WO2009083880A1

WO2009083880A1 - Transglutaminases binding fusion protein, compositions comprising thereof, micro-spheres comprising thereof uses and methods

Info

Publication number: WO2009083880A1
Application number: PCT/IB2008/055409
Authority: WO
Inventors: Héctor Eduardo CHULUYAN; Paulo Maffia; Nancy L. Tateosian; Diego Guerrieri; Nicolás O. AMIANO; Romina M. Reiteri; María Julieta COSTA; Sonia A. GÓMEZ; Verónica E. GARCÍA; Diego A. Chiappetta
Original assignee: Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET; Inis Biotech LLC
Current assignee: Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET; Inis Biotech LLC
Priority date: 2007-12-28
Filing date: 2008-12-18
Publication date: 2009-07-09
Anticipated expiration: 2010-06-28
Also published as: AR064713A1

Abstract

Transglutaminases binding fusion protein, compositions comprising thereof, micro-spheres comprising thereof, uses and methods. The fusion protein might comprise a transglutaminases binding domain, for example the cementoin domain and a serine proteases inhibitor, for example the secretory leucocitary proteases inhibitor (SLPI). The fusion proteins are useful for the preparation of medicaments for the treatment of, for example, autoimmune diseases, inflammatory disorders, cancer, infections and healing of wounds.

Description

Transglutaminases binding fusion protein, compositions comprising thereof, micro-spheres comprising thereof, uses and methods

The present invention refers to transglutaminases binding fusion proteins, compositions comprising thereof, micro-spheres comprising thereof, uses and methods. More specifically, this invention refers to fusion proteins comprising a transglutaminases binding domain, for example the cementoin domain and a serine protease inhibitor, for example the secretory leucocyte protease inhibitor (SLPI).

Background

Through decades, the purpose was to improve the pharmacological tools availability that allows controlling different diseases, for example inflammatory disorders, particularly the chronic inflammatory disorders. However, even today corticosteroids and non-steroid antiinflammatory drugs are being used as first selection drugs in the treatment of inflammatory disorders, in spite of the adverse effects observed, especially when they are chronically used. Presently, physiological antiinflammatory substances (different to glucocorticoids), as, for example the serine proteases inhibitors SLPI and ELAFIN, are under study. These serine protease inhibitors would act inhibiting not only the elastase activity produced by the polymorphonuclear leucocytes but also the translocation of the transcription factor NFkB to the core and the pro-inflammatory genes activation. Independently of this last action, the inhibition of the elastase activity results important, as the enzyme presents lethal effect at the labile parenchyma level, particularly the pulmonary parenchyma and it appears in pathologies such as COPD and fibrocystic disease, where an inefficient protection against the neutrophilic elastase is set out.

The SLPI (Secretory Leucocyte Protease Inhibitor) is a 11,7 KDa protein initially isolated from the salivary secretions of human parotid (R. C. Thompson, Proc. Natl. Acad. Sci 1986, 83:6692-6696). Later it was found in seminal liquid and in both nasal and bronchial cervical mucus. It is a potent serine proteases inhibitor, mainly elastase, cathepsin G, chymase and chymotrypsin of leucocytes. This proteolysis inhibitory action has a relevant effect in the control of tissue damage produced by these proteases in the inflammatory sites. A strict balance between proteases and the inhibitors thereof is indispensable in the healing of wounds and in all the inflammatory disorders. The unbalance between proteases and antiproteases occur in various inflammatory diseases, as the fibrocystic disease, the bronchopulmonary dysplasia, asthma, the chronic bronchitis, the emphysema associated to smoking, and the adult respiratory distress syndrome.

Cementoin is a polypeptide present in the ELAFIN molecule with the particularity of presenting the VKGQ sequence that confers to the molecule the possibility of polymerizing and binding to other molecules present in the interstitium by transglutamination . In this way, the molecule is retained in the site where the transglutaminase enzyme is found, particularly the transglutaminase-2.

On the other hand, it is known that therapies with SLPI usually present problems such as a relative short half-life in serum and lateral effects due to the fact that once administered it non-specifically directs to different sites, affecting healthy tissues. It is known that the repeated administration or large doses of proteases inhibitors may cause undesirable effects on the patient.

There exist different fusion proteins comprising SLPI, for example the SLIP-collagen fusion protein disclosed by the Patent Application US 2007/0031478 to KADLER KARL, that is used for the healing of wounds or the Patent Application US2003/0073217 to Barr et al . that discloses a fusion protein between two protease inhibitors: alAT and another useful inhibitor would be SLPI or TIMPl.

Since one of the major problems being presented by the treatment with SLPI or other serine proteases inhibitors is the non-specificity of the action thereof, there remains the need of said inhibitors specifically directing to the site or tissue where the therapeutic action thereof should be exerted.

BRIEF DESCRIPTION OF THE INVENTION

An object of the present invention is to provide a transglutaminase binding fusion protein comprising a transglutaminases binding domain linked to a serine protease inhibitor. In a preferred embodiment, the transglutaminase binding domain is the cementoin domain or parts thereof and the serine protease inhibitor is the secretory leucocyte protease inhibitor (SLPI). For example, the fusion protein may link to different transglutaminases as transglutaminase 1, 2, 3, 4, 5, 6 and 7. In another preferred embodiment the fusion protein comprises the sequence showed in SEC ID N⁰I. Another object of the present invention is to provide a pharmaceutical composition comprising a transglutaminases binding domain linked to a serine protease inhibitor and acceptable pharmaceutical excipients. In a preferred embodiment, the transglutaminases binding domain is the cementoin domain or parts thereof and the serine protease inhibitor is the secretory leucocyte protease inhibitor (SLPI). In a more preferred embodiment the composition comprises the fusion protein of the sequence showed in SEC ID N⁰I and excipients.

A further object of the present invention is to provide the use of the pharmaceutical composition comprising a transglutaminases binding domain linked to a serine protease inhibitor and acceptable pharmaceutical excipients for the preparation of a medicament for the treatment of, for example, autoimmune diseases, cancer, infections, inflammatory disorders, inflammatory diseases, infective diseases, rheumatic diseases, degenerative diseases and sepsis and inflammatory bowel diseases, or for the healing of wounds, or for preparation of a cosmetic.

