity in the right ear. OXA, oxazolone.. **P < 0.01 (means ± s.d.; n>3). (g) LxVP lentivirus protects against contact hypersensitivity. Image analysis of ear inflammation (luminol signal) in mice i.p. injected with LxVP or mutLxVP lentivirus. ***P < 0.001 (mean ± s.d.; n=2, 12 mice per group).

Figure 4. LxVP expression polarizes macrophages to an M2 anti-inflammatory phenotype. Multiplex analysis of (a) pro-inflammatory cytokines and (b) anti-inflammatory IL10 in culture supernatants of macrophages from mice i.p. injected with LxVP or mutLxVP lentivirus. (c) Real-time PCR analysis of Argl and iNOS mRNA in LxVP- and mutLxVP- transduced macrophages (mean ± s.d.; n=3). (d) Flow cytometry analysis of Mrcland SIGNRl expression in LxVP- and mutLxVP-transduced macrophages, (e) Macrophage presentation of antigen (ovalbumin) to B3Z T-cell hybridoma. (f) Phagocytosis of opsonized red blood cells, (g) Osteoclast differentiation, recorded as the number of multinucleated TRAP+ cells, (b) and (d) show representative experiments; all other data are means ± s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001. (h) Confocal immunofluorescence showing colocalization of GFP (green) and Mrclor IL10 (red) in sections of arthritic paws locally-injected with LxVP or mutLxVP lentivirus.

Figure 5. CN modulates the M2 phenotype of macrophages, (a) CN activity in total protein extracts of macrophages from mice i.p. injected with LxVP or mutLxVP lentivirus. *P<0.05 (mean ± s.d.; n=3). (b) NFAT transcriptional activity (relative luciferase units) in untreated and zymosan-stimulated (Zym) macrophages from NFAT-luc transgenic mice. ***P<0.001 (mean ± s.d.; n=5). CN activity (c) and NFAT transcriptional activity (d) in LPS-induced Ml macrophages and IL4+IL13-induced M2 macrophages. *P< 0.05; ***P<0.001 (mean ± s.d.; n=3). (e) Western blot showing CnB deletion and CnA destabilization in macrophages from Cnbl Δ/flox mice transduced with CRE-encoding lentivirus. Tubulin expression is shown as loading control, (f) ELISA analysis of IL10 protein in supernatants of CN-deleted and control macrophages. **P<0.01 (mean ± s.d.; n=3). (g) Representative flow cytometry analysis of iNOS protein in CN-deleted and control macrophages, (h) mRNA expression of Mrcland Argl in control and CN-deleted macrophages. *P<0.05. (i) CIA score in arthritic mice inoculated in the footpad with CN-deleted macrophages. Data are means ± s.e.m from a representative experiment; n= 10 mice per group. *P<0.05. (j) Immunofluorescence staining of Mrcl (top) and IL10 (bottom) in sections from paws inoculated with CN-deleted or control macrophages.

Figure 6. Cnbl®/floxLysMcre mice are resistant to inflammation, (a) CnB and CnA protein expression in Cnbl ®/flox and control macrophages, (b) IL10 in supernatants of macrophage cultures **P<0.01 (mean ± s.d.; n=3). (c,d)mRNA expression of Mrcl, Argl and iNOS . (e) Macrophage presentation of antigen to B3Z T cell hybridoma.. (f) (f,g) In vivo imaging analysis of inflammation in Cnbl A/floxLysMcre+ and Cnbl Δ/floxLysMcre- mice after (f) sensitization with oxazolone in the right ear and (h) zymosan inoculation in the right hindpaw .

Figure 7. Collagen-induced arthritis scores. Images of inflamed paws at different arthritic scores.

Figure 8. Local lentiviral treatment does not exacerbate arthritic symptoms. CIA score in mice inoculated in the footpads (right hindpaw) with vehicle (PBS) or control lentivirus (mutLxVP). n= 10 mice per group.

Figure 9. Lentivirus targets macrophages upon local injection into footpads. Confocal images show co- staining of GFP and Mac3 in paw sections from mice inoculated in the footpads with GFP-encoding lentivirus.

Figure 10. Systemic lentiviral treatment does not exacerbate arthritic symptoms. CIA score in mice inoculated i.p. with vehicle (PBS) or control lentivirus (mutLxVP). n= 10 mice per group

Figure 11. Macrophages migrate from the peritoneal cavity to inflammation sites, (a) In vivo imaging analysis of DIR signal in mice with zymosan-induced acute inflammation in the right paw; images were captured at different times after i.p. transfer of DIR-labeled macrophages, (mean ± s.d.; n≥3).

Figure 12. M2 phenotype of macrophages transduced with LxVP lentivirus. (a) mutLxVP and LxVP transduced phagocytic macrophages (GFP). red, erythrocytes and phalloidin. (b) Perimeter (nm) (left), total area (nm2) (central) and TRAP staining images (right) of LxVP or mutLxVP-transduced macrophages in osteoclast differentiation assays. **P < 0.01, ***P < 0.001 (means ± s.d.;n=3). (c) Non-transduced cells express Mrcl in LxVP-inoculated paws. Confocal immunofluorescence showing GFP (green) and Mrcl (red) in arthritic paws locally-injected with LxVP or mut LxVP lentivirus.

Figure 13. CN activity is diminished in M2 BMDM. CN activity (nmol free phosphate) in G CSF-induced Ml BMDM and MCSF-induced M2 BMDM. *P < 0.05 (mean ± s.d. n=2).

Figure 14. (a) and (b) CsA and FK506 do not induce M2 polarization.

Figure 15. Predicted binding mode of LxVP based on docking and molecular dynamic simulations. The LxVP peptide (sticks) is positioned parallel to the B subunit-binding helix of CN A and forms additional contacts with residues in CN B. In the zoom view, hydrogen bonds and salt bridges are shown as dashed lines. CN residues predicted by MD simulation to be involved in ligand binding are labeled and numbered. The interactions are detailed in 2D and are summarized in a table.

Figure 16. p38 MAPK activity mediates the induction of anti-inflammatory macrophages by specific CN targeting, (a) Representative western blot showing expression of phosphorylated (P)-p38 and P-ERK in peritoneal macrophages treated with CsA or FK506 or transduced with LxVP or control lentivirus. p38 and tubulin were used as loading controls, (b) P-p38 (top) and CNB (center) protein expression in peritoneal macrophages from Cnbl ^flox/flox LysMCre (control) and Cnbl °* LysMCre⁺ (CN KO) mice treated with CsA or FK506. Tubulin was used as loading control, (c) Effect of p38 inhibition by SB203580 (SB) treatment on Mrcl, Argl and IL10 mRNA levels in LxVP-transduced macrophages and (d) CN-KO macrophages. *P < 0.05, **P<0.01, ***P < 0.001.

DETAILED DESCRIPTION OF THE INVENTION

The present invention confronts the problem of promoting the resolution of inflammation in an inflammatory disease. In this sense, the inventors identified the CN-inhibitory LxVP peptide as a critical modulator of macrophage polarization that determines the course of inflammation by showing that the mechanism of LxVP-mediated CN inhibition involves a switch of macrophages toward an antiinflammatory phenotype.

