Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • 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
  • A61P35/00—Antineoplastic agents
• • 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
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/545—IL-1
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
  • C07K14/56—IFN-alpha
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

• the present invention relates to biologically active proteins comprising at least two domains wherein a first domain of said at least two domains comprises an amino acid sequence having and/or mediating said biological activity and a second domain of said at least two domains comprises an amino acid sequence consisting preferably of at least about 100 amino acid residues forming a random coil conformation whereby said random coil conformation mediates an increased in vivo and/or in vitro stability of said biologically active protein.
• nucleic acid molecules encoding the inventive biologically active proteins and vectors and cells comprising said nucleic acid molecules are disclosed.
• the present invention provides for compositions comprising the compounds of the invention as well as for specific uses of the biologically active proteins, nucleic acid molecules, vectors and cells of the invention.
• HSA human serum albumin
• Igs immunoglobulins
• FcRn neonatal Fc receptor
• most other proteins of pharmaceutical interest in particular recombinant antibody fragments, hormones, interferons, etc. suffer from rapid (blood) clearance. This is particularly true for proteins whose size is below the threshold value for kidney filtration of about 70 kDa (Caliceti (2003) Adv Drug Deliv Rev 55:1261-1277).
• the recycling mechanism of natural plasma proteins has been employed by producing fusion proteins with the Fc portion of Igs, for example Enbrel®, a hybrid between the extracellular domain of TNF ⁇ receptor and human IgGl (Goldenberg (1999) Clin Ther 21:75- 87) or with serum albumin, for example Albuferon®, a corresponding fusion of IFN ⁇ with HSA (Osborn (2002) J Pharmacol Exp Ther 303:540-548).
• Igs for example Enbrel®
• serum albumin for example Albuferon®
• Albumin with its high plasma concentration of 600 ⁇ M has also been utilized in an indirect manner, serving as carrier vehicle for biopharmaceuticals that are equipped with an albumin-binding function, for example via fusion with a bacterial albumin-binding domain (ABD) from Streptococcal protein G (Makrides (1996) J Pharmacol Exp Ther 277:534-542) or with a peptide selected against HSA from a phage display library (Dennis (2002) J Biol Chem, 277:35035-35043; Nguyen (2006) Protein Eng Des SeI 19:291-297).
• ABS bacterial albumin-binding domain
• fusion proteins comprising the Trypanosoma cruzi derived artificial repetitive sequence PSTAD have been reported to induce a prolonged plasma half-life of trans-sialidase (Alvarez (2004) PNAS 279:3375-3381). Yet, such Trypanosoma cruzi derived repeats have been reported to induce a humoral immune response (Alvarez (2004) loc. cit.). Accordingly, alternative means to prolong the action of biologically active proteins are desired.
• the technical problem underlying the present invention is the provision of biologically active proteins with an increased in vivo and/or in vitro stability.
• the solution to the above technical problem is achieved by providing the embodiments characterized in the claims.
• this invention relates to a biologically active protein comprising at least two domains wherein
• a first domain of said at least two domains comprises an amino acid sequence having and/or mediating said biological activity
• a second domain of said at least two domains comprises an amino acid sequence consisting preferably of at least about 100 amino acid residues forming random coil conformation.
• said second domain forming/adopting random coil conformation is capable of mediating an increased in vivo and/or in vitro stability of said biologically active protein. Said second domain, therefore, leads to an increased in vivo and/or in vitro stability of a given protein (or a fragment thereof) having a/or mediating a given biological activity, as defined herein below.
• intravenously administered biologically active proteins which are modified to comprise a random coil domain/part display an unexpected prolonged plasma half-life when compared to the unmodified biologically active proteins, i.e. which lacks said random coil domain.
• random coil relates to any conformation of a polymeric molecule, including amino acid polymers, in which the individual monomelic elements that form said polymeric structure are essentially randomly oriented towards the adjacent monomelic elements while still being chemically bound to said adjacent monomelic elements.
• a polypeptide or amino acid polymer adopting/having/forming "random coil conformation” substantially lacks a defined secondary and tertiary structure.
• the nature of polypeptide random coils and their methods of experimental identification are known to the person skilled in the art and have been described in the scientific literature (Cantor (1980) Biophysical Chemistry, 2nd ed., W. H. Freeman and Company, New York; Creighton (1993) Proteins - Structures and Molecular Properties, 2nd ed., W. H. Freeman and Company, New York; Smith (1996) Fold Des l :R95-R106).
• the biologically active proteins of the present invention comprise a domain (defined herein above as said "second domain” of the inventive biologically active protein) that adopts/ forms random coil conformation at physiological conditions.
• physiological conditions is known in the art and relates to those conditions in which proteins usually adopt their native conformation. More specifically, the term “physiological conditions” relates to the biophysical parameters as they are typically valid for higher forms of life and, particularly, in mammals, most preferably human beings.
• the term “physiological conditions” may relate to the biochemical and biophysical parameters as they are normally found in the body (in particular in body fluids) of mammals and in particular in humans. Said “physiological conditions” may relate to the corresponding parameters found in the healthy body as well as the parameters as found in sick mammals or human patients.
• a sick mammal or human patient may have a higher, yet "physiological” temperature condition when said mammal or said human suffers from fever.
• physiological conditions at which proteins adopt their native conformation/state, the most important parameters are temperature (37 0 C for the human body), pH (7.35 - 7.45 for human blood), osmolality (280 - 300 mmol/kg H 2 O), and, if necessary, protein content (66 - 85 g/1 serum).
• these parameters may vary, e.g.
• the temperature, pH, osmolality, and protein content may be different in given body or tissue fluids such as blood, liquor cerebrospinalis, peritoneal fluid and lymph (Klinke (2005) Physiologie, 5th ed., Georg Thieme Verlag, Stuttgart).
• the osmolality may be around 290 mmol/kg H 2 O and the protein concentration may be between 0.15 g/1 to 0.45 g/1.
• the pH may be around 7.4 and the protein content may be between 3 g/1 and 5 g/1.
• the biophysical parameters such as temperature, pH, osmolality and protein content may be different to the physiological conditions normally found in vivo. Temperatures between 1 °C and 42 °C or preferably 4 °C to 25 0 C may be considered useful to test and/or verify the biophysical properties and biological activity of a protein under physiological conditions in vitro.
• buffers in particular in experimental settings (for example in the determination of protein structures, in particular in circular dichroism (CD) measurements and other methods that allow the person skilled in the art to determine the structural properties of a protein/amino acid stretch) or in buffers, solvents and/or excipients for pharmaceutical compositions, are considered to represent "physiological solutions" / "physiological conditions" in vitro.
• buffers are, e.g. phosphate-buffered saline (PBS: 115 niM NaCl, 4 mM KH 2 PO 4 , 16 mM Na 2 HPO 4 pH 7.4), Tris buffers, acetate buffers, citrate buffers or similar buffers such as those used in the appended examples.
