WO2011107590A1

WO2011107590A1 - Cnn1 (cyr61) for prevention and therapy of inflammatory disease

Info

Publication number: WO2011107590A1
Application number: PCT/EP2011/053308
Authority: WO
Inventors: Wolfgang Poller; Carmen Scheibenbogen
Original assignee: Wolfgang Poller; Carmen Scheibenbogen
Priority date: 2010-03-04
Filing date: 2011-03-04
Publication date: 2011-09-09

Abstract

The invention relates to compounds for preventing or treating inflammatory diseases or dysfunctions of the immune system, comprising a polypeptide sequence of human CCN1 or a cyclic RGD peptide or a nucleic acids encoding a polypeptide sequence of human CCN1. It further relates to a pharmaceutical composition for use in preventing or treating of inflammatory diseases or dysfunctions of the immune system, comprising said compound. The use of such pharmaceutical composition in treating dilative cardiomyopathy of inflammatory etiology, autoimmune disorders, particularly autoimmune-myocarditis, or for preventing and treating of transplant rejection is provided.

Description

CCN1 (CYR61 ) for prevention and therapy of inflammatory disease Field of invention

This invention relates to polypeptides, peptides or nucleic acids for preventing and treating inflammatory diseases and dysfunctions of the immune system.

Progressive dilation and dysfunction of heart chambers, wall thinning, and tissue fibrosis are typical for dilated cardiomyopathy, a common final pathway of heart failure resulting from different etiologies, including monogenic defects in cardiac expressed genes and others triggered by exogenous factors (cardiotoxic drugs, cardiotropic viruses). Inflammatory cardiomyopathy (DCMi) represents an important DCM subtype and is often associated with virus-triggered heart-specific autoimmunity.

CCN1 , also known as CYR61 (cysteine-rich angiogenic inducer 61 ), is a member of the CCN gene family and has so far been functionally characterized in vitro. In cell cultures, it acts in a cell type- and context-dependent manner through binding to several integrins. CCN1 is involved in integrin-linked kinase mediated AKT and β-catenin-T cell factor/Lef signaling and stem cell differentiation. Several lines of evidence suggest that CCN1 plays a general role in the context of wound healing in vivo. It is locally up-regulated at injured sites, and coordinates in vitro a pattern of genes promoting extracellular matrix remodeling, cytokine induction, and angiogenesis, all of which are required for proper repair of tissue architecture. Furthermore, CCN1 belongs to the matricellular proteins and participates in mechanisms of cell adhesion, cell migration and angiogenesis.

EP0888452B1 shows the sequences of human CCN1 . The application of CCN1 for medical use has been suggested in different contexts; the indications are determined by the properties of CNN1 of positively influencing cell adhesion, cell migration and angiogenesis. US2004/0023910 shows the use of elevated expression of CCN1 in the treatment and diagnosis of human uterine leiomyomas. EP 202251 1A1 demonstrates the use of CCN 1 for coating implants. It has also been reported that CCN1 protects cardiomyocytes against oxidative stress, e. g. in case of infarction, via modulation of the beta1 -integrin-akt signaling pathway. WO 200198359A2 describes a method of treatment of breast cancer by inhibition of CCN1 . WO2004409391 1A1 shows the expression modulation of CCN1 for treatment of hypertrophy and neoplasms of the prostate.

In contrast, little is known about the influence of CCN1 on immunological mechanisms. In this context, CCN1 was shown to be an enhancer of the effect of the tumor necrosis factor alpha (TNFa) (Chen and Lau,. J. Cell. Commun. Signal. Nov 7, 2009). Furthermore, an increased expression of CCN1 is found in patients with morbus crohn and colitis ulcerosa.

Many diseases are caused by a disorder in processes connected to migration, activation and deactivation of immune cells. Rheumatoid arthritis, auto-immune myocarditis, colitis ulcerosa, morbus crohn or juvenile-onset diabetes are only some of the indications of medical and economic significance that are caused by misguided inflammatory reactions. Additionally, control or suppression of allogeneic transplant rejection is desirable from the medical point of view.

Different agents for treatment of inflammatory reactions are known in the art, but their application is accompanied by undesired side effects.

Summary of the invention

The objective of the present invention is to provide safe and efficacious means for preventing or treating inflammatory diseases or dysfunctions of the immune system. This objective is attained by the subject matter of the independent claims.

The present invention is based on the surprising finding that CCN1 exerts a chemotactic effect on different immune cells. Furthermore, monocytes become insensitive when exposed to CCN1 over a short time period. This CCN1 -induced insensitivity, however, is not limited to stimuli of CCN1 , but also to stimuli of other pathogenetic important chemokines, such as SDF-1 a (stromal cell-derived factor- 1 a), MCP-1 (monocyte chemoattractant protein-1 ) or MIP-1 a (macrophage inflammatory protein-1 a). Another surprising finding in the context of the present invention is that CCN1 circulates in the blood stream in physiologically relevant concentrations. Thus, CCN1 acts like a signal molecule modulating the functional state of the immune system and it is therefore suitable for therapeutic modulation of immune cell chemotaxis. The surprising properties of CCN1 can thus be used for means for pharmacological intervention in conditions related to inflammatory diseases or dysfunctions of the immune system.