Therefore, the present findings are thus based on the surprising discovery that CN inhibition via a mechanism of LxVP-mediated CN inhibition in macrophages induces a switch of this type of cells towards an M2 anti-inflammatory phenotype, characterized principally by the inhibition of pro-inflammatory cytokines and iNOS expression, and the up-regulation of IL10, Argl and Mrcl. These macrophages acquired M2 functional hallmarks, including reduced antigen presentation and increased phagocytosis, contributing to a faster resolution of the inflammatory response.

The present data highlights the attractiveness of macrophages as targets for anti-inflammatory gene and cell therapy. Thus, the identification of LxVP as a regulator of macrophage polarization opens new ways for macrophage-based therapy in inflammatory diseases. One of these new ways is the use of macrophages as targets for anti-inflammatory gene therapy. In this sense, the inventors have herein demonstrated that by administering lentiviruses encoding the CN- inhibitory LxVPcl peptide they significantly reduced the severity of inflammation in a variety of inflammatory in vivo settings.

In this regard, the inventors analyzed the effect of lentiviral vectors encoding LxVPcl in a mouse model of collagen-induced arthritis (CIA), wherein this disease was triggered by two intradermal collagen injections (Example 2). Footpad injection of LxVPcl lentiviruses before the first collagen exposure had a clear prophylactic effect, with arthritic scores significantly lower in LxVP-treated mice than in animals inoculated with AxAA control lentiviruses (mutLxVP) (Fig. la left). Moreover, LxVPcl delayed the onset of the disease and reduced disease incidence from 85% to 40% (Fig. la right). Histological analysis of joint sections confirmed the near absence of inflammation in LxVP-treated mice, while joints of control- transduced animals showed abundant inflammatory cell infiltrates and severe bone and cartilage damage (Fig. lb). Injection of control lentiviruses did not exacerbate disease symptoms (Fig. 8).

Characterization of GFP expression in tissue sections five days after the lentiviral footpad injection revealed that all transduced cells expressed F4/80 or Mac3, indicating that macrophages are the main lentiviral target. Analysis of lentivirus biodistribution in the lymphatic system showed that transduced cells were selectively located in draining popliteal lymph nodes (LN) (Fig. lc). Analysis of sorted GFP+ cells in the LN indicated that all transduced cells were F4/80+/CDllb+/CDllc+ macrophages (Fig. Id).

In addition, in order to evaluate whether lentiviral administration has a therapeutic effect at more advanced stages of CIA, the inventors injected lentiviruses after the first appearance of inflammation. In this sense, the inventors used a systemic administration route to better model the polyarticular nature of arthritis, and selected intraperitoneal (i.p.) injection as an efficient route for macrophage recruitment and transduction. LxVP-treated mice maintained the low arthritic scores recorded at the time of lentiviral injection, whereas arthritis in control animals increased in severity (Fig. 2 a, b). Injection (i.p.) of control lentivirus did not exacerbate disease symptoms (Fig. 10). Consistent with the outcome of local prophylactic treatment, the only transduced (GFP+) cells identified in the peritoneal exudate of non- arthritic mice five days after the lentiviral injection were F4/80+/CDllb+/CDllc+ macrophages (Fig. 2c lower panel). CD4+, CD8+ or B220+ cells did not show detectable GFP expression (Fig. 2c lowerupper). Analysis of the lymphatic system after i.p. lentiviral inoculation detected this transduced

F4/80+/CDllb+/CDllc+ population exclusively in the draining parathymic LN (Fig. 2d).

Based on these findings, the inventors concluded that CN-inhibited macrophages via a mechanism of LxVP-mediated CN inhibition, has potential applications in the development of new anti-inflammatory strategies, wherein one possible mechanism of inducing LxVP-mediated CN inhibition is by using lentiviruses. In this regard, lentiviruses can be used for the in vivo transduction of macrophages, particularly F4/80+/CDllb+/ CDllc+ macrophages, a triple-positive population associated with allergy- induced lung inflammation, adipose tissue from obese individuals, and tumors, but which is also found in non-pathological settings. Although the inventors have identified this macrophage population as the sole target of lentiviral infection in vivo, it cannot be excluded that other cell types are transduced at low frequency. Another possible mechanism of inducing LxVP-mediated CN inhibition is by using cell therapy. As regards cell therapy and as illustrated herein below, the preferential migration of macrophages to inflammation sites opens up the possibility of autologous cell therapy, in which macrophages from patients could be switched toward an M2 anti-inflammatory phenotype ex vivo, and then reintroduced systemically, resulting in selective migration to inflammatory foci. Several characteristics of macrophages support their potential for anti-inflammatory cell therapy. Macrophages are central players in disease progression and their infiltration of the inflammation site correlates with disease severity. Moreover, treatments that inhibit macrophage-derived pro-inflammatory cytokines effectively ameliorate the symptoms and impede the progression of disease. Key features, exploited in this invention, are the accessibility of macrophages and the ease with which their phenotype can be modulated, enabling control of the production of regulatory mediators and of their influence on other cell populations.

In this sense, the inventors first tested whether macrophages migrated from the peritoneal cavity toward sites of inflammation. To enable analysis of directed migration, the inventors generated a single inflammation focus by injecting zymosan into the right hind paws of mice. These mice were subsequently inoculated i.p. with donor macrophages labeled ex vivo with the fluorescent tracer DIR. Macrophages migrated selectively to the inflammation site, with no signal detected in control paws (Fig. 3a). Migration was already detected two hours after macrophage transfer, increased progressively for 2 days, and signal remained for at least 13 days (Fig. 3b and Fig. 11). LxVP- and control-transduced macrophages had the same migratory capacity as revealed by in vivo analysis of DIR signal and GFP staining in inflamed paws (Fig. 3c,d).

To verify that the transduced macrophages recruited to the inflammation sites are responsible for the beneficial effects of LxVP, the inventors performed cell therapy assays in the CIA model, in which transduced donor macrophages were injected directly into the footpads of arthritic mice. Paws inoculated with LxVP-transduced macrophages showed significantly lower arthritic scores than those inoculated with control-transduced macrophages (Fig. 3e), confirming the key role of LxVP-transduced macrophages in the resolution of inflammation.

Macrophages also migrated selectively to the inflammation site in an alternative inflammatory model of oxazolone-induced contact hypersensitivity in mouse ears. In these experiments, i.p.-injected DIR- labeled macrophages were only detected in the inflamed ear (Fig. 3f). Furthermore, direct i.p. injection of LxVP lentivirus into mice with contact hypersensitivity prevented ear inflammation (Fig. 3g). These results emphasize the anti-inflammatory action of calcineurin inhibition with LxVP peptide and suggest that LxVP-transduced macrophages have broad potential for the treatment of different inflammatory diseases.

The present findings moreover show that macrophages migrate specifically from the peritoneal cavity to inflammation sites. Several studies have described macrophage recruitment to the peritoneal cavity in response to an i.p inflammatory insult and to inflammation sites after intravenous injection; however, the migration of macrophages from the peritoneal cavity to peripheral inflamed locations has not been reported. The present results indicate that macrophages migrate rapidly after i.p. lentiviral infection, implying that transgene expression begins once transduced cells have arrived at the affected tissue, thus specifically localizing the action of the therapeutic molecule at the site of inflammation. Conventional treatments for inflammatory diseases are administered systemically, which can entail a strong immunosuppression leading to increased susceptibility to opportunistic infections or tumors. This is the case with the immunosuppressants (IS) CsA and FK50641. Moreover, IS/IP complexes alter CN- independent processes by interfering with the net mitochondrial uptake of ATP, an effect which is the basis of many of these compounds' undesired effects. In contrast, LxVP inhibits CN specifically in an immunophilin-independent manner, and therefore can suppress inflammatory responses without the serious toxic effects associated with IS/IP complexes.