• the pH of a buffer representing "physiological solution conditions” should lie in a range from 6.5 to 8.5, preferably in a range from 7.0 to 8.0, most preferably in a range from 7.2 to 7.7 and the osmolality should lie in a range from 10 to 1000 mmol/kg H 2 O, more preferably in a range from 50 to 500 mmol/kg H 2 O and most preferably in a range from 200 to 350 mmol/kg H 2 O.
• the protein content of a buffer representing physiological solution conditions may lie in a range from 0 to 100 g/1, neglecting the protein with biological activity itself, whereby typical stabilizing proteins may be used, for example human or bovine serum albumin.
• the random coil conformation as comprised in the above defined “second domain” of the inventive biologically active protein is maintained in pharmaceutical compositions, like liquid pharmaceuticals.
• pharmaceutical compositions like liquid pharmaceuticals.
• “physiological conditions” are to be used in corresponding buffer systems, solvents and/or excipients.
• the random coil conformation as comprised in the "second domain” of the inventive biologically active protein is transiently not present and/or can not be detected.
• said "second domain” will, in accordance with the present inventive protein constructs, adopt/form again its random coil after reconstitution in corresponding buffers/solutions/excipients/solvents.
• inventive protein constructs had been lyophilized or dried (e.g. in form of a pharmaceutical composition).
• inventive protein constructs had been lyophilized or dried (e.g. in form of a pharmaceutical composition).
• the random coil part/domain is again present and the corresponding inventive construct can be, e.g., administered to a mammal or human patient in need of medical intervention.
• the biologically active proteins of the present invention comprise a domain (defined herein above as said "second domain” of the inventive biologically active protein) that adopts/ forms random coil conformation at/under physiological conditions.
• denatured proteins are proteins that lost their functional conformation and may partially adopt random coil conformation as a result of said denaturation.
• Proteins can be denatured through various means including exposure to unphysiological temperature, pH and/or salt concentration or exposure to denaturing agents like urea/guanidinium chloride and detergents. Accordingly, the presence of compounds that are known to have a denaturing effect on proteins, such as urea, guanidinium chloride or sodium dodecyl sulphate, are to be avoided when studying a protein under physiological conditions.
• Urea may be tolerated up to concentrations of 10 mmol/1 or even 300 mmol/1 when investigating a protein for application under physiological conditions in human blood or urine, respectively.
• the amino acid sequence of the random coil domain (said "second domain") as comprised in the inventive protein construct natively adopts/has random coil conformation, in particular in vivo and when administered to mammals or human patients in need of medical intervention.
• the protein construct of the present invention may comprise the "second", random coil forming/adopting domain in form of the herein identified alanine, serine, and proline stretches (or other amino acid stretches that form/have/adopt random coil under physiological conditions), but may be (for example, in form of a specific composition, like a lyophylisate or dried composition) transiently or temporarily not in random coil form.
• a "second domain” of the inventive protein construct again adopts after, e.g., reconstitution in corresponding buffers (preferably “physiological” buffers/excipients and/or solvents), the herein defined random coil.
• Said "second domain” is, (after a corresponding reconstitution) capable of mediating an increased in vivo and/or in vitro stability of the inventive biologically active protein.
• the biologically active protein of this invention has a longer in vivo and/or in vitro half-life and stability in comparison to the same "protein of interest” / "first doman” that does not comprise the additional "second domain” as defined herein.
• domain relates to any region/part of an amino acid sequence that is capable of autonomously adopting a specific structure and/or function.
• a domain may represent a functional domain or a structural domain.
• the proteins of the present invention comprise at least one domain/part having and/or mediating biological activity and at least one domain/part forming random coil conformation.
• the proteins of the invention also may consist of more than two domains and may comprise e.g. an additional linker structure between the herein defined two domains/parts or another domain/part like, e.g.
• a protease sensitive cleavage site an affinity tag such as the His 6 -tag or the Strep-tag, a signal peptide, retention peptide, a targeting peptide like a membrane translocation peptide or additional effector domains like antibody fragments for tumour targeting associated with an anti-tumour toxin or an enzyme for prodrug-activation etc.
• CD spectroscopy represents a light absorption spectroscopy method in which the difference in absorbance of right- and left-circularly polarized light by a substance is measured.
• the secondary structure of a protein can be determined by CD spectroscopy using far-ultraviolet spectra with a wavelength between approximately 190 and 250 nm.
• CD spectrometry the skilled artisan is readily capable of determining whether an amino acid polymer forms/adopts random coil conformation at physiological conditions.
• Other established biophysical methods include nuclear magnetic resonance (NMR) spectroscopy, absorption spectrometry, infrared and Raman spectrometry, measurement of the hydrodynamic volume via size exclusion chromatography, analytical ultracentrifugation or dynamic/static light scattering as well as measurements of the frictional coefficient or intrinsic viscosity (Cantor (1980) loc. cit; Creighton (1993) loc. cit.; Smith (1996) loc. cit).
• the biologically active protein of the invention has a hydrodynamic volume as determined by analytical gel filtration (also known as size exclusion chromatography, SEC) of at least 70 kDa, preferably of at least 80 kDa, more preferably of at least 90 kDa, even more preferably of at least 100 kDa, particularly preferably of at least 125 kDa and most preferably of at least 150 kDa.
• analytical gel filtration also known as size exclusion chromatography, SEC
• SEC size exclusion chromatography
• the hydrodynamic volume of a globular polypeptide can be estimated by its molecular weight.
• the hydrodynamic volume of the proteins of the invention that comprise the above defined second domain i.e. the domain comprising at least 100 amino acid residues and having random coil conformation, are shown to have an unexpectedly high hydrodynamic volume in relation to the estimated hydrodynamic volume for a corresponding folded, globular protein based on their molecular weight.
• the amino acid sequence adopting/having/forming random coil conformation consists of at least about 100 amino acid residues, preferably of at least about 150 amino acid residues, more preferably of at least about 200 amino acid residues, even more preferably of at least about 250 amino acid residues, particularly preferably of at least about 300 amino acid residues, more particularly preferably of at least about 350 amino acid residues and most preferably of at least about 400 amino acid residues.
• the amino acid sequence forming random coil conformation consists of maximally about 1000 amino acid residues, preferably of maximally about 900 amino acid residues, more preferably of maximally about 800 amino acid residues, even more preferably of maximally about 700 amino acid residues, particularly preferably of maximally about 600 amino acid residues.
• the amino acid sequence forming random coil conformation may consist of maximally about 500 amino acid residues or of maximally about 450 amino acid residues. It is also envisaged herein that the amino acid sequence forming random coil conformation may consist of maximally about 1200, about 1500 and up to about 3000 amino acid residues. Accordingly, the amino acid sequence forming random coil conformation may consist of about 100 to about 3000 amino acid residues. In particular embodiments said amino acid sequence forming random coil conformation consists of about 100 to 1000 amino acid residues as characterized herein, i.e. comprising alanine, serine and proline as main or unique residues as defined below.
