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WO2009083880A1 - Protéines hybride liant les transglutaminases, compositions et microsphères les comprenant, utilisations et méthodes - Google Patents

Protéines hybride liant les transglutaminases, compositions et microsphères les comprenant, utilisations et méthodes Download PDF

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Publication number
WO2009083880A1
WO2009083880A1 PCT/IB2008/055409 IB2008055409W WO2009083880A1 WO 2009083880 A1 WO2009083880 A1 WO 2009083880A1 IB 2008055409 W IB2008055409 W IB 2008055409W WO 2009083880 A1 WO2009083880 A1 WO 2009083880A1
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Prior art keywords
inhibitor
slpi
micro
spheres
protein
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English (en)
Spanish (es)
Inventor
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
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Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Inis Biotech LLC
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Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Inis Biotech LLC
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Publication of WO2009083880A1 publication Critical patent/WO2009083880A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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).
  • SLPI secretory leucocyte protease inhibitor
  • 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
  • the SLPI 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.
  • proteases and the inhibitors thereof are 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.
  • 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.
  • fusion proteins comprising SLPI
  • 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.
  • 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.
  • the transglutaminase binding domain is the cementoin domain or parts thereof and the serine protease inhibitor is the secretory leucocyte protease inhibitor (SLPI).
  • the fusion protein may link to different transglutaminases as transglutaminase 1, 2, 3, 4, 5, 6 and 7.
  • the fusion protein comprises the sequence showed in SEC ID N 0 I.
  • Another object of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising a transglutaminases binding domain linked to a serine protease inhibitor and acceptable pharmaceutical excipients.
  • the transglutaminases binding domain is the cementoin domain or parts thereof and the serine protease inhibitor is the secretory leucocyte protease inhibitor (SLPI).
  • the composition comprises the fusion protein of the sequence showed in SEC ID N 0 I 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.
  • 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.
  • 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 ⁇ m and may be constituted by poly- ⁇ -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.
  • 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- ⁇ - caprolactone, polylactic acid (PPLA) or polylactic-glycolic acid (PLGA) .
  • the amount of SLPI per mg of micro-spheres is among 0.1 and 3000 ng.
  • 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 TNF ⁇ .
  • 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.
  • 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)
  • 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.
  • 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.
  • the fusion protein comprises the transglutaminase enzyme substrate (cementoin) y the secretory leucocyte protease inhibitor (SLPI) or some of the domains thereof.
  • the fusion protein has the amino acids sequence shown in SEC ID N 0 1.
  • 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.
  • nucleotide sequences coding for a fusion protein comprising a transglutaminases binding domain and a serine protease inhibitor or some of the domains thereof.
  • nucleotide sequence is the sequence shown in SEC ID N 0 2.
  • the fusion protein is also referred to as Cem-SLPI .
  • the 117 Clan IP Family 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 .
  • protein also refers to any polypeptide or amino acids chain, lineal branched, dimers or multimers.
  • the fusion protein keeps the inhibitory activity over serine proteases, for example elastase.
  • the fusion protein is the protein shown in SEC ID N 0 1 (Cem-SLPI), which shows an inhibitory activity greater than that of the individual SLPI protein (see figures 1 and 2 ) .
  • 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 TNF ⁇ cytokine, being the bond detected through human nati-SLPI monoclonal antibodies.
  • 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.
  • 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.
  • 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.
  • 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) .
  • the administration of said protein decreases the tumour size.
  • 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 .
  • the SLPI or the Cem-SLPI fusion protein may be formulated encapsulated in microspheres, for example the micro-spheres may be of PCL (poly- ⁇ -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 .
  • 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.
  • the micro-spheres containing SLPI have been used to treat rats with autoimmune orchitis.
  • 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.
  • ip intraperitoneal via
  • testicles were removed and the histopathology of each testicle was analyzed.
  • 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 .
  • 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.
  • sc subcutaneous injections
  • 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 .
  • micro-spheres of the invention carrying SLPI showed to have anti-trypsine activity.
  • micro-spheres may be of any size; preferably the micro-spheres are around 2 and 20 ⁇ m 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.
  • 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
  • Example 1 Preparation of the Cem-SLPI fusion protein and characterization thereof:
  • 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.
  • a pair of modified primers was used:
  • Reverse primer SEQ ID N 0 4 CTCGAGAGCTTTCACAGGGAAACGC
  • the cycling was carried out in a MJ Research thermocycler, the scheduled cycle was as follows: 2 min at 94 0 C, followed by 30 cycles of 1 min at 94 0 C, 1 min at 49 0 C and 1 min at 72 0 C and a final cycle of 10 min at 72 0 C.
  • 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 MgCl 2 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 PureLinkTM 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 15 0 C.
  • 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.
  • Reverse primer SEQ ID N 0 6 AAGCTTTTTGACTTTATCTTGACCTTTAA.
  • the cycling was carried out at an MJ Research thermocycler, the scheduled cycle was as follows: 2 min at 94 0 C, followed by 30 cycles of 1 min each at 94 0 C, 1 min at 6O 0 C and 1 min at 72 0 C and a final cycle of 10 min at 72 0 C.
  • 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 MgCl 2 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 .
  • fusion proteins cloned genes from SLPI and cementoin were amplified by PCR with 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 PureLinkTM Gel Extraction Kit was used, from Invitrogen, following the manufacturer's instructions.
  • 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 37 0 C 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.
  • Xhol enzyme Promega
  • 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 37 0 C 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 .
  • Ndel enzyme New England BioLab
  • E.coli TOPlO bacteria Invitrogen
  • 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) .
  • 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 37 0 C.
  • 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 28 0 C 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.
  • 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 4 0 C. 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:
  • 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:
  • the adhesion of the fusion protein to the surface of an epithelial cell monolayer (A549) stimulated with the TNFCC cytokine was quantified.
  • TNFCC cytokine was added at a concentration of 500 units per well, to the corresponding groups, being incubated with the cytokine for 24 hours.
  • 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 37 0 C.
  • the fusion protein adhesion was analyzed over the surface of A549 epithelial cells stimulated with the TNFCC cytokine .
  • 50OxIO 3 cells were plated in each well of 6 wells plate in 3 ml of HAM + 10% SBF medium. It was incubated at 37 0 C in a culture stove by gassing of CO 2 for 24hs.
  • anti SLPI(HBT), anti Cementoin (HBT) or an isotypical control IgGl (Caltag) were incubated for 45 minutes a 4 0 C, washed with cold PBS and a second antimouse antibody marked with FITC (Caltag) was added to each tube. After 45 minutes of incubation at 4 0 C cells were washed and analyzed in a flow cytometer.
  • 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 .
  • 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.
  • 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 wholesome 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 200 ⁇ l of RPMI 1640 (INVITROGEN, USA) .
  • Administration of the fusion protein Cem-SLPI (4 ⁇ g/ml), or dilution buffer was carried out by injecting 200 ⁇ l 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:
  • 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 37 0 C, 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:
  • Example 9 Preparation of the micro-spheres:
  • the PCL micro-spheres (poly- ⁇ -caprolactone) (M w 14.000, Sigma, St. Louis, Mo) containing SLPI were prepared using a water-in-oil-in-water emulsion (Ai/0/A 2 ) 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) .
  • PVA polyvinyl alcohol
  • the obtained emulsion Ai/0/A 2 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.
  • 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.
  • the prepared micro-spheres had an average size of 10.5 ⁇ m, with a protein content of 0.7 ⁇ g of SLPI/mg of microspheres .
  • the SLPI was encapsulated in the PCL micro-spheres.
  • 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:
  • 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:
  • EAO autoimmune orchitis