It is yet a further object of the present invention to provide micro-spheres for the preparation of a medicament or cosmetic, wherein said micro-spheres comprise an enveloped fusion protein comprising a transglutaminases binding domain linked to transglutaminases bound to serine protease inhibitor, or the SLPI protein or parts thereof. In a preferred embodiment the transglutaminases binding domain is the cementoin domain or parts thereof and the serine protease inhibitor is the secretory leucocyte protease inhibitor (SLPI). The fusion protein binds to, for example, to the transglutaminases 1, 2, 3, 4, 5, 6 or 7. Said micro-spheres may have a diameter between 2 and 20 μm and may be constituted by poly-ε-caprolactone, polylactic acid (PPLA) and polylactic-glycolic acid (PLGA) . The microspheres may be used for the preparation of a composition, medicament or a cosmetic. The medicament may be used for the treatment of autoimmune diseases, cancer, infections, inflammatory disorders, inflammatory diseases, infective diseases, rheumatic diseases, degenerative diseases, sepsis, and inflammatory bowel diseases and to prevent the transplant rejection; or for the healing of wounds.

It is a further object of the present invention to provide a method of treating patients suffering an autoimmune disease, wherein such a treatment comprises to administer an enough amount of the SLPI protein or parts thereof. The autoimmune disease may be autoimmune orchitis, AR, Systemic lupus erythematosus, thyroiditis, dermatomyositis, fibromyalgias, polimyositis, Hashimoto thyroiditis, Diabetes mellitus, hemolytic anemia, Encephalitis, Multiple sclerosis, primary liver cirrhosis, chronic active hepatitis, pemphigus, pemphigoid, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, miastenia gravis and Sjogren syndrome. The amount of SLPI protein to be administered may be among 100 mg and 0.001 ug, being the SLPI contained in a pharmaceutical composition, for example in controlled release micro-spheres. The micro-spheres may be of a size between 2 and 20 urn diameter and constituted by poly-ε- caprolactone, polylactic acid (PPLA) or polylactic-glycolic acid (PLGA) . In a preferred embodiment the amount of SLPI per mg of micro-spheres is among 0.1 and 3000 ng. DESCRIPTION OF FIGURES

Figure 1 illustrates a chart showing the inhibition of the serine protease or elastase activity from Cem-SLPI and SLPI alone, at 10 minutes after the reaction occurred.

Figure 2 illustrates a chart showing the results of the inhibition of the serine protease or elastase activity by a colorimetric test in real time.

Figure 3 illustrates a chart showing the percentage of healing of wounds in real time on mice treated with a buffer (control), only Cem-SLPI and SLPI.

Figure 4 illustrates the results of a test binding the SLPI and Cem-SLPI to cells expressing transglutaminases (A549 cells) in the presence or absence of TNFα.

Figure 5 shows the results of the Cem-SLPI adhesion (figure 5a) and SLPI alone (figure 5b) to A549 cells analyzed by the fluid cytometry technique. The clear zone of the chart corresponds to A549 cells not exposed to the protein (negative control). The chart should be interpreted in such a way that the colored zone non-superimposed to the clear zone is the A549 cells that are bond to the Cem-SLPI protein. Therefore, figure 5a shows that 89.7% of the A549 cells are bond to the Cem-SLPI protein. However, figure 5b shows that when cells are incubated only with SLPI, the protein is able to bind only 35% to the cells.

Figure 6 shows a chart of the flow cytometry of the popliteal lymph node cells of mice previously treated with Cem-SLPI by intravenous administration. The chart 6b shows the absence of adhesion of the fusion protein to the noninflammatory lymphatic node cells (superimposition between the color zone and the clear zone) and figure 6a shows the adhesion of the fusion protein to the lymphatic node cells inflamed by the administration of an inflamogen (absence of total superposition between the colored zone and the clear one) .

Figure 7 shows a chart representing the inhibition of the in vitro proliferation of mononuclear cells due to the SLPI or Cem-SLPI presence.

Figure 8 shows a chart representing the tumoral volume in mice that were given or not the Cem-SLPI fusion protein.

Figure 9 shows the cloning scheme and the preparation of the Cem-SLPI fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a fusion protein with serine protease activity being able to direct to and bind to transglutaminase (TG), having a long half life in the specific site of therapeutic action and a low plasmatic degradation. In a preferred embodiment the fusion protein comprises the transglutaminase enzyme substrate (cementoin) y the secretory leucocyte protease inhibitor (SLPI) or some of the domains thereof. In yet another preferred embodiment, the fusion protein has the amino acids sequence shown in SEC ID N⁰ 1. As from what is disclosed in the present application it is evident that other fusion proteins may be obtained which comprise different transglutaminase substrates or part thereof fused to different proteins with serine protease activity or parts thereof, being said embodiments within the scope of the present invention.

The fusion protein of the invention is able to anchor in the different sites where the transglutaminase (TG) is expressed. Since the fusion protein is able to selectively anchor to the tissues where the transglutaminase is expressed, it may therefore increase the inhibitor concentration in specific regions or tissues.

Also within the scope of the present invention there are nucleotide sequences coding for a fusion protein comprising a transglutaminases binding domain and a serine protease inhibitor or some of the domains thereof. In a preferred embodiment the nucleotide sequence is the sequence shown in SEC ID N⁰ 2.

Definitions: for the purpose of the present application and without being limited to, the fusion protein is also referred to as Cem-SLPI .

In the present application, when the 117 Clan IP Family is referred to, it refers to the MEROPS classification (MEROPS Database, htrp : / /merops . Sanger . ac . uk ) and when the WAP family is referred to (whey acidic protein) it refers to a series of very well characterized proteins presenting a core of four disulphur bridges, which is abundant in the murine milk serum. This structure is found in proteins such as protease inhibitors, neurophysins, vegetal agglutinins, adhesion molecules, scorpions' toxins, bacterial peptides and pollen proteins .

For the purpose of the present application, the term "protein" also refers to any polypeptide or amino acids chain, lineal branched, dimers or multimers.

Exemplary experiments were carried out to show that the fusion protein keeps the inhibitory activity over serine proteases, for example elastase. In a preferred embodiment the fusion protein is the protein shown in SEC ID N⁰ 1 (Cem-SLPI), which shows an inhibitory activity greater than that of the individual SLPI protein (see figures 1 and 2 ) .