• the gist of the present invention is, accordingly, the provision of amino acid polymers that form random coil conformation under physiological conditions and consist mainly of these three amino acid residues, whereby proline residues represent preferably about 4 % to about 40 % of the random coil forming domain.
• proline residues represent preferably about 4 % to about 40 % of the random coil forming domain.
• the alanine and serine residues comprise the remaining at least 60 % to 96 % of said random coil forming domain.
• said random coil forming domain may also comprise further amino acids differing from alanine, serine, and proline, i.e. as minor constituents.
• amino acid residues is not limited to the concise number of amino acid residues but also comprises amino acid stretches that comprise an additional 10% to 20 % or comprise 10% to 20 % less residues.
• amino acid residues may also comprise 80 to 100 and about 100 to 120 amino acid residues without deferring from the gist of the present invention.
• the "second domain" of the inventive biologically active protein(s)/polypeptide(s) comprises a maximal length of about 1000 amino acid residues.
• second domains are envisaged in context of the present invention, i.e.
• second domains providing for the desired random coil conformation under physiological conditions and comprising up to about 3000 amino acid residues.
• the term “about” in this context is not limited or restricted to the concise amount of amino acid residues but may also comprise +/- about 10% or +/- about 20% without deferring from this invention.
• amino acid polymers consisting mainly of alanine and serine residues or, in a preferred embodiment consisting mainly or uniquely of alanine, serine, and proline residues, form random coil conformation under physiological conditions.
• the present invention provides for modules/sequence units/polymer repeats/polymer cassettes/building blocks consisting of alanine, serine, and proline which can be used as (a) part(s) of the herein defined "second domain" of a biologically active protein/polypeptide.
• amino acid polymer also may form random coil conformation when other residues than alanine, serine, and proline are comprised as a minor constituent in said "second domain".
• minor constituent means that maximally 10% i.e. maximally 10 of 100 amino acids may be different from alanine, serine and proline, preferably maximally 8% i.e. maximally 8 of 100 amino acids may be different than alanine, serine and proline, more preferably maximally 6% i.e. maximally 6 of 100 amino acids may be different from alanine, serine and proline, even more preferably maximally 5% i.e.
• maximally 5 of 100 amino acids may be different from alanine, serine and proline, particularly preferably maximally 4% i.e. maximally 4 of 100 amino acids may be different from alanine, serine and proline, more particularly preferably maximally 3% i.e. maximally 3 of 100 amino acids may be different from alanine, serine and proline, even more particularly preferably maximally 2% i.e. maximally 2 of 100 amino acids may be different from alanine, serine and proline and most preferably maximally 1% i.e. maximally 1 of 100 of the amino acids that encode the random coil forming domain may be different from alanine, serine and proline.
• Said amino acids different from alanine, serine and proline may be selected from the group consisting of Arg, Asn, Asp, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and VaI.
• the amino acid polymers as disclosed herein and consisting of alanine, serine, and proline according to the invention were surprisingly found to adopt random coil conformation under physiological conditions. Therefore, they are advantageous molecules to provide for the herein defined "second domain" of the inventive biologically active protein(s)/polypeptide(s), i.e. a polypeptide stretche that forms under physiological conditions a random coil conformation and thereby mediates an increased in vivo and/or in vitro stability to biologically active ("functional") protein(s) or polypeptide(s).
• the hydrodynamic volume of a functional protein that is fused to said random coil domain is dramatically increased as can be estimated by using standard methods mentioned herein and also illustrated in the appended examples.
• random coil domain Since the random coil domain is thought not to adopt a stable structure or function by itself, the biological activity mediated by the functional protein of interest to which it is fused is essentially preserved. Moreover, the amino acid polymers that form random coil domain as disclosed herein are thought to be biologically inert, especially with respect to proteolysis in blood plasma, immunogenicity, isoelectric point/electrostatic behaviour, binding to cell surface receptors as well as internalisation, but still biodegradable, which provides clear advantages over synthetic polymers such as PEG.
• the amino acid polymers adopting random coil conformation under physiological conditions comprise a plurality of "amino acid repeats'V'amino acid cassettes'V'cassette repeats", wherein said "amino acid repeats'V'amino acid cassettes'V'cassette repeats" consist of Ala, Ser, and Pro residues (depicted herein as "PAS", or as "APS") and wherein no more than 6 consecutive amino acid residues are identical and wherein said proline residues constitute more than 4% and less than 40% of the amino acids of said second domain forming random coil.
• amino acid repeats'V'amino acid cassettes'V'cassette repeats consist of Ala, Ser, and Pro residues (depicted herein as "PAS", or as "APS") and wherein no more than 6 consecutive amino acid residues are identical and wherein said proline residues constitute more than 4% and less than 40% of the amino acids of said second domain forming random coil.
• Amino acid polymers adopting random coil conformation under physiological conditions may comprise a plurality of identical amino acid repeats / cassette repeats or a plurality of non-identical amino acid repeats.
• amino acid repeats "building blocks”, “modules”, “repeats”, “amino acid cassettes” etc. consisting of Ala, Ser and Pro residues are provided herein below; see SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28 or fragments or multimers of these sequences.
• a "fragment” comprises at least 3 amino acids and comprises at least one alanine, one serine and/or one proline.
• the amino acid repeat according to the present invention may consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein each repeat comprises (an) Ala, Ser, and Pro residue(s).
• the amino acid repeat according to the present invention does not comprise more than 100 amino acid residues.
• the amino acid repeat/cassette repeat as defined herein comprises more than about 4 %, preferably more than about 5 %, even more preferably more than about 6%, particularly preferably more than about 8%, more particularly preferably more than about 10%, even more particularly preferably more than about 15% and most preferably more than about 20% proline residues.
• Such an amino acid repeat/cassette repeat as defined herein preferably comprises less than about 40 % or less than about 35% proline residues; see also the herein below provided PAS constructs.
• amino acid polymers forming random coil conformation under physiological conditions have the formula (I):
• amino acid polymer according to formula (I) further comprises proline residues as defined herein and wherein x is independently selected from integer 0 to 6. Furthermore, for each n, y is independently selected from integer 1 to 6 and each z is independently selected from integer 1 to 6. n, finally, is any integer so that said second domain consists of at least about 100 amino acid residues, and in particular of at least about 100 to about 3000 amino acid residues, preferably to about 2000 and more preferably to about 1000 amino acid residues.
• the amino acid polymer comprising the above defined "amino acid repeats'V'amino acid cassettes"/"cassette repeats" forming random coil conformation comprises no more than 5 identical consecutive amino acid residues, more preferably no more than 4 identical consecutive amino acid residues and most preferably no more than 3 identical consecutive amino acid residues.