According to a first aspect of the present invention, a polypeptide is provided for preventing or treating inflammatory diseases or dysfunctions of the immune system, comprising a polypeptide sequence of human CCN1 (CRY61 ) (Seq. ID 001 ) or a polypeptide sequence functionally equivalent to CCN1 , with at least 85 % sequence identity, preferably 90 % sequence identity, more preferably 95 % sequence identity to CCN1 .

Sequence identity is a quantitative measure of the congruence of each of all elements of at least two sequences to be compared. Such an element can be an amino acid residue in a polypeptide sequence. Sequence identities are commonly indicated as a percentage, where a sequence identity of 90 % implies that 90 % of the elements of a sequence are equal to the elements of a second sequence. Methods to measure sequence identity are known to the skilled person; an overview is given in Raghava and Barton, BMC Bioinformatics. 2006, 7:415.

A polypeptide sequence is functionally equivalent to CCN 1 in the context of the present invention if such polypeptide sequence demonstrates substantially similar biological effects with respect to inducing refractivity to chemotactic stimulation and suppression of inflammatory disease and immunological dysfunction in vivo. Methods to measure such biological effects are given in, but not limited to, the examples of the present description.

According to a preferred embodiment of this first aspect of the invention, the polypeptide sequence is connected covalently to a stabilizing sequence. This stabilizing sequence is a polypeptide or protein. An example of such a stabilizing sequence is the Fc-domain of a gamma immune globulin. Examples of such stabilizing sequences and their use are shown in WO01/03737.

A polypeptide according to the first aspect of the invention may also be stabilized against proteolytic degradation by covalent modification of reactive groups, particularly reactive amino acid side chains, preferably with sialic acid, or by site-directed mutagenesis of protease cleavage sites.

According to a second aspect of the invention, a nucleic acid is provided for preventing or treating inflammatory diseases or dysfunctions of the immune system. The nucleic acid according to this second aspect of the invention comprises a nucleic acid sequence for expression of a polypeptide sequence according to the first aspect of the invention.

Gene expression in this context describes the process by which the information inherent in the coding frame of a nucleic acid sequence is used in the synthesis of a polypeptide or protein product. The term can be used to signify the reading of a gene, as in "expression of a nucleic acid" referring to the process of transcription, RNA processing and translation (including posttranslational processing where applicable) to render a polypeptide sequence encoded in the expressed nucleic acid. Similarly, "expression" can be used to refer to the making of a polypeptide product, as in "expression of a protein", describing the same process but from the point of reference of the product, not the genetic information used in obtaining the product.

According to a preferred embodiment of this second aspect of the invention, the nucleic acid sequence for expression of a polypeptide sequence is a DNA sequence. A preferred DNA sequence comprises a mammalian RNA polymerase-ll promoter. Likewise, any promoter operable in human cells is suitable for expressing the polypeptide sequence, e. g. the immediate-early CMV promoter or the SV40 promoter.

According to a preferred embodiment of this second aspect of the invention, the nucleic acid is provided as an isolated DNA construct, preferably a plasmid, a linear, covalently closed expression construct or a minichromosome. Such DNA constructs for eukaryotic expression of transgenes are readily applied for expression of CCN 1 -coding nucleic acids in the context of this aspect of the invention. So-called "naked" circular DNA plasmids containing genetic element for replication in bacteria, or linear or circular constructs comprising only the coding sequence and necessary promoter and terminator sequences (EP0967274; EP1631672) may be employed. Optionally, naked DNA vectors may be applied with help of carriers non-limiting examples such as liposomes, microspheres, nanocapsules or "ghost" bacterial shells,

According to another preferred embodiment of this second aspect of the invention, the nucleic acid is provided as a genetically modified virus, preferably an adeno-associated virus, a lentivirus or a herpesvirus. Modified viruses are known, which are able to act as vectors for therapeutic nucleic acids. Adenoviruses and adeno-associated-viruses (AAV) are most frequently used for this purpose. Vectors for gene transfer based on lentiviruses or herpes viruses are also known. Furthermore, nanoparticles based on AAV-vectors may be employed as vehicles for gene transfers.

According to a third aspect of the invention, a cyclic RGD-peptide is provided for preventing or treating inflammatory diseases or dysfunctions of the immune system.

According to a preferred embodiment of this third aspect of the invention, cyclo(-Ala-Arg-Gly-Asp-3- aminomethylbenzoyl) or a functionally equivalent cyclic RGD peptide based on the ARGD sequence is provided for prevention or treatment of inflammatory diseases or dysfunctions of the immune system. According to yet another aspect of the invention, a pharmaceutical composition is provided for treating and preventing inflammatory diseases or dysfunctions of the immune system, comprising a polypeptide, a nucleic acid or a cyclic RGD peptide according to any of the above aspects of the invention.