On the other hand, the healing capacity of the fusion protein of the invention was tested. As it is observed in Figure 3 upon three days of treatment the fusion protein kept and improved the healing activity with relation to the SLPI protein. In the test, equimolar amounts of each protein or control buffer solution were injected and the wound surface was periodically gauge measured.

The in vitro adhesion of the fusion protein of the invention to the cells surface expressing transglutaminase was also verified. The test was carried out on monolayers of A549 cells stimulated or not with the TNFα cytokine, being the bond detected through human nati-SLPI monoclonal antibodies. As it can be observed in Figure 4 the fusion protein of the invention binds to the A540 cells surface and the binding is even greater when the cells have been stimulated with the above mentioned cytokine.

The adhesion of the fusion protein of the invention was also analyzed through flow cytometry techniques. A549 Cells were incubated with Cem-SLPI or SLPI; then they were washed and marked with an antibody which detects the fusion protein. As it can be observed in figure 5 the fusion protein of the invention binds in a high rate to the cells (figure 5a) while said cells only bind a low amount of SLPI (figure 5b) .

The fusion proteins of the invention efficiently and selectively bind to the inflammation sites, for example lymphatic nodes. As it can be observed in figure 6 the fusion protein binds in a high rate to the inflamed lymphatic node (figure 6a) , while said binding is nearly nonexistent in non-inflamed nodes cells (figure 6b) .

The fusion protein of the invention inhibits the proliferation of in vitro mononuclear cells. The mononuclear cells of human peripheral blood were stimulated with IL-2 (8ng/ml) y then were cultivated in the presence of only SLPI or Cem-SLPI . The proliferation was quantified by the measurement of tritiated thymidine in cells. As it can be observed in Figure 7 both the SLPI alone as the fusion protein inhibits the proliferation of mononuclear cells. This inhibitory effect of the fusion proteins Cem- SLPI and SLPI results surprising and up to what is known to the inventors, it has never been described before.

The protein of the invention has anti-tumour activity (Figure 8) . In a murine model, the administration of said protein decreases the tumour size. In a preferred embodiment the tumour is a breast tumour and the administration via is intratumour.

Both the SLPI and the Cem-SLPI fusion protein may be formulated in compositions for therapeutic use. Said compositions may comprise solid, liquid, gel compositions, in micro-spheres of controlled release or any other form known in the art. Any formulation or composition comprising the fusion protein is within the scope of the present invention .

In a preferred embodiment the SLPI or the Cem-SLPI fusion protein may be formulated encapsulated in microspheres, for example the micro-spheres may be of PCL (poly- ε-caprolactone) , polylactic acid (PPLA) , polylactic- glycolic acid (PLGA) or any other micro sphere known in the art. Said micro-spheres carrying encapsulated the fusion protein or the SLPI may be used for the treatment of diseases such as cancer, autoimmune diseases, cancer, inflammatory diseases, infective diseases, rheumatic diseases, degenerative diseases, sepsis, inflammatory bowel diseases, and transplant rejection or for the healing of wounds. They may also be used for the preparation of cosmetics .

In a preferred embodiment the micro-spheres may be used for the treatment of autoimmune diseases, for example for the treatment of AR, systemic lupus erythematosus, dermatomyositis, uveitis, among others.

For example, the micro-spheres containing SLPI have been used to treat rats with autoimmune orchitis. Thus 100 mg/animal of said micro-spheres were administered to rats suffering artificial autoimmune orchitis by intraperitoneal via (ip) each week during 3 months or micro-spheres with SLPI as control. Subsequently, testicles were removed and the histopathology of each testicle was analyzed.

As it can be observed in the following Table, rats treated with the micro-spheres carrying SLPI did not show severe damages, the focal damage was higher and the total amount of damage was reduced in half.

Table 1.

Severe damage Focal damage Total damage

Control 60 % 20 % 80 %

SLPI 0 % 40 % 40 % The SLPI encapsulating micro-spheres or the fusion protein may be administered to patients who need them in amounts that range in accordance with the disease or disorder, for example in an amount among 100 mg and 0.001 ug .

The micro-spheres comprising for example SLPI were also tested as a formulation for the treatment of inflammatory disorders. Mice that were injected Pristane in their sole were treated with subcutaneous injections (sc) of said micro-spheres. Mice so treated showed a lesser size edema (p<0.05) with respect to controls treated with microspheres without SLPI. In brief, these results would indicate that, for example, the SLPI encapsulated in micro-spheres of, for example, PCL micro-spheres is released in sustained form during a long time, also maintaining both the in vitro and in vivo biological activity .

The micro-spheres of the invention carrying SLPI showed to have anti-trypsine activity.

Those skilled in the art know that the micro-spheres may be of any size; preferably the micro-spheres are around 2 and 20μm diameter.

The micro-spheres may comprise an amount of SLPI or of the fusion protein ranging around 0.1 and 3000 ng of SLPI or Cem-SLPI per mg of micro-spheres.

The Cem-SLPI fusion protein has a high bacterial effect, for example said fusion protein acts as an efficient bactericide against the M bovid BCG-Pasteur strain. In Table 2 exemplary results of the binding of the Cem-SLPI fusion protein and the SLPI alone to the bacteria and the bactericide activity of both proteins are shown. Table 2

Binding to BCG

Control 13 ± 1 %

SLPI 30 ± 3 %

Cem-SLPI 66 ± 1.8 %

Inhibition of the bacterial development

Control 0 ^£ό inhibition

Cem-SLPI 23 % inhibition

This invention is better illustrated by the following examples, which should not be interpreted as a limitation imposed to the scope thereof. On the other hand, it should be clearly understood that other embodiments, modifications and equivalents thereof may be introduced which after reading the present description, may advise those skilled in the art without moving away from the spirit of the present invention and/or the scope of the annexed claims.

Examples :

Example 1: Preparation of the Cem-SLPI fusion protein and characterization thereof:

Cloning of cDNA corresponding to the human mature SLPI protein.