• the amino acid polymer of the invention which forms random coil conformation comprises proline residues, wherein said proline residues constitute more than about 4 %, preferably more than about 5 %, even more preferably more than about 6%, particularly preferably more than about 8%, more particularly preferably more than about 10%, even more particularly preferably more than about 15% and most preferably more than about 20% of the amino acids constituting the random coil forming domain.
• Such an amino acid polymer of the invention which forms random coil conformation preferably comprises less than about 40 %, or less than about 35% of the amino acids constituting the random coil forming domain.
• the PAS#1P2 polymer with its smaller proportion of Pro residues shows a less pronounced minimum around 200 run in its CD spectrum, indicating a dependency of the random coil character of the amino acid polymers according to this invention upon the content of proline residues.
• the amino acid polymer comprising the above defined "amino acid repeats'V'amino acid cassettes'V'cassette repeats" forming random coil conformation comprises more than about 4 % but less than about 50 %, preferably more than about 10 % but less than about 50 % and most preferably more than about 20 % but less than about 50 % alanine residues of the amino acids constituting the random coil forming domain.
• the amino acid polymer forming comprising the above defined "amino acid repeats'V'amino acid cassettes'V'cassette repeats" random coil conformation comprises more than about 4 % and less than about 50 %, preferably more than about 10 % but less than about 50 % and most preferably more than about 20 % but less than about 50 % serine residues of the amino acids constituting the random coil forming domain.
• the amino acid polymer forming random coil conformation may comprise about 35 % proline residues, about 50 % alanine residues and about 15 % serine residues of the amino acids constituting the random coil forming domain.
• the amino acid polymer forming random coil conformation may comprise about 35 % proline residues, about 15 % alanine residues and about 50 % serine residues of the amino acids constituting the random coil forming domain.
• the term "about” as used herein above relates also to the precise value of the given percentage.
• amino acid polymers comprising the amino acid sequence selected from the group consisting of AAAASSASSASSSSSAAASA (piSA; SEQ ID NO: 2) AASAAASSAAASAAAASASS (SEQ ID NO: 4), ASASASASASASSAASAASA (SEQ ID NO: 6), SAASSSASSSSAASSASAAA (SEQ ID NO: 8), SSSSAASAASAAAAASSSAS (SEQ ID NO: 10), SSASSSAASSSASSSSASAA (SEQ ID NO: 12), SASASASASASAASSASSAS (SEQ ID NO: 14) and ASSAAASAAAASSAASASSS (SEQ ID NO: 16).
• AAAASSASSASSSSSAAASA piSA; SEQ ID NO: 2
• AASAAASSAAASAAAASASS SEQ ID NO: 4
• ASASASASASASSAASAASA SEQ ID NO: 6
• SAASSSASSSSAASSASAAA SEQ ID NO: 8
• SSSSAASAASAAAAASSSAS SEQ ID NO: 10
• the multimers of the described alanine-serine modules/sequence units may form random coil conformation in case the resulting amino acid sequence further comprises proline residues as defined herein above.
• These exemplified modules/sequence units may be encoded by nucleic acid molecules comprising the following sequences GCCGCTGCTGCATCCTCTGCAAGCTCCGCTTCTTCCTCTAGCTCCGCAGCTGCATC TGCT (SEQ ID NO: 1),
• the amino acid polymer forming random coil conformation comprises the amino acid sequence selected from the group consisting of ASPAAPAPASPAAPAPSAPA (PAS#1; SEQ ID NO: 18), AAPASP AP AAPSAP AP AAPS (PAS#2; SEQ ID NO: 20), SAPSSPSPSAPSSPSPASPS (modified PAS#3; modified SEQ ID NO: 22), APSSPSPSAPSSPSPASPSS (PAS#3, SEQ ID NO. 22, non-modified).
• a slightly modified, yet active PAS#3 may have the above recited sequence SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 63). This sequence corresponds to the herein provided SEQ ID No.
• this modified sequence according to the invention possess essentially the same internal repeating unit as multimers of the non-modified sequence, except for the very first and the very last residue. Accordingly, this modified PAS#3 (SEQ ID NO: 63) may be considered as an example of a further "module'V'building block" of the herein provided amino acid polymers in accordance with this invention.
• modules and (shorter) fragments or circularly permuted versions of the herein provided amino acid polymers may be used as “modules”, “repeats” and/or building blocks for the herein defined “second domain” of the provided biologically active protein.
• amino acid polymers forming random coil conformation may comprise amino acid sequences that may be selected from the group consisting SSPSAPSPSSPASPSPSSPA (PAS#4; SEQ ID NO: 24), AASPAAPSAPP AAASP AAPSAPPA (PAS#5; SEQ ID NO: 26) and ASAAAPAAASAAASAPSAAA (PAS#1P2; SEQ ID NO: 28).
• Such other and further examples of random coil conformation forming amino acid polymers to be used as building blocks ore modules of the herein defined "second domain" of the inventive biologically active protein(s)/polypeptide(s) may, inter alia, comprise combinations and/or fragments or circularly permuted versions of the specific "building blocks", "polymer cassettes" or “polymer repeats” shown above. Accordingly, the exemplified modules/sequence units/polymer repeats/polymer cassettes of the random coil domain may also provide for individual fragments which may be newly combined to form further modules/sequence units/polymer repeats/polymer cassettes in accordance with this invention.
• module(s) means for treating or preventing human diseases and conditions in which abnormal proteins are used to form the herein defined "second domain" of the inventive biologically active protein/polypeptide.
• Said second domain comprises an amino acid sequence consisting preferably of at least about 100 amino acid residues and forms a random coil conformation under physiological conditions.
• modules/sequence units/polymer repeats/polymer cassettes/building blocks of the random coil domain of the inventive biologically active proteins/polypeptides may be encoded by nucleic acid molecules comprising the following sequences
• a modified PAS#3 (modified SEQ ID NO: 22) as described herein above may be encoded by the following nucleic acid sequence:
• modules/sequence units/polymer repeats/polymer cassettes/building blocks of the random coil domain may be encoded by different nucleic acid sequences in accordance with the genetic code, which is of degenerate nature, i.e. different nucleotide triplet codons may encode the same amino acid residue.
• the terminal residues may differ, depending on the design of a nucleotide sequence cassette according to this invention and on the ligation strategy applied to obtain multimers thereof.
• the "module" PAS#1 as shown in SEQ ID NO: 18 and 30 may be encoded by nucleic acid sequences SEQ ID NO: 17 and 29, respectively.
• SEQ ID NO: 30 comprises an additional alanine at the C-terminus, whose codon may be deleted if individual nucleotide sequence cassettes are ligated via sticky ends as described in some of the appended Examples.
• the amino acid polymer forming random coil conformation may comprise a multimer consisting of either one of the amino acid sequences with SEQ ID NO: 18, 20, 22, 24, 26 or 28 as disclosed herein above or may comprise a multimer consisting of more than one of amino acid sequences SEQ ID NO: 18, 20, 22, 24, 26 and 28.