In addition, the inventors have come to the surprising result that even though known drugs such as CsA and FK506 bind the same Calcineurin protein binding pocket, namely the CN A-CN B composite surface, as LxVP peptides, they do not induce M2 macrophage polarization as shown in figure 14. The reason is because it is not enough for a compound to bind the CN A-CN B composite surface to induce M2 antiinflammatory macrophage polarization but it must also do so in an immunophilin independent manner and preferably activate p38 MAPK.

In this sense, the inventors of the present invention have surprisingly found that the anti-inflammatory actions of LxVP CN-targeted macrophages are mediated by activation of p38 MAPK. In this sense, the inventors analyzed whether the anti-inflammatory macrophage phenotype triggered by LxVP administration was mediated by p38 activity, and if p38 activation was implicated in the differences between LxVP and other known drugs such as CsA and FK506 that bind the same Calcineurin protein binding pocket.

In this regard, Figure 16 shows that LxVP-transduced, but not control or IS-drug-treated macrophages have a sustained activation of p38 (phospo p38 (P-p38), however no effect was detected in ERK activation (Fig. 16a,b). Moreover, treatment of LxVP-transduced macrophages with the p38-chemical inhibitor SB203580 (SB) reduced the expression of the anti-inflammatory markers Mrcl, Argl and ILIO to basal levels (Fig. 16c,d), indicating that p38 contributes to the anti-inflammatory phenotype of CN- targeted macrophages. Such result is not achieved by other known drugs such as CsA and FK506 that inhibit CN by binding to the same Calcineurin protein binding pocket.

Therefore, the inventors have surprisingly found that it is possible to induce a switch of a proinflammatory macrophage toward an anti-inflammatory phenotype by transforming or transducing said macrophage with an exogenous compound capable of inhibiting the CN-NFAT signaling pathway in an immunophilin independent manner by binding to the CN A-CN B composite surface or by allostericaliy modulating said surface and preferably capable of activating p38 MAPK, wherein the CN A-CN B composite surface is composed of amino acids 341 to 363 of the calcineurin catalytic subunit A (CN A) as set forth in SEQ ID No 4 and amino acids 113 to 127 of the calcineurin regulatory subunit B (CN B) as set forth in SEQ ID No 5. Thus, a first aspect of the present invention refers to a macrophage (from hereinafter "macrophage of the invention") comprising an exogenous compound (from hereinafter "compound of the invention") capable of inhibiting the CN-NFAT signaling pathway in an immunophilin independent manner by binding to the CN A-CN B composite surface or by allostericaliy modulating said surface, wherein the CN A-CN B composite surface is composed of amino acids 341 to 363 of the calcineurin catalytic subunit A (CN A) as set forth in SEQ ID No 4 and amino acids 113 to 127 of the calcineurin regulatory subunit B (CN B) as set forth in SEQ ID No 5, and wherein said exogenous compound induces a switch of the macrophage toward an anti-inflammatory phenotype.

In another preferred embodiment of the first aspect of the invention the exogenous compound capable of inhibiting the CN-NFAT signaling pathway in an immunophilin independent manner binds directly to the CN A-CN B composite surface.

It is noted that the compounds of the invention, with the above mentioned capabilities, may be produced by any person skilled in the art in a routine manner, without excessive experimentation, from the information provided in the present invention. As a way of example, these types of compounds could be identified in in vitro assays of calcineurin activity by their capacity to block the dephosphorylation of the calcineurin substrate in an immunophilin independent manner and preferably activate p38 MAPK. These compounds, in addition to inhibit the phosphatase activity of recombinant calcineurin should be able to efficiently compete the binding of the LxVP peptide to Calcineurin. In summary, these types of compounds should be able to:

a. Inhibit the phosphatase activity of calcineurin in the absence of immunophilins; and b. Compete the binding of the LxVP peptide to Calcineurin or be competed by LxVP for the binding to Calcineurin. As used in the specification and the appended claims, the CN A-CN B composite surface that serves as the protein binding pocket of the compounds of the invention is defined in figure 15.

As used in the specification and the appended claims the term "macrophage" must be understood as a white blood cell of the immune system differentiated from bone marrow derived monocytes.

Macrophages are characterized by their phagocytic activity and their antigen presentation capacity, in this way macrophages are key players in both the innate and adaptive immune responses.

Phenotypically macrophages express the surface marker F4/80 (Ly71) and may express also other surface markers such as CDllb (Macl), CDllc, CD14, CD40 or CD68. In a preferred embodiment of the invention the macrophage is positive for the following phenotypic markers: F4/80+, CDllb+ and CDllc+.

As used in the specification and the appended claims, the term "transformed" must be understood as the insertion of new genetic material into nonbacterial cells, including animal and plant cells; this term is synonymous to transfection. As used in the specification and the appended claims the term "transduction" must be understood as the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact (which occurs in conjugation), and it is DNAase resistant (transformation is susceptible to DNAase). Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome.

As used in the specification and the appended claims the term "anti-inflammatory phenotype" includes those macrophages that function in wound healing and tissue repair, and those that turn off immune system activation by producing increased levels of anti-inflammatory cytokines like IL-10 or TGFb but reduced levels of pro-inflammatory cytokines such as IL-12. In particular, this term includes those macrophages characterized by an increased expression of anti-inflammatory cytokine IL-10 and/or transforming growth factor beta (TGFP) and a reduced expression of at least a pro-inflammatory cytokine, preferably IL-12, in comparison to an untreated control macrophage.

In a preferred embodiment of the first aspect of the invention, the compound of the invention can be a nucleotide sequence, a gene construct or an expression vector capable of coding for a suitable LxVP peptide which in turn is capable of inhibiting the CN-NFAT signaling pathway. Such compounds may be used as drugs to transform human host macrophage cells in a procedure of treatment and prophylaxis of in vivo or ex vivo gene therapy of a human being affected by an inflammatory disease. The gene therapy procedures may be applied directly on the patient, administering one of these aforementioned compounds, such as nucleotide sequences, as a drug by using an appropriate vector capable of infecting macrophage cells (in vivo gene therapy), or macrophage cells could be extracted from a human being, and once ex vivo, they could be transformed with the nucleotide sequences and subsequently returned to the human being (cell therapy), or maintained ex vivo for subsequent uses.

Therefore, in a second aspect of the present invention the macrophage of the invention is transformed or transduced with a nucleotide sequence coding for a peptide capable of inhibiting the CN-NFAT signaling pathway, wherein said peptide comprises the following sequence:

(I) R1-L- 2-V-P-R3

As already stated above, the present invention is based on the fact that the inventors have discovered the surprising usefulness of LxVP peptides as critical modulators of macrophage polarization. Based on the amino acid structure of said main known LxVP modulators (LxVP peptides cl, c3 and c4

corresponding to sequences SEQ ID 1 to SEQ ID No 3), the inventors have come up with amino acid sequence (I) which forms the basic starting core for the setting-up of all those compounds capable of, on the one hand, inhibiting the CN-NFAT signaling pathway via a mechanism of LxVP-mediated CN inhibition and, on the other hand, switching macrophages towards an anti-inflammatory phenotype.