According to a preferred embodiment of any of the above aspects of the invention, said pharmaceutical composition, polypeptide, nucleic acid or cyclic RGD peptide may be used for the prevention or treatment of diseases that are associated with myocardial or cardiovascular inflammation. Such diseases associated with myocardial or cardiovascular inflammation may be, for example, cardiac viral infections and inflammatory cardiomyopathy, or acute myocardial infarct, dilative cardiomyopathy, especially of inflammatory etiology. Another preferred use of the present invention is the application in autoimmune diseases such as autoimmune-myocarditis, rheumatoid arthritis, lupus erythematosus and colitis ulcerosa. Another preferred use is in preventing transplant rejection.

According to yet another aspect of the invention, a dosage form is provided for preventing or treating inflammatory diseases or dysfunctions of the immune system, comprising a polypeptide, a nucleic acid or a cyclic RGD peptide according to any of the above aspects of the invention. Optionally, a pharmaceutical carrier or excipient may be present.

Said polypeptide, nucleic acid or cyclic RGD peptide can be applied to a subject in any form suitable to the intended treatment. Such a form may be an oral formulation, nasal inhalant, injection, a suppository or a topical formulation. An injection formulation is preferred.

Also within the scope of the present invention is a method for preventing or treating inflammatory diseases or dysfunctions of the immune system, comprising the administration of a polypeptide, a nucleic acid or a cyclic RGD peptide according to any of the above claims, to a subject in need thereof.

Similarly, a polypeptide, a nucleic acid or a cyclic RGD peptide according to any of the above claims is provided for the manufacture of a medicament for the prevention and treatment of inflammatory diseases or dysfunctions of the immune system. Medicaments according to the invention are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing.

Detailed description

The present invention provides for the first time evidence of a novel function of locally produced CCN1 acting as an immune signaling molecule at distant sites. A gene transfer approach was employed to overexpress CCN1 in mice, and to assess their susceptibility to experimental autoimmune myocarditis (EAM). Remarkably, CCN1 over-expression reduced EAM disease scores and cardiac immune cell infiltrations without changing cardiac chemokine expression, but strongly inhibited the migration of circulating immune cells. CCN1 gene transfer as demonstrated in the present invention for the treatment of cardiac inflammation, administration of recombinant CCN 1 protein, or CCN1 -mimetic drugs, may all have therapeutic potential in cardiac diseases associated with chronic pathogenic inflammation including autoimmune heart disease and cardiac transplant rejection.

The overexpression of CCN1 improves the clinical picture of experimental induced auto immune myocarditis (EAM). Animals treated with vectors expressing CCN1 show a significantly reduced immigration of monocytes and lymphocytes into the heart muscle. The levels of chemokine and receptor expression in heart muscle cells remain unchanged from CCN1 -expression. Also the recombinant CCN1 molecules do not bind to cells of the heart muscle. The mechanism of the CCN 1 - effect is a modulation of the reactivity of immune cells regarding chemotactic stimuli. This affects a broad spectrum of chemokines relevant to inflammations, such as SDF-1 , MCP-1 or MIP-1 a.

In this context, the action of CCN1 on cell migration proceeds in a biphasic mode. First, immune cells are stimulated by CCN1 . After a short period of exposure to the protein, the cells become refractory against chemotactic proteins, including CCN1 itself. Thus, CCN1 can be used for therapeutical purposes, including therapeutical modulation of immunopathogenic processes like auto immune myocarditis. The therapeutic effect is based on suppression of migration of immune cells contributing to pathogenicity during auto immune processes.

Furthermore, CCN1 may be used as a diagnostic or prognostic marker, because of its overexpression in damaged tissues and its occurrence in the blood stream. A suitable detection method could be ELISA.

In contrast to a wealth of data on effects of CCN1 in vitro, data on its functions in vivo are sparse and its therapeutic potential against cardiac diseases had not been addressed yet. The present invention provides direct evidence of a novel in vivo function of CCN1 acting as an immune signaling molecule at distant sites and upon distinct target cells. In a first series of experiments, expression and tracking of recombinant CCN1 protein in vivo shows that hepatic CCN 1 over-expression results in a circulating CCN1 pool binding to spleenic macrophages. This proof of existence for a "systemic" CCN1 pool complements the previous identification of "local" CCN1 pools generated at sites of injury. In a second set of experiments, it was investigated if chronic enhancement of the systemic CCN1 pool by gene therapy would influence the course of EAM, a local autoimmune-triggered inflammation associated with severe cardiac injury. EAM strongly up-regulates multiple chemokines and chemokine receptors in the heart and accordingly blockade of MIP-1 a and MCP-1 by antibodies, or a dominant negative protein, has reduced disease severity in rat autoimmune myocarditis. Unexpectedly, CCN1 gene therapy did not modulate cardiac expression of a broad spectrum of chemokines or chemokine receptors including MCP-1 and MIP-1 a known to be key mediators of EAM, nor of the cytokine IL-17 centrally involved in EAM pathogenesis. Instead, direct ex vivo analysis of circulating immune cells from mice during CCN1 gene therapy revealed that their migratory capacity ex vivo was strongly reduced. Although inhibition of the migration of vascular smooth muscle cells by CCN5 (WISP-2), a structurally distinct protein from the CCN protein, has been described, CCN1 has been considered so far as a protein stimulating cell migration. In accordance with its effect described on fibroblasts and CD34⁺ stem cells, it was observed in the context of the present invention that CCN1 has an early migration-supporting effect on monocytes. However, preincubation with CCN1 completely abrogated not only this early migration-stimulating effect of CCN1 , but also the cells' chemotactic response to MCP-1 , ΜΙΡ-1 α and SDF-1 a.