• fragments or circularly permuted versions of these exemplified sequences are used to build up further modules/sequence units/polymer repeats/polymer cassettes/building blocks of the random coil domain ("second domain") of the inventive biologically active protein(s)/polypeptide(s).
• the amino acid polymer forming random coil conformation may comprise a multimer consisting of a (circular) permutation of the amino acid sequence selected from the group consisting of ASPAAP APASPAAP APSAPA (SEQ ID NO: 18), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 20), APSSPSPSAPSSPSP ASPSS (SEQ ID NO: 22, or as modified sequence S-APSSPSPSAPSSPSPASPS (SEQ ID NO: 63), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 24), AASP AAPSAPP AAASPAAPSAPPA (SEQ ID NO: 26) and ASAAAP AAASAAASAPSAAA (SEQ ID NO: 28) or (a) multimers(s) of these (circular) permutated sequences.
• ASPAAP APASPAAP APSAPA SEQ ID NO: 18
• AAPASPAPAAPSAPAPAAPS SEQ ID NO: 20
• APSSPSPSAPSSPSP ASPSS SEQ ID NO: 22
• the amino acid polymer forming random coil conformation may comprise a multimer consisting of a fragment/part of the amino acid sequence selected from the group consisting of ASPAAP APASP AAP APSAPA (SEQ ID NO: 18), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 20), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 22; or as modified sequence S-APSSPSPSAPSSPSPASPS ((SEQ ID NO: 63)), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 24), AASP AAPSAPP AAASPAAPSAPPA (SEQ ID NO: 26) and ASAAAP AAASAAASAPSAAA (SEQ ID NO: 28) or (a) multimers(s) of these exemplified modules/sequence units/polymer repeats/polymer cassettes/building blocks.
• ASPAAP APASP AAP APSAPA SEQ ID NO: 18
• AAPASPAPAAPSAPAPAAPS SEQ ID NO: 20
• “Fragments” of these sequences to be employed in accordance with this invention for the identification of the "second domain" of the inventive biologically active schoolein/polypeptide may consist of at least 3, preferably of at least 4, more preferably of at least 5, even more preferably of at least 6, still more preferably of at least 8, particularly preferably of at least 10, more particularly preferably of at least 12, even more particularly preferably of at least 14, still more particularly preferably of at least 16, and most preferably of at least 18 consecutive amino acids of the amino acid sequence selected from the group consisting of said SEQ ID NOs: 18, 20, 22, 24, 26 and 28.
• modules/sequence units/building blocks etc. of the random coil domain are merely examples of the inventive amino acid polymer that forms random coil conformation under physiological conditions.
• these "modules”, “sequence units” and/or “repeats” comprise the above-identified content/fraction of alanine, serine and proline. Therefore, it is within the normal skill of the artisan to generate further such “modules”, “sequence units” and/or “repeats” in accordance with this invention.
• individual fragments of the herein identified inventive “modules”, “sequence units” and/or “repeats” may be combined to further individual “modules”, “sequence units” and/or “repeats”, as long as the above-identified rules for the overall distribution and amount of alanine, serine and proline are respected.
• these "modules”, “sequence units” and/or “repeats” may also comprise further amino acid residues, however only as minimal or minor constituents (maximally 10 %, preferably maximally 2 % of the individual "module", “sequence unit” and/or “repeat”).
• Said individual “module”, “sequence unit” and/or “repeat” consists, in accordance with this invention, of at least about 100 amino acid residues.
• Individual “modules”, “sequence units” and/or “repeats” may be combined in order to form longer random coil forming amino acid polymers, whereby a maximal length of the herein defined "second domain" of a biologically active protein is about 3000 amino acids.
• biologically active proteins that comprise at least two domains wherein a first domain as defined herein above of said at least two domains comprises an amino acid sequence having and/or mediating said biological activity; and a second domain of said at least two domains as defined herein comprises an amino acid sequence consisting preferably of at least about 100 amino acid residues and random coil conformation under physiological conditions.
• Said random coil conformation as provided herein and consisting mainly of alanine, serine, and proline mediates an increased in vivo and/or in vitro stability of said biologically active protein.
• Said second domain may be comprised of the individual "modules”, “sequence units” and/or “repeats” as provide herein or may comprise fragments or parts of these individual, illustrative "modules”, “sequence units” and/or “repeats”.
• said second domain may be build of further and or other individual “modules”, “sequence units”, “building blocks” and/or “repeats” which respect and follow the teachings provided herein above and which are exemplified herein below in the specification and the appended examples.
• the appended experimental part shows ample evidence that proteins comprising a herein defined, additional "second domain" providing for a random coil confirmation under physiological conditions (for example polymers consisting of about 200 or about 400 or about 600 amino acid residues and comprising PAS#1/SEQ ID NO. 18 , PAS#2/SEQ ID No. 20, PAS#3/SEQ ID NO22, PAS#5/SEQ ID NO. 26 and/or PAS#1P2/SEQ ID NO 28 as "building blocks”) have an advantageous serum stability or plasma half-life, even in vivo as compared to the non- modified biologically active protein.
• proteins comprising a herein defined, additional "second domain" providing for a random coil confirmation under physiological conditions (for example polymers consisting of about 200 or about 400 or about 600 amino acid residues and comprising PAS#1/SEQ ID NO. 18 , PAS#2/SEQ ID No. 20, PAS#3/SEQ ID NO22, PAS#5/SEQ ID NO. 26 and/or PAS#1P2/SEQ ID NO 28 as
• the in vivo stability of non-modified IFNa2b was compared to the in vivo stability of modified IFNa2b that comprised an additional "second domain" as defined herein, adopting a random coil conformation under physiological conditions.
• homo-polymers of most amino acids are usually insoluble in aqueous solution (Bamford (1956) Synthetic Polypeptides - Preparation, Structure, and Properties, 2nd ed., Academic Press, New York).
• Homo-polymers of several hydrophilic amino acids are known to form secondary structures, for example ⁇ -helix in the case of Ala (Shental-Bechor (2005) Biophys J 88:2391-2402) and ⁇ -sheet in the case of Ser (Quadrifoglio (1968) J Am Chem Soc 90:2760-2765) while poly-proline, the stiffest homooligopeptide (Schimmel (1967) Proc Natl Acad Sci USA 58:52-59), forms a type II trans helix in aqueous solution (Cowan (1955) Nature 176:501-503).
• polypeptides comprising mixtures of these three amino acids, of which each alone tends to form a homooligopeptide with defined secondary structure, adopt random coil conformation under physiological conditions.
• inventive polypeptides have a larger hydrodynamic radius than homo-polymers comprising the same number of GIy residues and they confer better solubility to the biologically active protein according to the invention.