It is noted that the inventors have already shown that the LxVPc2 peptide is a very weak competitor of CN binding to NFAT, suggesting that the NFATc2-CN interaction would be mainly driven by the PxlxlT motif. In contrast LxVP peptides based on NFATcl, c3 and c4 sequences (SEQ ID No 1 to SEQ ID No 3) proved to be very efficient competitors of the in vitro interaction of CN with NFATcl or NFATc2.

Expression of the LxVPc3 and LVPc4 peptides in macrophages might therefore be expected to have an effect on the inhibition of the CN-NFAT signaling pathway and therefore on the polarization of macrophages towards an anti-inflammatory phenotype. In this sense, it is additionally noted that the above amino acid sequence (I) has been constructed based on the fact that the residues conserved in the LxVP motif, Leucine, Valine and Proline are essential for the interaction of NFATcl, c3 and c4 with CN. In addition, this structure takes into account the fact that other residues present in said motif are also of importance for the protein-protein interaction. In this sense, beyond these three conserved residues NFATcl, NFATc3 and NFATc4 possess an aromatic amino acid adjacent to the nucleus of the LxVP motif (Tyrosine, Phenylalanine and Tyrosine, respectively). Furthermore, it is known that NFATc2 comprises a Proline residue adjacent to the C-terminal end of the nucleus of the LxVP motif, this gives rise to two consecutive Proline residues which the rest of the NFAT members lack. It is noted that, as stated above, LxVPc2 is not capable of selectively inhibiting the CN-NFAT signaling pathway and thus the reason why this residue is excluded from the basic starting core of the present invention.

In a preferred embodiment of the second aspect of the invention, amino acid sequence (I) is selected from a peptide comprising any of the following sequences:

a. SEQ ID No 1 (LxVPcl) or SEQ ID No 2 (LxVPc3) or SEQ ID No 3 (LxVPc4); or b. A variant of (a) which is at least 85%, preferentially 95% homologous to SEQ ID No 1 or to SEQ ID No 2 or to SEQ ID No 3 in terms of amino acid identity.

As used in the present description, the term "variant" is intended to include any amino acid sequence which may be isolated or constructed on the basis of the amino acid sequences shown in this invention, for example, by means of the insertion of amino acid substitutions, conservative or non-conservative, including the insertion of one or more amino acids, the addition of one or more amino acids, at any of the ends of the molecule, or the deletion of one or more amino acids, at any end or in the interior of the sequence, on condition that this shall not modify the basic nucleus of the invention (amino acid sequence (I)) and that it shall constitute a peptide capable of inhibiting the CN-NFAT signaling pathway.

In general, a variant is fundamentally homologous to the amino acid sequences mentioned above. Preferably, a variant is a sequence fundamentally homologous to the above mentioned amino acid sequences having a degree of amino acid identity of at least 85%, or more preferably, of at least 95%.

a. SEQ ID No 1 (LxVPcl);

b. SEQ ID No 2 (LxVPc3); or

c. SEQ ID o 3 (LxVPc4). In yet another preferred embodiment of the invention, the macrophage is at least positive for the phenotypic marker F4/80+, more preferably the macrophage is at least positive for the following phenotypic markers: F4/80+, CDllb+ and CDllc+.

Any of the compounds of the invention comprising an amino acid sequence, may be produced by the gene expression of nucleotide sequences which permit the encoding of their residues, likewise by means of their chemical synthesis. Therefore, the amino acid sequences of the peptides of this invention and the nucleotide sequences which encode the same are comprised of a series of sequences which any person skilled in the art may produce in a routine manner, without excessive experimentation, from the information provided in this invention. Replacements or modifications, suitable for carrying out the various aspects of this invention may be determined by means of routine experimentation in order to produce peptides with the structural and functional properties described, for example by comparing the protein-protein interactions by means of in vitro or in vivo competition assays using CN with any one of the NFATs cl-c4.

In general, the gene expression of the nucleotide sequences of the present invention is produced through expression vectors. Examples of suitable expression vectors may be selected according to the conditions and needs of each particular use among cell expression plasmids which may also contain markers which may be used for selecting the cells transfected or transformed with the gene or genes of interest. The choice of vector will depend on the host cell and on the type of use desired.

Therefore, in accordance with a particular embodiment of this invention, said vector is a plasmid or a viral vector capable of infecting macrophage cells. This vector, preferably a viral vector, coding for any of the above mentioned amino acid sequences may be used as a drug in order to transform human macrophage cells in a procedure of treatment and prophylaxis of in vivo gene therapy of a human being affected by an inflammatory disease.

Thus, a third aspect of the invention refers to a vector (from hereinafter "vector of the invention") comprising a compound of the invention comprising a polynucleotide sequence, preferably coding for amino acid sequence (I), which is capable of transporting or delivering said polynucleotide sequence into the macrophages of a subject.

The term "subject" means all mammals including humans. Examples of subjects include, but are not limited to, humans, cows, dogs, cats, goats, sheep, pigs, and rabbits. Preferably the term subject refers to humans.

a. SEQ ID No 1 (LxVPcl) or SEQ ID No 2 (LxVPc3) or SEQ ID No 3 (LxVPc4); or

In another preferred embodiment of the third aspect of the invention, the vector comprises a polynucleotide sequence coding for a peptide consisting of any of the following sequences: a. SEQ ID No 1 (LxVPcl);

b. SEQ ID o 2 (LxVPc3); or

c. SEQ ID No 3 (LxVPc4).

In another preferred aspect of the invention, the vector is a viral vector encoding for an amino acid sequence as defined in any of the embodiments of the second aspect of the invention, wherein preferably the viral vector is a lentivirus. Preferably, said lentivirus are pseudotyped lentiviral vectors consisting of vector particles bearing glycoproteins (GPs) derived from other enveloped viruses. Among the first and still most widely used GPs for pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G), due to the very broad tropism and stability of the resulting pseudotypes. Lentiviruses particles bearing the vesicular stomatitis virus GP (VSV-G) have been found herein to predominantly infect macrophages.

In addition to the above, the compounds of the invention can be also delivered to the macrophage cells in other ways. For example, the compounds can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on whether the delivery is occurring for example in vivo or in vitro. Thus, the compounds can comprise, in addition to the disclosed nucleic acids or vectors, for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting of macrophages, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987); U.S. Pat. No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule or a nanoparticle that can be targeted to specific cell types, in this specific case to macrophages.

Thus, a further aspect of the invention refers to a non-viral vector (from hereinafter"non-viral vector of the invention") comprising, but not limited to, nanoparticles or lipids for the delivery of a compound of the invention into the macrophages of a subject. More preferably, said non-viral vector is used for the delivery of any of the following compounds:

a. A peptide comprising the following sequence R1-L-R2-V-P-R3, wherein said sequence is as

defined above; or

b. An amino acid sequence selected from any of the following:

a. SEQ ID No 1 (LxVPcl) or SEQ ID No 2 (LxVPc3) or SEQ ID No 3 (LxVPc4); or

b. A variant of (a) which is at least 95% homologous to SEQ ID No 1 or to SEQ ID No 2 or to SEQ ID No 3 in terms of amino acid identity. In addition, a further embodiment of the invention refers to the use of the non-viral or viral vectors of the invention for the ex vivo transformation or transduction of macrophages.