For the cloning of the corresponding gene to the SLPI protein, in a first step messenger RNA was extracted from HeLa cells, developed on a monolayer in RPMI+10% SBF, by using Trizol® ( Invitrogen) . Then the retro-transcription to cDNA was carried out using oligo-dT with MMLV-RT enzyme (Promega, Madison, WI) in accordance with supplier's instructions. In order to carry out PCR amplification of the cDNA corresponding to the mature SLPI gene, with the complete open reading frame thereof, a pair of modified primers was used:

Forward primer SEQ ID N⁰ 3: AAGCTTTCTGGAAAGTCCTTCAAAGC .

Reverse primer SEQ ID N⁰ 4: CTCGAGAGCTTTCACAGGGAAACGC

The cycling was carried out in a MJ Research thermocycler, the scheduled cycle was as follows: 2 min at 94⁰C, followed by 30 cycles of 1 min at 94⁰C, 1 min at 49⁰C and 1 min at 72⁰C and a final cycle of 10 min at 72⁰C. The reaction mixture used in said PCR reaction contained 0.2uM of each primer, 200 uM of each nucleotide, Ix PCR Buffer containing 50 mM KCl, 10 mM Tris-HCl pH 8.9, 2.5 mM MgCl₂ y 1 unit of Taq DNA polymerase (Promega) at a total volume of 25 ul .

The PCR product was subjected to a run of 2% agarose gel electrophoresis, and the band of 341 bp was cut therefrom and DNA was purified using the purification kit PureLink™ Gel Extraction Kit, (Invitrogen) following the manufacturer's recommendations. Subsequently, it was cloned in a cloning vector pGem T easy Vector (Promega, Madison, WI) using the T4 DNA ligase enzyme (Promega) . The binding buffer contained: 30OmM Tris-HCl (pH 7.8), 10OmM MgC12, 10OmM DTT and 1OmM ATP, and was incubated for 18 hours at 15⁰C. Then, 5 ul of the binding reaction was used for electrophoration of competent JM109 bacteria using a Biorad E.coli electrophorator . Subsequently, 100 ul of bacteria were plated in LB agar with ampycillin. A group of isolated colonies were analyzed to find the cloning gene in the correct direction, using the colony PCR technique (Molecular Cloning: A Laboratory Manual (3-Volume Set) by Joseph Sambrook, David W. Russell, Publisher: Cold Spring Harbor Laboratory; 3rd edition (January 15, 2001) ISBN: 0879695773). The selected clones were developed in LB ampycillin and then the plasmid was purified using the commercial kit Wizard SV Plasmid DNA Purification Systems (Promega) .

The so purified plasmids were sequenced in the DNA Automatic Sequencing Service - Instituto de Bioquimica y Biologia Molecular (IBBM) at La Plata Nacional University, Buenos Aires. For comparison and analysis of sequences the EMBL data base service was used, Swissprot, and GenBank, thorough the National Center for Biotechnology Information, NLM, Bethesda, MD.

Cloning of the cDNA corresponding to cementoin.

The mRNA was purified from HeLa cells as it was described for SLPI. The cDNA retro-transcription was carried out using oligo-dT as primers, along with MMLV-RT enzyme (Promega, Madison, WI) in accordance with the manufacturer's instructions. The gene corresponding to the cementoin peptide was amplified by PCR from cDNA using a pair of modified primers.

Forward primer SEQ ID N⁰5: GTTCTACATATGGCTGTCACGGGAGTT

Reverse primer SEQ ID N⁰6: AAGCTTTTTGACTTTATCTTGACCTTTAA.

The cycling was carried out at an MJ Research thermocycler, the scheduled cycle was as follows: 2 min at 94⁰C, followed by 30 cycles of 1 min each at 94⁰C, 1 min at 6O⁰C and 1 min at 72⁰C and a final cycle of 10 min at 72⁰C. The reaction mixture used in said PCR reaction contained 0.2uM of each primer, 200 uM of each nucleotide, Ix PCR Buffer containing 50 mM KCl, 10 mM Tris-HCl pH 8.9, 2.5 mM MgCl₂ y 1 Taq unit. The PCR product of 133 bp was purified and cloned in n vector pGem T easy Vector (Promega, Madison, WI). Then, JM109 bacteria were electrophored with this construction and were plated in LB agar ampycillin. Positive clones were selected and sequenced in the same manner as SLPI .

Generation of fusion proteins: cloned genes from SLPI and cementoin were amplified by PCR with modified primers.

These mutations will confer the new gene cementoin a splicing site with the restriction enzyme Ndel and the translation initiation site ATG (Met) at the 5' end, while at the 3 ' end a site was added for the splicing with HindiII enzyme and amino acids Lys and Leu were added, this last one was inevitable due to the genetic engineering adopted, though it is considered that these minimum changes will not affect the activity nor the new protein folding.

In the case of the SLPI gene amplified by PCR with the modified primers, a splice site was added at the 5' end for the HindIII enzyme and an Xhol site at the 3' end, and amino acids Leu and GIu were added.

Each of the plasmids (pGEM-T-SLPI and pGEM-T cementoin) were electrophored and amplified in E.coli JM109 strain (Stratagen). Then, they were digested with the Apal and HindIII enzymes (Promega) . Both digestions were seeded in a 1,5% agarose gel and the corresponding strands to the pGemT-SLPI plasmid and the cementoin fragment were recovered, respectively; in order to recover the gel DNA the kit PureLink™ Gel Extraction Kit was used, from Invitrogen, following the manufacturer's instructions.

Subsequently, products 1 and 2 (see Figure 9) were incubated together at equimolar concentrations in a binding reaction; T4 DNA ligase enzyme (Promega) was used. The final construction was thus obtained, in the same reading frame, of the gene cementoin-SLPI as fusion protein in a pGEMT-Easy plasmid.

5 ul of this binding was electrophored in electro competent bacteria using an electrophorer for bacteria (Biorad) . Then, this electrophoration was plated in LB agar ampycillin medium, incubated at 37⁰C and 20 isolated colonies were checked using the Colony PCR technique. From 5 positive clones from E. coll JM 109 with pGEM T+cementoin- SLPI plasmid in a correct sense, the isolation, development and purification of plasmid was carried out, using the commercially available Wizard SV Kit from Promega, to be sent then to the above mentioned sequencing service.

A clone was selected (clone 23) and digestions to release the fusion protein gene were carried out.