The migration-inhibiting effect of CCN1 on circulating immune cells taken from mice during CCN 1 gene therapy could not be explained by previously published data on CCN1 functions and prompted a third series of experiments on immune cells in vitro to elucidate the mechanism of inhibition and to compare it with those of known immune cell migration and chemotaxis modulating agents. Of note, none of these agents is an endogenous protein, but they are either small molecule drugs or antibodies. A peculiar bi-phasic action profile of CCN1 on primary human monocytes and THP-1 cells was observed which is likely to result in a highly dynamic influence of CCN1 on leucocyte trafficking. Importantly, the in vitro transwell migration experiments as conducted do not involve any immune cell- endothelium interactions, but reflect direct effects of CCN1 on the immune cells themselves. Clearly, under such in vitro conditions it is the direct interaction of CCN1 with one or more of the known receptors on the immune cells that results in their "reprogramming" by prolonged exposure. This must be assumed to occur also in vivo during CCN1 therapy. Given the fact that CCN1 is an evolutionary highly conserved endogenous protein, the bi-phasic profile is likely to confer important homeostatic functions including an optimized response of inflammatory effector cells to tissue injury.

As CCN1 is known to exert most of its functions by binding to various integrins, one aspect of the present invention was to compare its effects on immune cell migration with that of the integrin binding cyclo-RGD peptides currently developed as antiangogenic and anti-proliferative agent for cancer therapy. With respect to the comparison of cyclo-RGD peptides with CCN1 (Fig. 4B) it is of interest that these synthetic compounds bind selectively to one type of integrins only, whereas the complex four-domain protein CCN1 binds to a broad spectrum of integrins including α₆β-ι , α_νβ₃, α_νβ₅, and α_Μβ2, and to heparan sulfate proteoglycans. Remarkably, a direct comparison of CCN1 with an integrin- interacting cyclo-RGD peptide showed similar migration-inhibiting effect, although chemotaxis towards SDF-1 a was not blocked as efficiently as with CCN1 . Thus, the present invention describes a novel migration-inhibiting effect for cyclo-RGD peptides which may be of considerable relevance in both anticancer and anti-inflammatory treatment. Although the chemokine target spectrum of CCN1 appears to be broader, evaluation of cyclo-RGD peptides for immunomodulation and the treatment of inflammatory diseases seems warranted based on these data. One may consider CCN1 as a new, endogenous "parent compound" for immune cell chemotaxis modulation, and cyclo-RGD peptides as synthetic, partially CCN1 -mimetic drugs with immediate potential for clinical investigation since they have already been allowed into the clinical for other indications.

In addition to these therapeutic perspectives, CCN1 modulation in vivo is interesting from an analytical viewpoint, since CCN1 functions are strongly altered by multiple factors not commonly present in vitro. These include numerous interacting proteins, homo-dimerization via CT domains, and multimer formations via VWC domains. Whereas isolated functions of the four CCN domains have been clarified to a considerable degree at molecular and cell level, the complexity of their known molecular interactions makes it impossible to predict their overall effect within an intact organism. Therefore, CCN1 tracking and modulation in disease models should contribute to a more comprehensive picture of its physiologic and pathogenic functions.

Most likely, different anti-inflammatory therapeutic strategies are required for distinct target diseases, with CCN1 expanding the spectrum of tools for chemotaxis modulation and offering new therapeutic perspectives for important cardiac and autoimmune disorders. The present invention provides CCN1 as a new, endogenous "parent compound" for chemotaxis modulation. It further provides cyclo-RGD- peptides as a first class of partially CCN1 -mimetic drugs with immediate potential for clinical evaluation. CCN1 gene transfer for the treatment of cardiac inflammation, administration of recombinant CCN1 protein, or CCN1 -mimetic drugs, may all have therapeutic potential in cardiac diseases associated with chronic pathogenic inflammation including autoimmune heart disease and cardiac transplant rejection. Brief description of the figures

Figure 1 shows overexpression and tracking of recombinant CCN1 protein in vivo

Figure 2 shows that systemic CCN1 therapy attenuates experimental autoimmune myocarditis

Figure 3 shows that preincubation with CCN1 inhibits immune cell migration in vitro