• WO 2006/081249 describes protein conjugates comprising a biologically active protein coupled to a polypeptide comprising 2 to 500 units of an amino acid repeat having GIy, Asn, and GIn as a major constituent and Ser, Thr, Asp, GIn, GIu, His, and Asn as a minor constituent. Said protein conjugates are described to have either an increased or a decreased plasma half-life when compared to the unconjugated biologically active protein. WO 2006/081249, however, does not provide any teaching to predict whether a specific amino acid repeat reduces or augments the plasma half-life of the conjugate.
• WO 2006/081249 does not teach or suggest that the plasma half-life of proteins can be increased when the conjugated protein comprises an amino acid repeat that forms random coil conformation as shown in the present invention.
• the amino acid repeats disclosed in WO 2006/081249 comprise at least two residues selected from GIy, Asn, and GIn, which is in clear contrast with the polypeptide repeats of the present invention that preferentially consist of Ala, Ser, and Pro residues.
• biological activity describes the biological effect of a substance on living matter.
• biologically active protein or “polypeptide having and/or mediating biological activity” as used herein relate to proteins or polypeptides that are capable of inducing a biological effect in living cells/organisms that are exposed to said protein or polypeptide.
• biologically active protein relates to the whole protein of the invention which both comprises an amino acid sequence having and/or mediating said biological activity and an amino acid sequence forming random coil conformation.
• amino acid sequence having and/or mediating biological activity or “amino acid sequence with biological activity” as used herein relate to the above-defined “first domain" of the biologically active protein of the invention, mediating or having or being capable of mediating or having the above defined “biological activity”.
• amino acid sequence having and/or mediating biological activity or “amino acid sequence with biological activity” also relate to a “biologically active polypeptide” or “biologically active polypeptide stretch" of the invention and relating to the "first domain" of said biologically active protein.
• amino acid sequence having and/or mediating biological activity or “amino acid sequence with biological activity” are functional fragments of any protein of interest, the half-life of which, either in vivo or in vitro, needs to be prolonged.
• the amino acid sequence having and/or mediating biological activity in accordance with the present invention may be deduced from any "protein of interest", i.e. any protein of pharmaceutical or biological interest or any protein that is useful as a therapeutic/diagnostic agent.
• the biologically active proteins in accordance with the present invention may comprise a biologically active amino acid sequence which is derived from naturally produced polypeptides or polypeptides produced by recombinant DNA technology.
• the protein of interest may be selected from the group consisting of binding proteins, immunoglobulins, antibody fragments, transport proteins, signaling proteins/peptides such as cytokines, growth factors, hormones or enzymes.
• binding protein relates to a molecule that is able to specifically interact with (a) potential binding partner(s) so that it is able to discriminate between said potential binding partner(s) and a plurality of different molecules as said potential binding partner(s) to such an extent that, from a pool of said plurality of different molecules as potential binding partner(s), only said potential binding partner(s) is/are bound, or is/are significantly bound.
• Methods for the measurement of binding of a binding protein to a potential binding partner are known in the art and can be routinely performed e.g. by using ELISA, isothermal titration calorimetry, equilibrium dialysis, pull down assays or a Biacore apparatus.
• Exemplary binding proteins which are useful in the context of the present invention include, but are not limited to antibodies, antibody fragments such as Fab fragments, F(ab') 2 fragments, single chain variable fragments (scFv), isolated variable regions of antibodies (VL- and/or VH-regions), CDRs, single domain antibodies, CDR-derived peptidomimetics, lectins, lipocalins or various types of scaffold-derived binding proteins as described, for example, in Skerra (2000) J MoI Recognit 13:167-187 or Binz (2005) Nat Biotechnol 23:1257-1268.
• Fab fragments fragments
• F(ab') 2 fragments single chain variable fragments
• VL- and/or VH-regions isolated variable regions of antibodies
• CDRs single domain antibodies
• CDR-derived peptidomimetics lectins
• lipocalins or various types of scaffold-derived binding proteins as described, for example, in Skerra (2000) J MoI Recognit 13:167-187
• exemplary biologically active proteins of interest which are useful in the context of the present invention include, but are not limited to granulocyte colony stimulating factor, human growth hormone, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, tumor necrosis factor, erythropoietin, coagulation factors such as coagulation factor VIII, gpl20/gpl60, soluble tumor necrosis factor I and II receptor, thrombolytics such as reteplase, exendin-4, interleukin-1 receptor antagonists such as anakinra, interleukin-2 and neutrophil gelatinase- associated lipocalin or those listed in Walsh (2003) Nat Biotechnol 21 :865-870 or Walsh (2004) Eur J Pharm Biopharm 58:185-196.
• coagulation factors such as coagulation factor VIII, gpl20/gpl60, soluble tumor necrosis factor I and II receptor
• thrombolytics such as reteplase, exendin
• the neutrophil gelatinase-associated lipocalin (NGAL; also called human neutrophil lipocalin, 24p3, uterocalin, siderocalin, or neu-related lipocalin) as mentioned herein above is a member of the lipocalin family of binding proteins, which was first identified as a neutrophil granule component. NGAL and was shown to tightly bind the catecholate-type siderophore FeIII»enterochelin/enterobactin (Goetz (2002) MoI Cell 10:1033-1043) as well as some other siderophores of mycobacteria, including M. tuberculosis carboxymycobactins (Holmes (2005) Structure 13:29-41).
• the present invention relates to the biologically active protein of the invention, wherein said first domain comprising an amino acid sequence that encodes a polypeptide having and/or mediating said biological activity and said second domain that forms random coil conformation are connected by a polypeptide linker.
• This polypeptide linker inserted between said first and said second domains, preferably comprises plural, hydrophilic, peptide-bonded amino acids that are covalently linked to these domains.
• said polypeptide linker comprises a plasma protease cleavage site which allows the controlled release of said first domain comprising a polypeptide having and/or mediating a biological activity. Linkers of different types or lengths may be identified without undue burden to obtain full functional activity of specific polypeptides.
• the biologically active proteins of the present invention are fusion proteins.
• a fusion protein as described herein is meant to comprise at least one domain which mediates a biological activity and at least one other domain which forms random coil conformation in a single multi-domain polypeptide.
• the biologically active protein in accordance with the present invention may represent a protein conjugate wherein a protein of interest or a polypeptide/polypeptide stretch/amino acid sequence having and/or mediating biological activity is conjugated via a non-peptide bond to an amino acid sequence which forms random coil conformation.
• Non-peptide bonds that are useful for cross-linking proteins are known in the art and may include disulfide bonds, e.g.
• Cys side chains thioether bonds or non-peptide covalent bonds induced by chemical cross-linkers, such as disuccinimidyl suberate (DSS) or sulfosuccinimidyl 4-[p- maleimidophenyljbutyrate (Sulfo-SMPB), as well as non-covalent protein-protein interactions.
• DSS disuccinimidyl suberate
• Sulfo-SMPB sulfosuccinimidyl 4-[p- maleimidophenyljbutyrate
• biologically active protein of the present invention may also comprise more than one "amino acid sequence having and/or mediating a biological activity", i.e. the herein defined "first domain" of the biologically active protein is not limited in context of this invention to one single biological activity of interest.