The first splice was made with the Xhol enzyme (Promega), incubating in an eppendorff tube: 16,3 ul of deionized sterile water; 2ul of RE 1OX Buffer, 0,2 ul of acetylated BSA lOug/ml, 1 ul of plasmid DNA lug/ml, 1 ul (5 units) of enzyme, in a final volume of 20 ul, and was incubated at 37⁰C for 4 hours. Then it was undergone to an electrophoresis in agarose gel 1% and the band corresponding to the spliced plasmid was cut and purified. Then this plasmid with the Ndel enzyme (New England BioLab) was directed under the following digestion conditions: 1 ug of DNA, 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM Magnesium Acetate, 1 mM Dithiothreitol, pH 7.9, 5 units of enzyme, incubation temperature 37⁰C for 4 hours and the fragment release of 450 bp was checked in a 1.5% agarose gel electrophoresis. This was purified from gel and bound in the pET22b+ (Stratagen) expression vector previously spliced with Ndel and Xhol .

Then E.coli TOPlO bacteria (Invitrogen) were electrophored, a clone with the right bound construction was sought and the pET-Cem-SLPI vector was purified, which finally was used to electrophore E.coli expression bacteria Origami (Novagen) .

Five clones were checked to compare the expression of the fusion protein, and one was selected.

Purification

From a bacteria stock stored at -7O⁰C producing the fusion protein a preculture is obtained by inoculating 7.5 ml of sterile LB medium with the ampycillin and chloramphenicol antibiotics. The culture was incubated overnight with constant stirring at 37⁰C.

The following day, 500 ml of LB medium was inoculated with 5 ml of the previous preculture. This culture was allowed to develop at a temperature of 37° with constant stirring to reach an optical density of 0.6 measured at 600 nm.

Once this value was reached, the recombinant protein expression was induced by adding IPTG at a final concentration of 1 mM. Induction was kept for 3 hours at a temperature of 28⁰C with constant stirring. The culture was centrifuged at 7,000 RPM for 7 minutes and the pellet was divided and stored at-20°C for further purification. Pellets were resuspended in 2 ml of cold lysis buffer (NaH2PO4 50 mM, NaCl IM, Imidazole 10 mM; pH 8 ) . Lysozyme was added up to reach a final concentration of 1 mg/ml . DNAsa y RNAsa were also incorporated at a final concentration of lOug/ml. These enzymes were allowed to act for 30 minutes. The full procedure was carried out onto ice to avoid the proteolysis.

Finally, bacteria were lysed by ultrasound using 7 pulses of 60 Watts for 30 seconds in a sonicator equipped with microtip. Then the lisate was centrifuged at 10,00Og for 30 minutes at a temperature of 4⁰C. The soluble fraction was recovered and 800 ul were added to the specific resin of the purification system: a column with nickel-agarose resin for the Hist-Taq purification that allows obtaining the protein of interest with a high purity level and in a unique chromatographic step.

Example 2: Tests on the inhibitory activity of the fusion protein over serine proteases or elastase:

In an ELISA plate equimolar quantities of SLPI or the fusion protein (2pmo/ul) were added to each well together with proteins, human leucocitary elastase (SIGMA) was added at a concentration of 4pmol/ul diluted in 15 ul of Tris-HCl buffer pH 7.5. At the same time, the colorimetric substrate N-methoxysuccinyl-Al-Ala-Pro-Val p-nitro-anilide (Sigma Aldrich) was added to every plate well at a final concentration of 0,6 mM. The colorimetric reaction produced by the proteolytic cleavage specific of the substrate was read in an ELISA reader at 405 nm after 10 minutes.

Example 3: Tests on checking of in vivo healing activity: Balb/c mice anesthetized with sulfuric ether were given a superficial injury (epidermic) using a 6 mm diameter punch. Immediately after the injury, mice were given, in the injury zone, 150 ul of a 300 ug/ml de Cem- SLPI, SLPI or buffer solution. The injury area was gauged measured each 24 hours

Example 4: Adhesion analysis by ELISA and by flow cytometry of the fusion protein:

Analysis of the in vitro adhesion. ELISA over monolayer .

The adhesion of the fusion protein to the surface of an epithelial cell monolayer (A549) stimulated with the TNFCC cytokine was quantified.

On day 1 3OxIO³ cells were plated per well in a flat bottom 96 wells plate. 200 ul of HAM +10%SBF medium(GIBCO) were added to each well.

On day 2 TNFCC cytokine was added at a concentration of 500 units per well, to the corresponding groups, being incubated with the cytokine for 24 hours.

On day 3 the treatment with the fusion protein or with SLPI was carried out. In each corresponding well a concentration of 4 ug/ml of each protein was added and were incubated at 37⁰C for 1 hour. Then, they were washed with PBS and the antibodies were added: anti-SLPI and anti- cementoin (HBT) at a final concentration of 0,8ug/ml, each one diluted in RPMI 5% SBF. They were incubated for 1 hour 30 min at 37⁰C.

Then, the plate was washed twice with RPMI+5% SBF + 0,1% sodium azide and twice more with RPMI alone. Then, the plate was incubated with an anti-mouse antibody marked with peroxidase (Biorad) diluted 1:1500 in RPMI+5%SBF. It was incubated for 1 hour at 37⁰C.

Then, it was washed three times with physiological solution and revealed with 50 ul/well of TMB solution. The reaction was stopped with 50 ul of sulphuric acid and was read at 450 nm in an ELISA reader.

Flow Cytometry.

The fusion protein adhesion was analyzed over the surface of A549 epithelial cells stimulated with the TNFCC cytokine .

50OxIO³ cells were plated in each well of 6 wells plate in 3 ml of HAM + 10% SBF medium. It was incubated at 37⁰C in a culture stove by gassing of CO₂ for 24hs.

The following day the TNFCC cytokine at a final concentration of 500 U/ml was added to the corresponding group .

On the third day cells were raised with a scrapper and were partitioned among the different groups. Then SLPI or the fusion protein was added at a final concentration of 4ug/ml and was incubated at 4° C for 1 hour. Then it was washed three times with physiological solution and resuspended in cold PBS in FACS tubes for the marking thereof .