Figure 4 shows that Cyclo-RGD Peptides are partially CCN1 -Mimetic Drugs

Figure 5 shows cell culture results of THP-1 cells

Figure 6 shows additional cell culture results for THP-1 -cells

Examples

Example 1 : Overexpression and tracking of recombinant CCN1 protein in vivo

Recombinant adenoviral vector AdV-mCCN1 was generated according to the structure of the upper map of Fig. 1 A with ligation of a full-length mouse CCN1 -cDNA into the transfer plasmid pZS2, followed by its ligation to the long arm of adenovirus mutant RR5. The product was transfected into HEK293 cells and a recombinant adenoviral clone AdV-CMV-mCCN 1 was amplified and purified by CsCI ultracentrifugation. For HA-tagged CCN1 vector (HA = hemagglutinin epitope YPYDVPDYA from influenza virus A), CCN1 -cDNA was amplified from mouse muscle mRNA after reverse transcription. The amplification product was cloned into the pRK5 plasmid. CCN1 -HA was then amplified as a PCR product and cloned into pJet1 .2. pJetl . 2-mCCN1 -HA was digested and mCCN1 -HA was cloned into pRK5. pRK5-mCCN1 -HA was digested and CCN1 -HA was cloned into the adenovector transfer plasmid pZS2. AdV-mCCN1 -HA expresses the 397-amino-acid CCN1 linked to a carboxy-terminal 9- amino-acid HA-tag (Fig. 1 A, lower map). Both vectors are under control of a CMV promoter and contain a bovine growth hormone termination signal (bGH) which was employed in Northern blots (panel D) to distinguish endogenous from endogenous CCN1 -mRNA.

The endothelial Eahy.926 cell line was cultured in 6-well-plates with 5x10⁵ cells per well in 3 mL DMEM medium with Glutamax-I, 10% fetal calf serum, 1 % penicillin, 1 % streptomycin, and 1 xHAT. Cells were transduced after 24h with 3x10³ particles of vector AdV-C/WV-mCCN1 per cell. 72h later cell protein was isolated and culture supernatants collected and both analyzed by Western blotting using an a-mouse-CCN1 primary antibody and a secondary HRP antibody. Human and bovine CCN 1 were detected using a biotinylated a-CCN1 antibody, followed by streptavidin/HRP conjugate. AdV-CCN1 - transduced EAhy.926 cells showed a 40 kDa band (representing CCN1 ) in the cell lysates as shown in lanes 5/6 of the Western blot (Fig. 1 B). Lysates of untreated (NC, lanes 1 /2) or control vector-treated (RR5, lanes 3/4) cells showed no CCN1 -specific band. In the supernatants, a full-length 40 kDa protein, and a 20 kDa fragment were detected in AdV-CCN1 -transduced cells (lanes 5/6). EAhy.926 cells transduced after 24h with 3x10³ particles of AdV-mCCN1 -HA were analyzed by flow cytometry to assess the distribution of the recombinant HA-tagged CCN1 protein between the intracellular compartment and the cell membrane (Fig. 1 C). 7.5 ng/mL Brefeldin A was added after 48h for intracellular staining. After 72h cells were harvested and stained for FACS analysis with a-HA- FITC. The blue curve shows non-transfected cells and the red curve represents CCN-HA transfected cells (one of two independent experiments).

Northern blot analysis of recombinant CCN1 in mouse livers shows the stability of AdV-mCCN1 expression in vivo after i.v. administration of 3x10¹⁰ vector particles/animals (Fig 1 . D). Shown are untreated animals (NT), CCN1 -transduced animals, and mice that received the control (RR5) vector (n=2-3). The blots were hybridized with a probe detecting the bGH termination sequence of recombinant CCN1 only, over a period of 40 days (upper blot). Quantification versus β-actin is shown in the lower graph.

For FACS analyses, livers, spleens and hearts of AAV-CCN1 -HA vector-transduced mice and non- transfected control mice were taken on day 25. Single-cell suspensions from all organs were suspended in FACS buffer (PBS with 2% Flebogamma) and analysed by flow cytometry using a FACS Canto II analyzer and a-HA-FITC antibody. Flow cytometric analysis of mice treated with AdV- mCCN1 -HA showed no HA-tagged protein bound to the cell membrane of cardiac cells in animals treated with CCN1 -HA vector (blue curve) compared to control vector (RR5)-treated mice (red curve) 3 weeks after gene transfer (Fig. 1 E.). Membrane bound HA-tagged protein was detected in the splenic CD1 1 b⁺ cells by use of an a-HA antibody (one of two independent experiments).

Fig. 1 F shows the situation 5 weeks after AdV-mCCN1 vector injection. Western blot analysis of mouse sera, after ethanol precipitation of serum albumin by using 0.1 M NaCI in ice-cold sera and 95% ethanol, showed mCCN 1 specific bands which correspond to glycosylated full-length CCN 1 ~ 50 kDa, an unglycosylated form at 40 kDa, and a NH₂-terminal plasmid degradation fragment at 21 kDa.