In any case, this method also includes a method of increasing the efficacy of other agents given for the same disease, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the vector or transformed macrophage of the invention; and, optionally, a pharmaceutically acceptable carrier, thereby increasing the efficacy of the other agent or agents.

Therefore, a further aspect of the invention refers to a composition (from hereinafter composition of the invention), preferably a pharmaceutical composition (from hereinafter "pharmaceutical composition of the invention"), which comprises the vector and/or the macrophage of the invention and optionally a pharmaceutically acceptable carrier.

As used in the specification and the appended claims, the pharmaceutical composition comprising the vector and/or macrophage of the invention can be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" a material is meant that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as it would be well known to one of skill in the art.

A fourth aspect of the invention refers to the pharmaceutical composition of the invention, the composition of the invention, the macrophage of the invention, the vector of the invention or the non- viral vector of the invention for its use in therapy.

A fifth aspect of the invention refers to the pharmaceutical composition of the invention, the composition of the invention, the macrophage of the invention, the vector of the invention or the non- viral vector of the invention for its use in the treatment or prevention of an inflammatory disease.

As used herein, the term "prevention" refers to the methods to avert or avoid a disease or disorder or delay the recurrence or onset of one or more symptoms of an inflammatory disorder in a subject resulting from the administration of the pharmaceutical composition of the invention.

In a preferred embodiment of the fifth aspect of the invention, the inflammatory disease can be selected from the list consisting of rheumatoid arthritis, encephalomyelitis, lupus erythematosus, psoriasis, atopic dermatitis, allergies , contact hypersensitivity, hepatitis, atherosclerosis, obesity, diabetes and Crohn^'s disease. Effective dosages and schedules for administering the pharmaceutical compositions of the invention disclosed herein may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions, are those large enough to produce the desired anti-inflammatory effect in the disorder. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.

Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

The pharmaceutical composition, the composition of the invention, the macrophage of the invention, the vector of the invention or the non-viral vector of the invention provided by this invention may be administered by any suitable administration route; to this end, said composition shall be formulated in the pharmaceutical format suited to the administration route chosen.

The pharmaceutical composition of this invention, the composition of the invention, the macrophage of the invention, the vector of the invention or the non-viral vector of the invention may be used in a method of treatment in an isolated manner or jointly with other pharmaceutical compounds.

The following examples merely serve to illustrate the present invention. EXAMPLES

Example 1. Materials and methods used to conduct the examples of the present invention.

1. Animals: Male DBA1J mice (7-10 weeks old) and C57BL6 mice (6-8 weeks old) were purchased from Charles River. NFAT-luc transgenic mice and Calcineurin Bl conditional knock out mice were kindly provided by Prof. Jeffery D. Molkentin and Prof. Gerald R. Crabtree respectively. Animals were housed in a dedicated pathogen-free facility and were fed and watered ad libitum.

Animal studies were approved by the local ethics committee. All animal procedures conformed to EU Directive 86/609/EEC and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under Real Decreto 1201/2005.

2. Lentivirus production and cell infection. Lentiviruses expressing GFP-peptide fusion proteins and luciferase were previously described. Cre recombinase encoding lentiviruses were generated by cloning a Cre PCR product in pHRSIN lentiviral vector. Lentiviruses were produced by transient calcium phosphate transfection of H EK-293 cells, employing a three plasmid HIV-derived and VSV pseudotyped lentiviral system kindly provided by M. K. Collins, U niversity College London, U K. HEK293T cells were cultured in Dulbecco's Modified Eagle medium (Sigma) supplemented with

10% fetal bovine serum, L-glutamine (2mM) and penicillin (lOOU/ml) and streptomycin

(lOOU/ml). Cells were plated at 30% confluence and transfected the next day. At 48 h and 72 h after transfection, supernatants were collected, concentrated by ultracentrifugation (26000 rpm for 2 h at 4 °C) and stored at -80 °C. For infection of peritoneal macrophages, mice were i.p. inoculated with 300 μΙ concentrated lentiviral supernatant (7x108-2x109 TU/ml).

Isolation and culture of peritoneal macrophages. Peritoneal macrophages elicited by the i.p. injection of thyoglicoiiate (Difco) or lentivirus were collected by peritoneal lavages with PBSIX ( 2 x 10 ml) after 3 or 5 days respectively and immediately cultured in AlphaMEM (Lonza) supplemented with 10% fetal bovine serum, L-glutamine (2mM) and antibiotics. After overnight culture, non-adherent cells were removed.

Collagen-induced arthritis and lentiviral treatments. Male DBA1J mice were inoculated intradermal^ at day 0 and 21 with 200 μg of type II collagen from chicken (Sigma) emulsified (1/1) in complete Freund^'s adjuvant (Difco) plus Mycobacterium tuberculosis (Difco) . Limbs were graded by blinded examiners for arthritic scores as follows: grade 0= no swelling; grade 1= slight swelling and erythema; grade2= moderate swelling and edema; grade 3= extreme swelling and pronounced edema; grade 4= joint rigidity. Joint sections were analyzed by Masson^'s trichrome staining. For prophylactic treatments, lentiviral vectors were injected five days before the first immunization with collagen (day -5). For therapeutic treatments vectors were injected on day 28. Lentiviral particles (7x108-2x109 TU/ml) were administered in the footpad (30 μΙ) or in the peritoneal cavity (300 μΙ)

Flow cytometry and sorting. Peritoneal cells were washed and suspended in PBS containing 1% BSA and 0.5% EDTA. Before primary antibody labeling, cells were incubated with Fc receptor- blocking antibody (BD Pharmingen). Cells (2x105) were incubated with fluorophore-coupled primary antibodies anti-F4/80, anti-CDllb, anti-CDllc, anti-B220, anti-CD4 and anti-CD8 (1/100) (all from BD Pharmingen) for 30 min at 4°C and then washed twice. For staining with antibodies to Mrcl and SIGNR1 (Serotec), cells were fixed and permeabilized with methanol and incubated with primary antibody at RT for lh. Cells were incubated with secondary antibodies for 15 min at 4 °C and washed for cytometric analysis. Data were analyzed withy FlowJo software 1.6.

GFP-positive cells in lymph nodes (LN) were separated by FACS (Aria cell sorter)

Real time qRT-PCR. Total RNA was extracted from macrophages using TriPure reagent (Roche). Complementary DNA was synthesized using M-MLV retro transcriptase (Invitrogen). Argl, iNOS, IL10 and Mrcl mRNA transcripts were analyzed by SYBR Green gene expression assay (Applied Biosystems).