To each FACS tube 5 ul of some of the following antibodies were added: anti SLPI(HBT), anti Cementoin (HBT) or an isotypical control IgGl (Caltag) , were incubated for 45 minutes a 4⁰C, washed with cold PBS and a second antimouse antibody marked with FITC (Caltag) was added to each tube. After 45 minutes of incubation at 4⁰C cells were washed and analyzed in a flow cytometer.

Analysis of the in vivo adhesion.

The in vivo adhesion of the fusion protein to the popliteal lymph nodes of rats was analyzed. 100 ul of Pristane were injected in the sole of the right leg of three male rats, the left leg was kept as non-inflammated control. At 24 hours, the dose was repeated. After 3 hours, 200 ul of a 330 ug/ml of the fusion protein solution were injected through the penis dorsal vein or intraperitoneal via .

After 2 hours animals were sacrificed and right and left popliteal ganglions were obtained. Subsequently ganglions were disintegrated with a metallic mesh in cold PBS; they were washed and incubated for 40 minutes in PBS+10%SBF to block Fc receptors and marking and analysis by flow cytometry was carried out.

Example 5: Test of the anti-inflammatory activity of the fusion protein:

The anti-inflammatory activity of the fusion protein was analyzed in a in an inflammation model of the mice leg.

10 ul of Pristane + 10 ul of buffer were injected to the sole of the right leg of three mice and 10 ul Pristane + 10 ul of Cem-SLPI (300 ug/ml) were injected to the left leg. At 12 and 24 hours the leg size was gauge measured.

Example 6: Test of the anti-tumoral activity of the fusion protein: BalB/c female adult mice were purchased from the biotery at the Veterinary Faculty of La Plata University (UNLP) and kept in the biotery of the Microbiology Chair, Parasitology and Immunology of the Medicine Faculty at the University of Buenos Aires so as to carry out the experiments. The tumoral activity of the F3II cells (breast adenocarcinoma) , was evaluated in mice injected by subcutaneous via in the right flank with 800,000 cells, suspended in 200μl of RPMI 1640 (INVITROGEN, USA) . The tumors size was evaluated twice or three times per week using a gauge and the volume calculated from the formula: V= (LxW²) /2, wherein L is the length and W is the width of the tumor. Administration of the fusion protein Cem-SLPI (4 μg/ml), or dilution buffer was carried out by injecting 200 μl by subcutaneous via in the inoculation zone of the tumoral cells since the onset of the experiment and into the tumoral mass as the tumor started to be palpable along the experiment.

Example 7: Tests of the bacterial activity of the fusion protein:

Cultures of M bovis BCG Pasteur bacteria were developed in the surface of films in a Sauton synthetic medium for two weeks, bacteria were decanted and mixed with metallic spheres, and films were mildly homogenized (Huygen K, Abramowicz D, Vandenbussche P, Jacobs F, De Bruyn J, Kentos A, Drowart A, Van Vooren JP, Goldman M. Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice. Infect Immun. 1992 JuI; 60 ( 7) : 2880-6 ). Once the stationary phase was reached, the bacteria mass and the concentration were determined, and the aliquots thereof were preserved at -7O⁰C in glycerol up to the use thereof.

Antibacteria Test: 50 ml of M bovis BCG-Pasteur bacteria under the logarithmic phase at 0,5 mg/ml in Sauton buffer were added to 96 wells micro plates together with 50 ml of different concentrations of the fusion protein Cem-SLPI . After an overnight incubation at 37⁰C, the amount of CFU was determined by serial dilutions in 7H11 - OADC plates .

Example 8: Inhibition tests of the mononuclear cells proliferation in the presence SLPI or Cem-SLPI:

In a U bottom 96 wells culture micro plate 100,000 mononuclear cells were placed, resuspended in culture medium in 200 ul, obtained from the peripheral blood of a healthy donor. Cells were stimulated with IL-2 (8ng/ml) in the presence or absence of Cem-SLPI (4 μg/ml) for five days at 37⁰C and under a 5% atmosphere of CO₂. Finally, proliferation was quantified by introduction of ³H- Thymidine in a liquid scintillation counter.

Example 9: Preparation of the micro-spheres: The PCL micro-spheres (poly-ε-caprolactone) (M_w 14.000, Sigma, St. Louis, Mo) containing SLPI were prepared using a water-in-oil-in-water emulsion (Ai/0/A₂) based on the solvents evaporation technique. Briefly, 400 mg of PCL were dissolved in 10 ml of dichloromethane (O solution, Anedra) of HPLC grade. One ml of a solution containing 0.8 mg SLPI (Ai) and 30 mg of manitol were added to the oil solution (O), mixed with a vortex for 1 min. The first emulsion (Ai/0) was then added by constant stirring to 100 ml of an aqueous solution of 2% polyvinyl alcohol (PVA, Riedel-de Haen) . The obtained emulsion Ai/0/A₂ was kept at room temperature for 4 hours up to the complete evaporation of the methylene chloride. The micro-spheres were then collected by centrifugation (5000rpm at room temperature) and washed three times with distilled water. Finally, the micro-spheres were lyophilized.

Morphology and distribution of the micro-spheres size

Morphological characteristics of the micro-spheres were evaluated through a scanning electronic microscope (JEOL, JSM-35C, Japan) , equipped with a digital system of images acquisition (SemAfore). Samples were adhered with a double phase adhesion tape to a metallic microscope slide coated with gold and using a VE-300 VEECO evaporator under argon atmosphere. The size determination of the microspheres was carried out using a light microscope (Arcano XSZ-107E, Argentina) and measuring the diameters of 300 particles. Size distribution of the particles was adjusted in accordance with the frequency percentage.

Micro-spheres Characteristics

The prepared micro-spheres had an average size of 10.5 μm, with a protein content of 0.7 μg of SLPI/mg of microspheres .

SLPI Encapsulation:

The SLPI was encapsulated in the PCL micro-spheres. In order to evaluate the SLPI in Vitro release, 30 mg of micro-spheres were resuspended in PBS and a specific ELISA assay was carried out. The SLPI was release for a period of 30 days with an exploratory phase on day 15.

Example 10: Tests of the anti-inflammatory activity of the micro-spheres:

In vivo Experiments:

Sole model: The micro-spheres anti-inflammatory activity was evaluated in tests made in vivo. Balb/c mice were treated with pristane in the sole and the microspheres with SLPI were simultaneously administered by s.c. via .