Example 2: Systemic CCN1 therapy attenuates experimental autoimmune myocarditis.

To evaluate the effect of systemic CCN1 overexpression CCN 1 or RR5 control vectors), as 3x10¹⁰ particles per animal, were injected i.v. into female BALB/c mice one week prior to immunization with a 1/1 emulsion of 100 g/mouse of MyHC-a peptide (the cardiac autoantigen) together with CFA (Fig. 2 A. Histological analysis by HE staining showed leukocyte infiltration of mouse hearts 3 weeks after 2nd immunization. AdV-CMVmCCN1 -HA vector or RR5 as control vector were injected intravenously in the same doses into 10 weeks old mice. Cardiac immune cell infiltration was strongly reduced in the hearts of mice treated with the vector Ad V-CCN1 in comparison to RR5- treated mice (examples out of 2 independent experiments).

Female BALB/c wildtype mice were immunized with MyHC-a peptide together with CFA at days 0 and 7, control mice with MyHC-a/CFA only. All analyses were performed at the peak of inflammation occurring 21 days post MyHC-a/CFA immunization. Myocarditis severity was assessed (Fig. 2 B) on haematoxylin-eosin sections and graded in a semi-quantitative score from 0 to 4 (0: no inflammatory infiltrates; 1 : small foci of inflammatory cells between myocytes; 2: larger foci of 100 inflammatory cells; 3: more than 10% of a cross section involved; and 4: more than 30% of a cross section involved). The disease severity scores were significantly (** p< 0.01 ) reduced by AdV-CCN1 vector treatment (CCN1 , n=10) compared to control vector- treated animals (RR5, n=12).

Total RNA extraction and reverse-transcription (RT) reaction were performed for the cytokines IFNy, IL17a and the chemokines MIP1 a and MCP1 , respectively. Reverse transcriptase PCR analysis was performed for hearts of non-treated mice (Fig. 2 C), mice that had received MyHC-a/CFA alone (n=6), and mice that had received MyHC-a/CFA (n=6) with either AdV-mCCN1 (n=10) or RR5 vector (n=12). Expression of IFN-γ (upper left), IL-17a (upper right), MIP-1 a (lower left), and MCP-1 (lower right) was calculated as the expression level compared to HPRT. There was no significant reduction of chemokine or cytokine expression levels in CCN1 - treated compared to RR5-treated mice. Hearts were taken at the peak of inflammation.

Single cell suspensions were made from spleens of RR5 transfected and CCN1 transfected Balb/c mice. Erythrocytes were lysed using ACK buffer. Splenocytes were added to the upper well and cells in lower chamber were harvested after 24 h and resuspended in 100 μΙ_ PBS +2% Flebogamma. 30 μΙ_ of 1 :10 dilution of polystyrol beads were added to each approach. Counting was performed by FACS analysis by gating on the bead population and uptake of exactly 20.000 beads per approach. Counted was the number of cells that were collected in parallel after excluding the bead population. Increase was estimated as ratio to control samples without stimulation. Dead cells were estimated by gating strategy. Migration assays of splenocytes isolated from mice at peak inflammation, 3 weeks after the 2nd immunization, showed a significantly (** p<0.01 ) reduced migration of both monocytes (left) and lymphocytes (right), in AdV-CMV-CCN1 (n=6) treated animals as compared to RR5-treated controls (n=6), in culture medium containing serum after 24h (Fig. 2 D). The experiments were performed in culture dishes with stacked medium volumes, separated by a permeable membrane (Transwell dishes).

Viability of splenocytes during CCN1 therapy was assessed by FACS gating strategy (Fig. 2 E). There is no difference in absolute numbers (not shown)) and frequency of dead splenocytes ex vivo after the migration assay of mice treated with CCN1 or RR5 vector (n.s. - not significant)

Example 3: Preincubation with CCN1 inhibits immune cell migration in vitro

To further elucidate the mechanism of inhibition of immune cell migration in vitro experiments with human immune cell were performed. The basal migration (cell numbers) of freshly isolated human CD14+ cells (n=12) is significantly (** p< 0.01 ) reduced during the migration in culture medium containing 10% serum for cells that were pre-incubated with CCN1 at 200 ng/mL for 24h (Fig. 3 A). Peripheral blood mononuclear cells (PBMCs) of healthy human donors (male and female aged 25 to 50) were cultivated in IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 10% AB serum, 1 % each of penicillin and streptomycin. 37 °C, 5 % C0₂. Lower wells of 96-well plates, 8 μιη Transwell Permeable Supports contained 235 μΙ_ of IMDM medium +10% AB serum alone or additionally 200 ng/mL MCP1 , MIP1 a or rec. human CCN1 . 75 μΙ_ of 1 ,5x10⁵ PBMCs from healthy human donors purified by Ficoll gradient were suspended in culture medium +10% AB serum and added to the upper well (Cells were incubated at 37°C, 5% C02 for 42h. Cells in lower chamber were harvested, stained for CD14 (aCD14 APC antibody) and proceeded as described before. In pre- incubation experiments cells were stimulated with recombinant human CCN1 for 24h, then washed and re-stimulated as described above. Each approach was performed in triplicate.