Argl: Foward: SEQ ID No 6

Reverse: SEQ ID No 7

iNOS: Foward: SEQ ID No 8

Reverse: SEQ ID No 9

IL10: Foward: SEQ ID No 10

Reverse: SEQ ID No 11

Mrcl: Foward: SEQ ID No 12

Reverse: SEQ ID No 13 Cell lysates and immunoblotting. Cells (2.5x106) were incubated with lysis buffer (ΙΟΟμΙ) (20 mM Tris HCI pH 7.5, 5 mM MgCI2, 50 mM NaF, 10 mM EDTA, 500 mM NaCI, 1% Triton x-100 and protease inhibitors) with shaking for 15 min at 49C. Debris was removed by centrifugation (14000 r.p.mJ42C/15 min.) and protein was quantified by the Bradford assay (Biorad). Proteins were separated by SDS-PAGE in 15% gels and transferred to nitrocellulose membranes for immunodetection. Membranes were probed with anti-CNB (l/1000)(Upstate), anti-CNA (1/1000) (Chemicon) or anti-tubulin (1/40000) (Sigma) primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG (CNB and tubulin) and goat anti-rabbit IgG (CNA) (Thermo Scientific). The enhanced chemiluminiscence system (GE

Healthcare) was used to detect peroxidase activity.

Cytokine measurements. Cytokines were measured in supernatants of 48 h macrophage cultures by IL10 ELISA (Diaclone) or with the mouse 10 plex kit Flowcytomix (Bender Medsystems).

Immunohistochemistry and immunofluorescence. For immunohistochemistry, paw fragments were collected after mice perfusion through the left ventricle with PBS1X and fixed in 10% formalin at 4⁹C for 24 h. Fixed tissues were dehydrated through a graded ethanol series for paraffin embedding. 5 μιτι tissue sections were obtained for staining. Endogenous peroxidase was blocked with 3% hydrogen peroxide and putative endogenous avidin-binding sites were blocked with the Avidin/Biotin Blocking kit (Vector laboratories). Slide-mounted sections were incubated overnight at 4 ⁹C in anti-GFP primary antibody (Invitrogen) diluted 1:250 in PBS, 1% horse serum, 5% BSA. After washes, slides were incubated with biotinilated secondary antibody diluted in the same solution for 1 h at RT. Slides were then incubated with preformed Avidin- Biotin peroxidase complexes (ABC Kit, Vector Laboratories) and stain was developed by incubation with the peroxidase substrate diaminobenzidine For immunofluorescence, paw fragments were removed after mice perfusion with PBS1X and snap-frozen in O.C.T.embedding medium (Tissue-Tek). Anti-GFP was used as before; anti-Mrcl (Serotec), anti-F4/80 (AbD Serotec) and anti-Mac3 (Santa Cruz) were diluted 1:50 and anti-ILlO was diluted 1:100.

CN activity assay. Calcineurin enzyme activity in total macrophage extracts (5 g) was determined with the Calcineurin Cellular Activity Assay Kit (Biomol)

Reporter gene assays. Peritoneal macrophages from NFAT-luc transgenic mice were stimulated with zymosan (100 μg/ml) (Sigma) or vehicle for 5 h. Cell homogenates were lysed according to the Luciferase Assay System (Promega) and luciferase activity was measured with a luminometer (Berthold detection systems, Sirius). Relative luciferase units (R.L.U.) were normalized to protein content. LN from animals infected with lentivirus encoding luciferase were homogenized and filtered through 70 μηη nylon mesh. Cell homogenates were analyzed as above.

Macrophage migration. Thyoglicolate-elicited peritoneal macrophages were collected by peritoneal lavage and labeled ex vivo (5min RT) with the fluorescent lipophilic tracer DIR (Invitrogen). Labeled peritoneal macrophages were reinjected into the peritoneal cavity of receptor mice with zymosan-induced acute inflammation in the right hindpaws (180 μg Zym (Sigma)/footpad) or oxazolone-induced contact hypersensitivity in the right ears (see below). DIR signal was tracked in vivo with the IVIS system (ICG/ICG BCK filters) (Xenogen). Images were analyzed with Living image 3.1. When indicated, mice were inoculated with 300 μΙ lentivirus- enriched supernatant (7x108-2x109 TU/ml) Antigen presentation. Macrophages were cultured in 6-well plates in the presence of 2 mg/ml of Ovalbumin (OVA) antigen for 2 h. After several washes with PBS, macrophages were cultured for 5 h in regular culture medium after which 2 x 106 β-galactosidase-expressing B3Z T cells were added and incubation continued overnight. Antigen presentation was quantified by assessing the hydrolysis of chlorophenol red- -D-galactopyranoside (CPRG) (Calbiochem) in a

spectrophotometer (O.D. 595-655nm)

Phagocytosis. In vivo infected macrophages were collected by peritoneal lavage and plated on crystal overnight. (1x106 total cells/ crystal). Non-adherent cells were removed and

macrophages were serum-starved for 2 h. Sheep red blood cells were untreated or opsonized with rabbit IgG and then added to the macrophage culture for 15 min. Cells were fixed, permeabilized and stained with phalloidin TxRed (Invitrogen) and Alexa 647 goat anti-rabbit IgG . Phagocytosed red blood cells were counted under a confocal microscope.

Osteoclast differentiation. In vivo infected peritoneal macrophages were cultured in AlphaMEM with RANKL (100 ng/ml) and MCSF (20 ng/ml) (both from Peprotech) for seven days. Cells were stained with tartrate-resistant acid phosphatase (TRAP) (Sigma) on day 7 and multinucleated

TRAP+ cells were counted. Cell perimeter and area were analyzed with Metamorph software Ex vivo M1/M2 macrophage polarization. Peritoneal macrophages were cultured for 24 h in the presence of LPS (100 ng/ml) for Ml polarization or of IL4 (20 ng/ml) plus IL13 (20 ng/ml) (Peprotech) for M2 polarization. Bone marrow (BM) cells were obtained from femurs and tibias. After lysis of erythrocytes, cell homogenates were plated in AlphaMEM supplemented with 10% fetal bovine serum, HEPES (lOmM), β-mercaptoethanol (50μΜ), L-glutamine (2mM) and antibiotics. Cells were cultured for six days in the presence of GMCSF (5000U/ml) for Ml differentiation or MCSF (5000U/ml) (both from Peprotech) for M2 differentiation. When indicated, lentivirus was added to culture medium on day two of differentiation and maintained for 48 h. On day six macrophages were stimulated with LPS (lOOng/ml) and culture supematants were collected the next day for IL10 ELISA (Diaclone).

Cell therapy. For experiments with macrophages transduced with lentivirus encoding LxVP or mutLxVP, transduced cells were obtained from donor mice as described above. In experiments with CN KO cells, macrophages from Cnbl Δ /flox mice were infected ex vivo with CRE recombinase-encoding lentivirus to induce CN deletion. Macrophages (4x104) were injected into the footpads of receptor mice one day before the second immunization with collagen. Mice were assessed daily for arthritic symptoms.

Zymosan-induced acute inflammation. Acute inflammation was induced in mouse paws by injection of 180 g of zymosan (30μΙ) (Sigma) in the footpads. Stock zymosan solution was prepared at 30 mg/ml in endotoxin-free saline by boiling twice and subsequent sonication.

Working solution was prepared by diluting in saline.

Oxazolone-lnduced contact hypersensitivity. Mice were sensitized on the abdomen with 2% 4- ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) (Sigma) in absolute ethanol. For migration assays DIR-labeled macrophages were injected i.p six days later, and after 5 h hypersensitivity was elicited in the right ear with 1% oxazolone in absolute ethanol. In experiments to assess the therapeutic effect of lentivirus, mice were inoculated i.p. on day five with LxVP or mutLxVP lentiviral particles. Hypersensitivity was elicited the next day in the right ear with 1% oxazolone and ear inflammation was quantified on day nine with the MS imaging system after i.p. injection of luminol (200mg/kg body weight) (Sigma). Luminescence images were analyzed with Living image 3.1.