Example 11: Assays on the use of the micro-spheres for the treatment of immune diseases:

Experimental model of autoimmune orchitis (EAO) : EAO group's rats were immunized with a testicular homogenate (TH). The TH (0,4 ml) emulsified with 0,4 ml of complete Freund adjuvant (CFA) were intradermically injected in multiple sites of the sole near the ganglion regions. Previously, 100 mg/animal of the micro-spheres with or without SLPI were i.p. injected. Administration of the micro-spheres was weekly repeated during all the experiment. Rats were sacrificed and weighed, one of the testis were removed, weighed and frozen to carry out the immunohistochemical tests. The other testis was fixed in Bouin for histopathological studies. The histopathology of the testis was made over paraffin sections obtained from three different levels and dyed with hematoxiline-eosine . SEQUENCE LIST <110> Conicet

<120> Fusion protein, pharmaceutical compositions, micro-spheres,

Uses and methods <130> C25 <160> 6

<170> Patentln version 3.2

<210> 1

<211> 156

<212> PRT

<213> Artificial

<220>

<223> Fusion protein cemetoin-SLPI

<400> 1

Met Ala VaI Thr GIy VaI Pro VaI Lys GIy GIn Asp Thr VaI Lys GIy 1 5 10 15

Arg VaI Pro Phe Asn GIy GIn Asp Pro VaI Lys GIy GIn VaI Ser VaI 20 25 30

Lys GIy GIn Asp Lys VaI Lys Lys Leu Ser GIy Lys Ser Phe Lys Ala 35 40 45

GIy VaI Cys Pro Pro Lys Lys Ser Ala GIn Cys Leu Arg Tyr Lys Lys 50 55 60

Pro GIu Cys GIn Ser Asp Trp GIn Cys Pro GIy Lys Lys Arg Cys Cys 65 70 75 80

Pro Asp Thr Cys GIy lie Lys Cys Leu Asp Pro VaI Asp Thr Pro Asn 85 90 95

Pro Thr Arg Arg Lys Pro GIy Lys Cys Pro VaI Thr Tyr GIy GIn Cys 100 105 110

Leu Met Leu Asn Pro Pro Asn Phe Cys GIu Met Asp GIy GIn Cys Lys 115 120 125

Arg Asp Leu Lys Cys Cys Met GIy Met Cys GIy Lys Ser Cys VaI Ser 130 135 140 Pro VaI Lys Ala Leu GIu His His His His His His 145 150 155

<210> 2

<211> 468

<212> DNA

<213> Artificial

<220>

<223> Nucleotides Sequence of the fusion protein cementoin-SLPI

<400> 2 atggctgtca cgggagttcc tgttaaaggt caagacactg tcaaaggccg tgttccattc

60 aatggacaag atcccgttaa aggacaagtt tcagttaaag gtcaagataa agtcaaaaag 120 ctttctggaa agtccttcaa agctggagtc tgtcctccta agaaatctgc ccagtgcctt 180 agatacaaga aacctgagtg ccagagtgac tggcagtgtc cagggaagaa gagatgttgt 240 cctgacactt gtggcatcaa atgcctggat cctgttgaca ccccaaaccc aacaaggagg 300 aagcctggga agtgcccagt gacttatggc caatgtttga tgcttaaccc ccccaatttc 360 tgtgagatgg atggccagtg caagcgtgac ttgaagtgtt gcatgggcat gtgtgggaaa 420 tcctgcgttt cccctgtgaa agctctcgag caccaccacc accaccac 468

<210> 3

<211> 26

<212> DNA

<213> Artificial

<220>

<223> Forward primer

<400> 3 aagctttctg gaaagtcctt caaagc 26

<210> 4

<211> 25

<212> DNA

<213> Artificial

<220>

<223> Reverse primer

<400> 4 ctcgagagct ttcacaggga aacgc 25 <210> 5

<211> 27

<212> DNA

<213> Artificial

<220>

<223> Forward primer

<400> 5 gttctacata tggctgtcac gggagtt 27

<210> 6

<211> 29

<212> DNA

<213> Artificial

<220>

<223> Reverse primer

<400> 6 aagctttttg actttatctt gacctttaa 29

Claims

CLAIMSHaving the nature of the present invention so especially described and determined as well as the way it has to be carried on into practice, it is hereby declared the following claims as of ownership and of exclusive right :

1. A transglutaminases binding fusion protein, characterized by comprising a transglutaminases binding domain bound to a serine proteases.

2. The protein according to claim 1, characterized because the transglutaminases binding domain is the cementoin domain or parts thereof.

3. The protein according to claim 1, characterized because the serine proteases inhibitor is selected from the 117 Clan IP family y the WAP family.

4. The protein according to claim 3, characterized because the serine proteases inhibitor is selected from the group consisting of the secretory leucocitary proteases inhibitor (SLPI), specific elastase inhibitor ELAFIN, αl- anti-tripsine, serum acid protein-2, unit 2 of the quelonianina inhibitor, unit 1 of the mucosal peptidases inhibitor, unit 1 of the peptidases inhibitor WFIKKN, unit 1 of the peptidases putative inhibitor WFIKKNRP 1, unit 1 of the eppin inhibitor, and parts thereof.

5. The protein according to claim 3, characterized because the serine proteases inhibitor is the secretory leucocitary proteases inhibitor (SLPI).

6. The protein according to claim 1, characterized because the transglutaminase is selected from the group consisting of transglutaminase 1, 2, 3, 4, 5, 6 and 7.

7. The protein according to claim 1, characterized by comprising the sequence shown in SEQ ID N⁰I.

8. A pharmaceutical composition, characterized by comprising a fusion protein, wherein said fusion protein comprises a transglutaminases binding domain bound to a serine proteases inhibitor, and pharmaceutically acceptable excipients .

9. The composition according to claim 8, characterized by the transglutaminases binding domain is the cementoin domain or parts thereof.

10. The composition according to claim 8, characterized because the serine proteases inhibitor is selected from the group consisting of the secretory leucocitary proteases inhibitor (SLPI), specific elastase inhibitor ELAFIN, αl-anti-tripsine, serum acid protein-2, unit 2 of the quelonianina inhibitor, unit 1 of the mucosal peptidases inhibitor, unit 1 of the peptidases inhibitor WFIKKN, unit 1 of the peptidases putative inhibitor WFIKKNRP 1, unit 1 of the eppin inhibitor, and parts thereof .