A prolonged CCN1 exposure (+CCN1 24hPre) resulted in abrogation of the chemotaxis-stimulating effects of CCN1 itself, and of the chemokines MCP-1 and MIP-1 a as shown in Fig. 3 B. After 24 hours of 200 ng/mL CCN1 preincubation, the chemokines were no longer able to stimulate monocyte chemotaxis (* p<0.05, ** p<0.01 ) (compare this in vitro finding to the ex vivo data shown in Fig. 2D).

Similar to the situation with primary human monocytes, monocytic THP1 cells were stimulated with 200 ng/mL CCN1 , SDF-1 a, MCP-1 , and MIP-1 a for 24h with (grey bars) and without (black bars) 24h 200 ng/mL CCN1 pre-incubation (CCN1 -Pre). * denotes a significant p<0.05 change in migration (n=9) compared to control (medium). ** denotes a significant p<0.05 change of the effect after CCN1 pre- incubation compared without pre-incubation (Fig. 3 C). The in vitro chemotaxis assay with THP-1 cells was performed as describes for primary cells. Cells in lower chamber were incubated with MTT 5mg/ml (tetrazolium-bromide) in a 1 :10 dilution for 4h at 37°C. The 0,04N HCL in isopropanol was added 1 :1 into the lower well to dissolve the dark blue crystals. Plates were read at a wavelength of 570 nm against blank without cells and Migration was calculated at ratio to unstimulated cells. Cells were stimulated with CCN1 , MIP1 a, MCP1 , or SDF1 a and preincubated with CCN1 or cRGD 4772.

THP1 cells were allowed to adhere to fibronectin coated plates for 5 min, 10 min, and 30 min, respectively (Fig. 3 D, left panel). THP1 cells were pre-incubated for 2h or 24h with 200 ng/mL of CCN1 , and further stimulated for 30 min during the adhesion on fibronectin. Compared were untreated cells to cells that were treated with 200 ng/mL of CCN1 for 30 min, and cells that had been preincubated with CCN1 (n=8). Neither for stimulation nor for preincubation with CCN1 a significant change was detected.

Intracellular signal transduction proteins linked to integrins were analyzed by Western blot analysis. 30 pg of THP-1 lysate (20 mM Tris, pH 8, 10 mM NaCI, 0.5% (v/v) Triton X-100, 5 mM EDTA, 3 mM MgCI2) was boiled 95°C for 5 min and proteins were separated on NuPAGE 4-12% Bis-Tris gels under denaturing and reducing conditions and transferred onto a PVDF membrane. Membranes were incubated with the primary antibodies a-GAPDH, a-Nck2, a-PINCH, and a-ILK, and secondary antibody Ig-HRP. Antibodies were incubated for 1 h or overnight in dry milk. For detection Rodeo™ ECL Western Blot Detection kit was used. The left side of Fig. 3 E shows the protein expression levels of integrin-linked kinase (ILK), NCK2, PINCH, and β-arrestin 2 that blocks G protein-mediated signaling, at various time points (5 min, 30 min , 3h , 8h , and 24h) after addition of 200 ng/mL MCP-1 . The blots on the right side show the same analyses, but with addition of MCP-1 after a preceding preincubation with CCN1 at 200 ng/mL for 24h (one of at least two independent experiments). No single significant change of protein levels was associated with prolonged CCN1 exposure.

Example 4: Cyclic RDG-peptides are CCN1 -mimeting agents With respect to the similarities of integrin binding the effects of CCN 1 and cyclo-RDG peptides were compared. CCN1 , SDF-1 a, and MCP-1 are chemotactic for monocytic THP1 cells at concentrations of 200 ng/mL. In contrast, preincubation with 200 ng/mL of CCN1 (CCN 1 -Pre) resulted in a significant (* p<0.05, ** p<0.01 ) abrogation of the chemotaxis-stimulating effects of CCN1 , SDF-1 a, and MCP-1 after 1 h or 24h of CCN1 preincubation, respectively (left panel). Comparable results were obtained for a 1 h or 24h preincubations with 10 μΜ of cRGD peptide (right panel) (n=9-12).

Dose-dependency of cRGD preincubation is shown in Fig. 4 B for a preincubation with 0.1 μΜ, 1 .0 μΜ, 10 μΜ, and 100 μΜ of cRGD peptide, respectively, of cells stimulated with 200 ng/mL CCN1 , SDF-1 a, and MCP-1 , respectively. The cRGD peptide preincubation resulted in a significantly (* p<0.05) impaired reduction of the subsequent migration stimulation by SDF-1 a, in comparison to CCN1 preincubated cells (grey bars) (n=6-9).