21. Statistical analysis. The significance of statistical differences was assessed by unpaired two-tailed Student^'s t-test using Graphpad Prism software 5.01. Differences were considered statistically significant at P<0,05.

Example 2. Macrophage-mediated effects of LxVP peptide delay the onset and reduce the severity of collagen-induced arthritis

To evaluate the anti-inflammatory potential of the CN-inhibitory LxVPcl peptide the inventors analyzed the effect of lentiviral vectors encoding this motif in a mouse model of collagen-induced arthritis (CIA), in which disease is triggered by two intradermal collagen injections. Footpad injection of LxVPcl lentivirus before the first collagen exposure had a clear prophylactic effect, with arthritic scores significantly lower in LxVPcl-treated mice than in animals inoculated with AxAA control lentivirus (mutLxVP) (Fig. la left). Moreover, LxVPcl delayed the onset of disease and reduced disease incidence from 85% to 40% (Fig. la right). Histological analysis of joint sections confirmed the near absence of inflammation in LxVPcl- treated mice, while joints of control-transduced animals showed abundant inflammatory cell infiltrates and severe bone and cartilage damage (Fig. lb).

Characterization of GFP expression in tissue sections five days after lentivirus footpad injection revealed that all transduced cells expressed F4/80 or Mac3, indicating that macrophages are the main lentiviral target (Fig. lc). Analysis of lentivirus biodistribution in the lymphatic system showed that transduced cells were selectively located in draining popliteal lymph nodes (LN) (Fig. lc). Analysis of sorted GFP+ cells in the LN indicated that all transduced cells were F4/80+/CDllb+/CDllc+ macrophages (Fig. Id).

To evaluate whether lentiviral administration has a therapeutic effect at more advanced stages of CIA, the inventors' injected lentivirus after the first appearance of inflammation. They used a systemic administration route to better model the polyarticular nature of arthritis, and selected intraperitoneal (i.p.) injection as an efficient route for macrophage recruitment and transduction. LxVPcl-treated mice maintained the low arthritic scores recorded at the time of lentiviral injection, whereas arthritis in control animals increased in severity (Fig. 2 a, b). Injection (i.p.) of control lentivirus did not exacerbate disease symptoms (Fig. 10).

Consistent with the outcome of local prophylactic treatment, the only transduced (GFP+) cells identified in the peritoneal exudate of non-arthritic mice five days after lentivirus injection were

F4/80+/CDllb+/CDllc+ macrophages (Fig. 2c lower panel). CD4+, CD8+ or B220+ cells did not show detectable GFP expression (Fig. 2c upper panel). Analysis of the lymphatic system after i.p. lentiviral inoculation detected this transduced F4/80+/CDllb+/CDllc+ population exclusively in the draining parathymic LN (Fig. 2d).

Example 3. Macrophages mediate anti-inflammatory effects after migration from the peritoneal cavity to inflammation sites

To elucidate the mechanism underlying the effect of LxVP-transduced peritoneal macrophages, the inventors first tested whether macrophages migrated from the peritoneal cavity toward sites of inflammation. To enable analysis of directed migration, the inventors generated a single inflammation focus by injecting zymosan into the right hind paws of mice. These mice were subsequently inoculated i.p. with donor macrophages labeled ex vivo with the fluorescent tracer DIR. Macrophages migrated selectively to the inflammation site, with no signal detected in control paws (Fig. 3a). Migration was already detected two hours after macrophage transfer, increased progressively for 2 days, and signal remained for at least 13 days (Fig. 3b). LxVPcl- and control-transduced macrophages had the same migratory capacity as revealed by in vivo analysis of DIR signal and GFP staining in inflamed paws (Fig. 3 c, d).

To verify that the transduced macrophages recruited to the inflammation sites are responsible for the beneficial effects of LxVP, the inventors performed cell therapy assays in the CIA model, in which transduced donor macrophages were injected directly into the footpads of arthritic mice. Paws inoculated with LxVP-transduced macrophages showed significantly lower arthritic scores than those inoculated with control-transduced macrophages (Fig. 3e), confirming the key role of LxVPcl-transduced macrophages in the resolution of inflammation.

Macrophages also migrated selectively to the inflammation site in an alternative inflammatory model of oxazolone-induced contact hypersensitivity in mouse ears. In these experiments, i.p.-injected DIR- labeled macrophages were only detected in the inflamed ear (Fig. 3f). Furthermore, direct i.p. injection of LxVPcl lentivirus into mice with contact hypersensitivity prevented ear inflammation (Fig. 3g). These results emphasize the anti-inflammatory action of calcineurin inhibition with LxVPcl peptide and suggest that LxVPcl-transduced macrophages have broad potential for the treatment of different inflammatory diseases.

Example 4. CN controls macrophage polarization

LxVPcl-transduced macrophages showed down-regulated expression of several inflammatory cytokines, including IL17, TNFct, IFNy and IL6 (Fig. 4a), and up-regulated the expression of the anti-inflammatory cytokine IL10 (Fig. 4b), suggesting that LxVPcl induces macrophages toward an M2 phenotype. Analysis of additional Ml and M2 phenotypic markers showed that LxVPcl-transduced macrophages have a typical M2 expression profile, including upregulation of Argl mRNA, down-regulation of iNOS mRNA and increased cell surface expression of Mrcl and SIGNR1 (Fig.,4c,d). LxVPcl-transduced macrophages moreover showed reduced antigen presentation capacity, increased phagocytic activity, and impaired differentiation to osteoclasts (Fig. 4 e-g and Fig. 12 a, b), all functional hallmarks of M2 macrophages. Mrcl MRCland IL10 were both up-regulated in inflamed paws inoculated with LxVP lentiviruses, confirming that this anti-inflammatory phenotype is also present in vivo under pro-inflammatory conditions (Fig. 4h). CN phosphatase activity was inhibited in LxVPcl-transduced peritoneal

macrophages, indicating that the LxVPcl-induced M2 polarization is mediated through an effect on CN (Fig. 5a). Consistent with this, LxVPcl-transduced peritoneal macrophages from NFAT-luciferase reporter transgenic mice showed decreased NFAT activity both under basal conditions and in response to the NFAT-inducer zymosan (Fig. 5b). To examine the implication of CN signaling in LxVPcl-independent macrophage polarization, the inventors polarized peritoneal macrophages ex vivo with physiological stimuli. Macrophages driven toward M2 with IL4 plus IL13 showed markedly lower CN and NFAT activities than cells polarized toward Ml with LPS (Fig. 5 c, d). Similar differences were observed between bone-marrow-derived macrophages polarized to M2 with MCSF and Ml with GMCSF (Fig. 13). Further evidence demonstrating the role of CN in M1/M2 polarization was obtained using CN-depleted macrophages. Since the inventors did not have access to an inducible CRE system for macrophages, the inventors transduced CnBl <8>/flox macrophages ex vivo with CRE-encoding lentivirus, which efficiently and specifically blocks CN production (Fig. 5e) while avoiding compensatory phenomena that can occur in conventional knockout mice. CN-deleted macrophages up-regulated IL10 expression and down- regulated expression of iNOS (Fig. 5 f, g). Other M2 markers such as Mrcl and Argl were also increased in CN depleted macrophages (Fig. 5h). In cell therapy experiments in the CIA model, footpad-injected CN- deleted macrophages had a beneficial effect on disease severity (Fig. 5i), mimicking the action of LxVPcl- transduced macrophages (Fig. 3e). Paws of mice inoculated with CN-deleted macrophages also showed increased expression of Mrcl and IL10 (Fig.5j).