11. The composition according to claim 10, characterized because the serine proteases inhibitor is the secretory leucocitary proteases inhibitor (SLPI).

12. The composition according to claim 8, characterized because the transglutaminase is selected from the group consisting of transglutaminase 1, 2, 3, 4, 5, 6 and 7.

13. The composition according to claim 8, characterized by comprising the sequence shown in SEQ ID N⁰I.

14. The composition according to claim 8, characterized because said composition is in the form selected from the group consisting of emulsions, liquid forms, tablets, lozenges, capsules, micro-spheres, gels and patches .

15. The composition according to claim 14, characterized because said composition is of controlled release .

16. The use of the composition of claim 8, characterized because said composition is used for the preparation of a medicament.

17. The use according to claim 16, characterized because the medicament is used for the treatment of diseases selected from the group consisting of autoimmune diseases, cancer, infections, inflammatory disorders, inflammatory diseases, infective diseases, rheumatic diseases, degenerative diseases, sepsis, inflammatory bowel diseases and transplant rejection.

18. The use according to claim 16, characterized because the medicament is used for the healing of wounds.

19. The use of the composition of claim 8, characterized because said composition is used for the preparation of a cosmetic.

20. Micro-spheres for the preparation of a medicament or cosmetic, characterized by comprising encapsulated a protein selected from the group consisting of a fusion protein comprising a transglutaminases binding domain bound to a serine proteases inhibitor and a SLPI proton or parts thereof .

21. The micro-spheres according to claim 20, characterized by comprising encapsulated the fusion protein, wherein said fusion protein comprises a transglutaminases binding domain bound to a serine proteases inhibitor.

22. The micro-spheres according to claim 20, characterized by comprising encapsulated the SLPI protein or parts thereof.

23. The micro-spheres according to claim 20, characterized because the transglutaminases binding domain is the cementoin domain or parts thereof.

24. The micro-spheres according to claim 20, characterized because the serine proteases inhibitor is selected from the group consisting of the secretory leucocitary proteases inhibitor (SLPI), specific elastase inhibitor ELAFIN, αl-anti-tripsine, serum acid protein-2, unit 2 of the quelonianina inhibitor, unit 1 of the mucosal peptidases inhibitor, unit 1 of the peptidases inhibitor WFIKKN, unit 1 of the peptidases putative inhibitor WFIKKNRP 1, unit 1 of the eppin inhibitor, and parts thereof .

25. The micro-spheres according to claim 24, characterized because the serine proteases inhibitor is the secretory leucocitary proteases inhibitor (SLPI).

26. The micro-spheres according to claim 20, characterized because the transglutaminase is selected from the group consisting of transglutaminase 1, 2, 3, 4, 5, 6, 7.

27. The micro-spheres according to claim 20, characterized because the fusion protein comprises the sequence shown in SEQ ID N⁰I.

28. The micro-spheres according to claim 20, characterized by having a diameter size between 2 and 20 μm.

29. The micro-spheres according to claim 20, characterized by being formed by polymers selected from the group consisting of poly-ε-caprolactone, polylactic acid (PPLA) and polylactic-glycolic acid (PLGA) .

30. The use of the micro-spheres of claim 20, characterized because said micro-spheres are used for the preparation of a medicament.

31. The use of the micro-spheres of claim 20, characterized because said micro-spheres are used for the preparation of a cosmetic composition.

32. The use according to claim 30, characterized because the medicament is used for the treatment of diseases selected from the group consisting of autoimmune diseases, cancer, infections, inflammatory disorders, inflammatory diseases, infective diseases, rheumatic diseases, degenerative diseases, sepsis, inflammatory bowel diseases and transplant rejection.

33. The use according to claim 30, characterized because the medicament is used for the healing of wounds.

34. The use of the fusion protein of claim 1, characterized because said fusion protein is used for the preparation of a medicament.

35. The use de Ia fusion protein of claim 1, characterized because said fusion protein is used for the preparation of a cosmetic.

36. The use according to claim 34, characterized because the medicament is used for the treatment of diseases selected from the group consisting of autoimmune diseases, cancer, infections, inflammatory disorders, inflammatory diseases, infective diseases, rheumatic diseases, degenerative diseases, sepsis, inflammatory bowel diseases and transplant rejection.

37. The use according to claim 34, characterized because the medicament is used for the healing of wounds.

38. A method of treating patients suffering from an autoimmune disease, characterized by comprising the administration of a required amount of the SLPI protein or parts thereof.

39. The method according to claim 38, characterized because the autoimmune disease is selected from the group consisting of autoimmune orchitis, AR, systemic lupus erythematosus, thyroiditis, dermatomyositis, fibromyalgias, polimyositis, Hashimoto thyroiditis, diabetes mellitus, Hemolytic anemia, encephalitis, multiple sclerosis, primary liver cirrhosis, active chronic hepatitis, pemphigus, pemphigoid, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, scleroderma, miastenia gravis and Sjogren syndrome.

40. The method according to claim 38, characterized because the SLPI protein is administered by the via selected from the group consisting of intravenous, subcutaneous, intradermal, intramuscular, intraperitoneal, intratumoral, intranasal, peridural, intraventricular and oral .

41. The method according to claim 38, characterized because the SLPI protein is administered near to the autoimmune injury.

42. The method according to claim 38, characterized because the SLPI protein is administered in the autoimmune injury .

43. The method according to claim 38, characterized because the required amount ranges around 100 mg and 0.001 ug of the SLPI protein.

44. The method according to claim 38, characterized because the SLPI protein is administered encapsulated into micro-spheres .

45. The method according to claim 44, characterized because the micro-spheres has a diameter size between 2 and 20 μm.

46. The method according to claim 44, characterized because the micro-spheres are formed by polymers selected from the group consisting of poly-ε-caprolactone, polylactic acid (PPLA) and polylactic-glycolic acid (PLGA)

47. The method according to claim 44, characterized because the amount of SLPI per mg of micro-spheres is around 0.1 and 3000 ng.