The used cyclic RDG-peptide is Cylco(-Ala-Arg-Gly-Asp-3-aminomethylbenzol), available at Bachem No. : H-4772). A known synonym for this peptide is XJ735.

Example 5:Cell culture result with THP-1 cells

Monocytic THP-1 cells were stimulated for 24h either directly with 200 ng/mL CCN1 , (grey bar) or after 1 h of pre-incubation with 200 ng/mL CCN1 (CCN1 -Pre), the PI3K inhibitor Ly294_002 at 10 μΜ, ΡΙ3Κγ inhibitor at 10 μΜ, the Akt inhibitor Triciribin at 10 μΜ, Rho kinase / ROCK inhibitor at 10 μΜ, MEK inhibitor PD98059 at 50 μΜ, and cRGD peptide 1 at 100 μΜ (H4772) and cRGD peptide 2 at 100 μΜ (H2574), respectively (Fig. 6A). Fig 5 B shows the resuls for SDF-1 a-stimulation, Fig. 5C for MCP-1 , and Fig. 5D for MIP-1 a stimulation. * p<0.05, ** p<0.001 are the statistical significances for the difference in the stimulation of THP-1 migration rate after preincubation with the respective agent, as compared to the stimulation of migration without preincubation.

Example 6: Additional cell culture result for THP-1 cells

THP1 cells were allowed to adhere to fibronectin coated plates for 5 min, 10 min, and 30 min, respectively (Fig. 5A). THP-1 cells were washed in PBS with calcium and magnesium at a concentration of 2.5 x 10⁶ cells/mL. Calcein AM was added to the cells at a final concentration of 12.5 μΜ. Cells were incubated at room temperature for 30 minutes, washed and resuspended at a concentration of 1 x 106 cells/mL. 100 μί of cell suspension was added to each well of a 96-well fibronectin coated plate in triplicate. Plates were then incubated for 30 minutes at 37° C and measured "before wash" using a fluorescence plate reader at excitation wavelength 485 nm and emission wavelength 520 nm. Finally non-adherent cells were washed away and the "after wash" fluorescence was read again and the average percent adhesion using the following formula: [(RFU after wash)/(RFU before wash)] x 100 was calculated.

THP1 cells were pre-incubated for 2h and 24h with 200 ng/mL CCN1 and further stimulated for 30 min during the adhesion on fibronectin (Fig. 5B). Untreated cells, cells treated with 200 ng/mL CCN1 for 30 min and cells that were prior preincubated (n=8) were compared. Neither for stimulation nor for preincubation with CCN 1 a significant change was detected.

Claims

s

1 . A polypeptide for preventing or treating inflammatory diseases and/or dysfunctions of the immune system, comprising a polypeptide sequence of human CCN1 (Seq. ID 001 ), or a polypeptide sequence functionally equivalent to CCN1 , said functionally equivalent polypeptide sequence having at least 85 % sequence identity, preferably 90 % sequence identity, more preferably 95 % sequence identity to CCN1 .

2. A polypeptide according to claim 1 , characterized in that said polypeptide sequence is

covalently linked to a stabilizing sequence, and/or in that said polypeptide is stabilized by covalent modification of reactive groups with sialic acid, or by site-directed mutagenesis of protease cleavage sites.

3. A nucleic acid for preventing or treating inflammatory diseases and/or dysfunctions of the immune system, comprising the nucleic acid sequence encoding the polypeptide sequence according to any of claim 1 or 2.

4. A nucleic acid according to claim 3, characterized in that the nucleic acid sequence is under expression control of a RNA-polymerase-ll promoter operable in human cells.

5. A cyclic RDG-peptide for preventing or treating inflammatory diseases and/or dysfunctions of the immune system.

6. A cyclic RDG-peptide according to claim 5, characterized in that said cyclic RGD peptide is cyclo(-Ala-Arg-Gly-Asp-3-aminomethylbenzoyl).

7. A pharmaceutical composition for preventing or treating inflammatory diseases and/or dysfunctions of the immune system, comprising a polypeptide, a nucleic acid or a cyclic RGD peptide according to any of the above claims.

8. A pharmaceutical composition according to claim 7 for preventing or treating of dilative cardiomyopathy, particularly dilative cardiomyopathy of inflammatory etiology, autoimmune disorders, particularly autoimmune-myocarditis rheumatoid arthritis, lupus erythmetatosus, colitis ulcerosa or preventing of transplant rejection.

9. A dosage form for preventing and treating inflammatory diseases and/or dysfunctions,

comprising a comprising a polypeptide, a nucleic acid or a cyclic RGD peptide according to any of the above claims 1 -7.

10. A method for preventing and treating inflammatory diseases and/or dysfunction of the immune system, comprising the administration of a polypeptide, a nucleic acid or a cyclic RGD peptide according to any of the above claims 1 -7 to a subject in need thereof.

1 1 . A polypeptide, a nucleic acid or a cyclic RGD peptide according to any of the above claims 1 -7 for the manufacturing of a medicament for preventing and treating inflammatory diseases and/or dysfunctions of the immune system