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Abstract

La présente invention concerne les protéines hybrides liant les transglutaminases, les compositions les comprenant, les microsphères les comprenant, les utilisations et les méthodes. Les protéines hybrides peuvent comprendre un domaine de liaison aux transglutaminases, par exemple de domaine cementoïne et un inhibiteur de la serine protéase, par exemple l'inhibiteur sécrétoire de la protéase leucocytaire (SLPI). Les protéines hybrides sont utiles pour la préparation de médicaments pour le traitement, par exemple, de maladies auto-immunes, de troubles inflammatoires, du cancer, d'infections et la cicatrisation de plaies.
PCT/IB2008/055409 2007-12-28 2008-12-18 Protéines hybride liant les transglutaminases, compositions et microsphères les comprenant, utilisations et méthodes Ceased WO2009083880A1 (fr)

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ARP070105988 2007-12-28

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011107505A1 (fr) * 2010-03-03 2011-09-09 Proteo Biotech Ag Nouvelles utilisations de l'élafine
RU2639526C2 (ru) * 2011-06-28 2017-12-21 ИНХИБРКС ЭлЭлСи Слитые полипептиды, содержащие домен wap, и способы их применения
CN110152496A (zh) * 2018-01-30 2019-08-23 广州达济医学科技有限公司 一种白细胞过滤膜及其制备方法
US10400029B2 (en) 2011-06-28 2019-09-03 Inhibrx, Lp Serpin fusion polypeptides and methods of use thereof
US10723785B2 (en) 2011-06-28 2020-07-28 Inhibrx, Inc. Serpin fusion polypeptides and methods of use thereof
WO2023122120A1 (fr) * 2021-12-22 2023-06-29 The Regents Of The University Of California Compositions et méthodes de cicatrisation de plaie

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WO2007011363A2 (fr) * 2004-08-11 2007-01-25 Trubion Pharmaceuticals, Inc. Proteines de fusion a domaine de liaison

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011107505A1 (fr) * 2010-03-03 2011-09-09 Proteo Biotech Ag Nouvelles utilisations de l'élafine
GB2492908A (en) * 2010-03-03 2013-01-16 Proteo Biotech Ag Novel uses of elafin
RU2639526C2 (ru) * 2011-06-28 2017-12-21 ИНХИБРКС ЭлЭлСи Слитые полипептиды, содержащие домен wap, и способы их применения
US9914765B2 (en) 2011-06-28 2018-03-13 Inhibrx Lp WAP domain fusion polypeptides and methods of use thereof
US10400029B2 (en) 2011-06-28 2019-09-03 Inhibrx, Lp Serpin fusion polypeptides and methods of use thereof
US10723785B2 (en) 2011-06-28 2020-07-28 Inhibrx, Inc. Serpin fusion polypeptides and methods of use thereof
US10730929B2 (en) 2011-06-28 2020-08-04 Inhibrx Lp Serpin fusion polypeptides and methods of use thereof
US11046752B2 (en) 2011-06-28 2021-06-29 Inhibrx, Inc. Serpin fusion polypeptides and methods of use thereof
US11827691B2 (en) 2011-06-28 2023-11-28 Inhibrx, Inc. Serpin fusion polypeptides and methods of use thereof
US11965017B2 (en) 2011-06-28 2024-04-23 Inhibrx, Inc. Serpin fusion polypeptides and methods of use thereof
CN110152496A (zh) * 2018-01-30 2019-08-23 广州达济医学科技有限公司 一种白细胞过滤膜及其制备方法
WO2023122120A1 (fr) * 2021-12-22 2023-06-29 The Regents Of The University Of California Compositions et méthodes de cicatrisation de plaie

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