To analyze the role of macrophage-expressed CN in the inflammatory response in vivo, the inventors generated Cnbl ®/flox LysM-Cre Rosa26 YFP mice which induce constitutive CN-deletion specifically in the myeloid lineage. CN expression was efficiently suppressed in macrophages (Fig. 6a), and these cells showed increased expression of IL10, decreased expression of iNOS and increased Argl and Mrcl mRNA (Fig. 6 b-d). M2 functionality of CN-deleted macrophages was evidenced by their impaired antigen presentation capacity (Fig 6e). Remarkably, examination of the inflammatory response of Cnbl

®/floxLysM-Cre mice revealed a resistance to oxazolone-induced contact hypersensitivity and zymosan- induced footpad inflammation (Fig.6 f,g).

Claims

34 CLAIMS

1. A macrophage comprising an exogenous compound capable of inhibiting the CN-NFAT signaling pathway in an immunophilin independent manner by binding to the CN A-CN B composite surface or by aliosterically modulating said surface, wherein the CN A-CN B composite surface is composed of amino acids 341 to 363 of the calcineurin catalytic subunit A (CN A) as set forth in SEQ ID No 4 and amino acids 113 to 127 of the calcineurin regulatory subunit B (CN B) as set forth in SEQ ID No 5, and wherein said exogenous compound induces a switch of the macrophage toward an anti-inflammatory phenotype.

2. The macrophage of claim 1, wherein the exogenous compound capable of inhibiting the CN- NFAT signaling pathway in an immunophilin independent manner directly binds to the CN A-CN B composite surface.

3. The macrophage of any one of claims 1 or 2, wherein the exogenous compound is further

capable of activating p38 MAPK.

4. The macrophage of any one of claims 1 to 3, wherein the anti-inflammatory phenotype is

characterized by an increased expression of anti-inflammatory cytokine IL-10 or transforming growth factor beta (TGFP) and a reduced expression of at least a pro-inflammatory cytokine, preferably IL-12, in comparison to an untreated control macrophage.

5. The macrophage of any of claims 1 to 4, wherein the exogenous compound comprises a

nucleotide sequence coding for a peptide which comprises the following sequence:

R1-L-R2-V-P- 3

wherein Rl is an aromatic aminoacid of the Tyrosine or Phenylalanine type, R2 is an Alanine or Serine-type amino acid, R3 is a Glutamine, Serine or Alanine-type amino acid , L is Leucine, V is Valine and P is Proline.

6. The macrophage of claim 5, wherein the nucleotide sequence codes for a peptide which

comprises any of the following sequences:

a. SEQ ID No 1 (LxVPcl) or SEQ ID No 2 (LxVPc3) or SEQ ID No 3 (LxVPc4); or

b. A variant of (a) which is at least 85% homologous to SEQ ID No 1 or to SEQ ID No 2 or to SEQ ID No 3 in terms of amino acid identity.

7. The macrophage of claim 5, wherein the nucleotide sequence codes for a peptide which consists of any of the following sequences:

a. SEQ ID No 1 (LxVPcl);

b. SEQ ID No 2 (LxVPc3); or 35 c. SEQ ID o 3 (LxVPc4).

8. A macrophage transformed or transduced with an exogenous compound, preferably a vector, comprising a nucleotide sequence coding for a peptide capable of inhibiting the CN-NFAT signaling pathway, which comprises the following sequence:

R1-L-R2-V-P-R3

wherein Rl is an aromatic aminoacid of the Tyrosine or Phenylalanine type, R2 is an Alanine or Serine-type amino acid, R3 is a Glutamine, Serine or Alanine-type amino acid, L is Leucine, V is Valine and P is Proline, and wherein said transformation or transduction induces a switch of the macrophage toward an anti-inflammatory phenotype.

9. The macrophage of claim 8, wherein the peptide comprises any of the following sequences: a. SEQ ID No 1 (LxVPcl) or SEQ ID No 2 (LxVPc3) or SEQ ID No 3 (LxVPc4); or

10. The macrophage of claim 8, wherein the peptide consists of any of the following sequences: a. SEQ ID No 1 (LxVPcl);

b. SEQ ID No 2 (LxVPc3); or

c. SEQ ID No 3 (LxVPc4).

11. The macrophage of any one of claims 8 to 10, wherein the anti-inflammatory phenotype is characterized by an increased expression of anti-inflammatory cytokine IL-10 or Transforming growth factor beta (TGF ) and a reduced expression of at least a pro-inflammatory cytokine, preferably IL-12, in comparison to an untreated control macrophage.

12. The macrophage of any of the previous claims, wherein the macrophage is further defined by being positive for the following phenotypic markers: F4/80+, CDllb+ and CDllc+.

13. A composition comprising the macrophage as defined in any of claims 1-12.

14. A vector which comprises a compound as defined in any of claims 1-12, which is capable of transporting or delivering said compound into the macrophages of a subject.

15. A vector which comprises a polynucleotide sequence coding for an amino acid sequence as defined in any of claims 5 to 10, which is capable of transporting or delivering said

polynucleotide sequence into the macrophages of a subject.

16. The vector of any one of claims 14 to 15, wherein the vector is a viral vector or a non-viral vector such as a nanoparticle. 36

17. The viral vector of claim 16, wherein the viral vector is a lentivirus.

18. Use of the vector as defined in any of claims 14 to 17, for the in vitro transformation or

transduction of macrophages.

19. A pharmaceutical composition comprising a vector as defined in any of claims 14 to 17 or a macrophage as defined in any of claims 1 to 12.

20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier.

21. The vector as defined in any of claims 14 to 17 or the macrophage as defined in any of claims 1 to 12, for its use in therapy.

22. The vector as defined in any of claims 14 to 17 or the macrophage as defined in any of claims 1 to 12, for its use in the treatment of an inflammatory disease selected from the list consisting of rheumatoid arthritis, encephalomyelitis, lupus erythematosus, psoriasis, atopic dermatitis, allergies , contact hypersensitivity, hepatitis, atherosclerosis, obesity, diabetes and Crohn^'s disease.

23. The pharmaceutical composition of any of claims 19 or 20 or the composition of claim 13, for its use in therapy.

24. The pharmaceutical composition of any of claims 19 or 20 or the composition of claim 13 for its use in the treatment of an inflammatory disease selected from the list consisting of rheumatoid arthritis, encephalomyelitis, lupus erythematosus, psoriasis, atopic dermatitis, allergies , contact hypersensitivity, hepatitis, atherosclerosis, obesity, diabetes and Crohn^'s disease.

25. The pharmaceutical composition of any of claims 19 or 20 or the composition of claim 13 for its use in combination therapy with a further active pharmaceutical ingredient.