• first domain of the biologically active protein
• the person skilled in the art is aware that the "amino acid sequence having and/or mediating a biological activity” and the "random coil domain/part” as comprised in the biologically active proteins of the invention may be organized in a specific order.
• a non-limiting example of a "biologically active protein" of the present invention comprising one random coil domain/part i.e.
• the domain order may be: "amino acid sequence having and/or mediating first biological activity” - "random coil domain/part” - "amino acid sequence having and/or mediating second biological activity”.
• first and second domain of the inventive biologically active polypeptide may be arranged in an order, whereby said "first domain” (i.e. protein of interest; “amino acid sequence having and/or mediating said biological activity") is located at the amino (N-) terminus and said "second domain” (i.e. the domain that comprises an amino acid sequence consisting of at least about 100 amino acid residues forming/adopting random coil conformation) is located at the carboxy (C-) terminus of the inventive polypeptide.
• first domain i.e. protein of interest; "amino acid sequence having and/or mediating said biological activity
• second domain i.e. the domain that comprises an amino acid sequence consisting of at least about 100 amino acid residues forming/adopting random coil conformation
• amino acid sequence having and/or mediating said biological activity is located in/at the carboxy (C-) terminus and said "second domain” (i.e. the domain that comprises an amino acid sequence consisting of at least about 100 amino acid residues forming/adopting random coil conformation) is located in/at the amino (N-) terminus of the inventive polypeptide.
• said "second domain” i.e. the domain that comprises an amino acid sequence consisting of at least about 100 amino acid residues forming/adopting random coil conformation
• said "random coil stretch” may, therefore, be located between the two domains having and/or mediating the desired biological activity.
• said domain(s) comprising an amino acid sequence having and/or mediating the said biological activity may also be a biologically active fragment of a given protein with a desired biological function. Therefore, the herein defined "second domain” (an amino acid sequence consisting of at least about 100 amino acid residues forming a random coil) may also be located between two biologically active fragments of a protein of interest or between biologically active fragments of two proteins of interest. Yet, also when more than one domain "having and/or mediating a biological activity" are to be comprised in the biologically active protein of this invention, the herein defined
• second domain i.e. the amino acid sequence consisting of at least about 100 amino acid residues forming a random coil conformation, may be located at the N- or C-terminus of the biological active protein of this invention.
• second domain i.e. the amino acid sequence consisting of at least about 100 amino acid residues forming a random coil conformation.
• amino acid sequence having and/or mediating first biological activity - "random coil domain/part” - "amino acid sequence having and/or mediating second biological activity” or
• amino acid sequence having and/or mediating first biological activity - amino acid sequence having and/or mediating second biological activity
• random coil domain/part or
• the corresponding order(s) is/are also envisaged when the representation starts from the C- terminus of the biologically active protein/polypeptide of the present invention.
• random coil domain/part as used herein in the representations above corresponds to the "second domain” as defined herein, i.e. to an amino acid sequence consisting of at least about 100 amino acid residues that adopts/has random coil conformation under physiological conditions.
• amino acid sequence having and/or mediating first biological activity is not limited to full-length polypeptides that have and/or mediate said biological activity or function, but also to biologically and/or pharmacologically active fragments thereof.
• first domains as defined herein are comprised in the inventive "biologically active protein”
• these "first domains” are or represent different parts of a protein complex or fragments of such parts of protein complex.
• first domains being amino acid stretches that have and/or mediate a biological activity of interest or desire may be located between two "second domains” (i.e. domains that comprise an amino acid sequence consisting of at least about 100 amino acid residues forming/adopting random coil conformation). Therefore "random coil stretches" may be located both N-terminally and C-terminally of the domain having and/or mediating the desired biological activity.
• the biologically active proteins of the invention which are modified to comprise a random coil domain surprisingly exhibit an increased in vivo and/or in vitro stability when compared to unmodified biologically active proteins that lack said random coil domain.
• in vivo stability relates to the capacity of a specific substance that is administered to the living body to remain biologically available and biologically active. In vivo, a substance may be removed and/or inactivated due to excretion, aggregation, degradation and/or other metabolic processes.
• biologically active proteins that have an increased in vivo stability may be less well excreted through the kidneys (urine) or via the feces and/or may be more stable against proteolysis, in particular against in vivo proteolysis in biological fluids, like blood, liquor cerebrospinalis, peritoneal fluid and lymph.
• the increased in vivo stability of a biologically active protein manifests in a prolonged plasma half-life of said biologically active protein.
• biologically active proteins may be specifically detected in the blood plasma using western blotting techniques or enzyme linked immunosorbent assay (ELISA).
• ELISA enzyme linked immunosorbent assay
• other methods may be employed to specifically measure the plasma half-life of a protein of interest. Such methods include, but are not limited to the physical detection of a radioactively labelled protein of interest. Methods for radioactive labelling of proteins e.g. by radioiodination are known in the art.
• the term "increased in vitro stability" as used herein relates to the capacity of a biologically active protein to resist degradation and/or aggregation and to maintain its original biological activity in an in vitro environment. Methods for measuring the biological activity of biologically active proteins are well known in the art.
• nucleic acid molecules encoding the biologically active proteins as described herein.
• said nucleic acid molecule may comprise a nucleic acid sequence encoding a polypeptide having biological activity and a nucleic acid sequence encoding an amino acid sequence which forms/adopts random coil conformation.
• said nucleic acid molecule may comprise a nucieic acid sequence encoding one of the herein disclosed amino acid sequences that form/adopt random coil conformation.
• nucleic acid molecule is intended to include nucleic acid molecules such as DNA molecules and RNA molecules. Said nucleic acid molecule may be single-stranded or double-stranded, but preferably is double- stranded DNA.
• said nucleic acid molecule may be comprised in a vector.
• the present invention relates to nucleic acid molecules which upon expression encode the biologically active proteins of the invention.
• the present invention relates to nucleic acid molecules which upon expression encode the herein disclosed polypeptides that, entirely or in part, form/adopt random coil conformation under physiological conditions.
• Said nucleic acid molecules may be fused to suitable expression control sequences known in the art to ensure proper transcription and translation of the polypeptide as well as signal sequences to ensure cellular secretion or targeting to organelles.
• Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
• the nucleic acid molecule of the invention is comprised in a recombinant vector in which a nucleic acid molecule encoding the herein described biologically active protein is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells.
• Expression of said nucleic acid molecule comprises transcription of the nucleic acid molecule into a translatable mRNA.
• Regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lambda PL, lac, trp, tac, tet or T7 promoter in E. coli.
• Possible regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells or yeast, are well known to those skilled in the art.
• regulatory elements usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript.
• Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally- associated or heterologous promoter regions. Examples for regulatory elements permitting expression in eukaryotic host cells are the AOXl or GALl promoter in yeast or the CMV, SV40, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer or a globin intron in mammalian and other animal cells.
• regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the coding region.
• the present invention also relates to vectors, particularly plasmids, cosmids, viruses, and bacteriophages that are conventionally employed in genetic engineering comprising a nucleic acid molecule encoding the biologically active protein of the invention. Therefore, the present invention also relates to vectors comprising the nucleic acid molecule of this invention.
• said vector is an expression vector and/or a gene transfer or targeting vector.
• Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses or bovine papilloma virus may be used for delivery of the polynucleotides or vector of the invention into targeted cell populations.
• the vectors containing the nucleic acid molecules of the invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. Accordingly, the invention further relates to a cell comprising said nucleic acid molecule or said vector. Such methods, for example, include the techniques described in Sambrook (1989), loc. cit. and Ausubel (1989), loc. cit.. Accordingly, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see Sambrook (1989), loc. cit.). As a further alternative, the nucleic acid molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
• the nucleic acid molecule or vector of the invention which is present in host cell may either be integrated into the genome of the host cell or it may be maintained extra- chromosomally. Accordingly, the present invention also relates to a host cell comprising the nucleic acid molecule and/or the vector of this invention.
• Host cells for the expression of polypeptides are well known in the art and comprise prokaryotic cells as well as eukaryotic cells , e.g. E.
• coli cells coli cells, yeast cells, invertebrate cells, CHO-cells, CHO-Kl -cells, HeIa cells, COS-I monkey cells, melanoma cells such as Bowes cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines and the like.
• the present invention comprises methods for the preparation of the biologically active proteins of the invention comprising culturing the (host) cell of this invention and isolating said biologically active protein from the culture as described herein.
• the inventive biologically active protein comprising a random coil domain may be produced by recombinant DNA technology, e.g. by cultivating a cell comprising the described nucleic acid molecule or vectors which encode the inventive biologically active protein and isolating said biologically active protein from the culture.
• the inventive biologically active protein may be produced in any suitable cell-culture system including prokaryotic cells, e.g. E. coli BL21 or JM83, or eukaryotic cells, e.g.
• Pichia pastoris yeast strain X-33 or CHO cells are further suitable cell lines known in the art are obtainable from cell line depositories, like the American Type Culture Collection (ATCC).
• ATCC American Type Culture Collection
• the term "prokaryotic” is meant to include bacterial cells while the term “eukaryotic” is meant to include yeast, higher plant, insect and mammalian cells.
• the transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
• the present invention relates to a process for the preparation of a biologically active protein described above comprising cultivating a cell of the invention under conditions suitable for the expression of the biologically active protein and isolating the biologically active protein from the cell or the culture medium.
• the biologically active protein of the invention can be isolated from the growth medium, cellular lysates or cellular membrane fractions.
• the isolation and purification of the expressed polypeptides of the invention may be performed by any conventional means (Scopes (1982), "Protein Purification", Springer- Verlag, N. Y.), including ammonium sulphate precipitation, affinity columns, column chromatography, gel electrophoresis and the like and may involve the use of monoclonal or polyclonal antibodies directed, e.g., against a tag fused with the biologically active protein of the invention.
• the protein can be purified via the Strep-tag II using streptavidin affinity chromatography (Skerra (2000) Methods Enzymol 326:271-304) as described in the appended examples.
• Strep-tag II using streptavidin affinity chromatography (Skerra (2000) Methods Enzymol 326:271-304) as described in the appended examples.
• Substantially pure polypeptides of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred, for pharmaceutical uses.
• the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
• the invention further relates to the use of the biologically active protein of the invention, the nucleic acid molecule of the invention, the vector of the invention or the (host) cell of the invention for the preparation of a medicament, wherein said biologically active protein has an increased in vivo and/or in vitro stability.
• the present invention relates to a method for the treatment of diseases and/or disorders that benefit from the improved stability of said biologically active protein, comprising administering the biologically active protein as described herein to a mammal in need of such treatment.
• the skilled person is readily capable of determining which disease/disorder is to be treated with a specific biologically active protein of the invention.
• the present invention also relates to the use of the nucleic acid molecules, vectors as well as transfected cells comprising the nucleic acid molecules or vectors of the present invention in medical approaches, like, e.g. cell based gene therapy approaches or nucleic acid based gene therapy approaches.
• the inventive biologically active protein comprising the herein defined "first" and “second" domains (or the nucleic acid molecule or the vector or the host cell of the present invention) of the invention is part of a composition.
• Said composition may comprise one or more of the inventive biologically active proteins or nucleic acid molecules, vectors or host cells encoding and/or expressing the same.
• Said composition may be a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier and/or diluent.
• the present invention relates to the use of the herein described biologically active protein for the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of diseases which require the uptake of such a pharmaceutical composition.
• the composition as described herein may be a diagnostic composition, optionally further comprising suitable means for detection, wherein said diagnostic composition has an increased in vivo and/or in vitro stability.
• compositions of the invention may be in solid or liquid form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). Furthermore, it is envisaged that the medicament of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition.
• compositions may be effected by different ways, e.g., by parenteral, subcutaneous, intraperitoneal, topical, intrabronchial, intrapulmonary and intranasal administration and, if desired for local treatment, intralesional administration.
• Parenteral administrations include intraperitoneal, intramuscular, intradermal, subcutaneous intravenous or intraarterial administration.
• the compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like a specifically effected organ.
• Suitable pharmaceutical carriers examples include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. Suitable carriers may comprise any material which, when combined with the biologically active protein of the invention, retains the biological activity of the biologically active protein (see Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed). Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions).
• the buffers, solvents and/or excipients as employed in context of the pharmaceutical composition are preferably "physiological" as defined herein above.
• non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
• Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
• Intravenous vehicles may include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present including, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
• the pharmaceutical composition of the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin.
• compositions can be administered to the subject at a suitable dose.
• the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Pharmaceutically active matter may be present in amounts between 1 ⁇ g and 20 mg/kg body weight per dose, e.g. between 0.1 mg to 10 mg/kg body weight, e.g. between 0.5 mg to 5 mg/kg body weight. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg per kilogram of body weight per minute. Yet, doses below or above the indicated exemplary ranges also are envisioned, especially considering the aforementioned factors.
• the pharmaceutical composition of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition.
• further biologically active agents may be e.g. antibodies, antibody fragments, hormones, growth factors, enzymes, binding molecules, cytokines, chemokines, nucleic acid molecules and drugs.
• compositions to be used in research or as diagnostic(s) are envisaged.
• biologically active proteins comprising a random coil domain as defined herein, are used in a diagnostic setting.
• inventive biologically active protein of this invention comprising the herein defined “first" and “second” domain, may be detectably labelled.
• Such labels comprise, but are not limited to radioactive labels (like [ 3 H]hydrogen [ 125 I]iodide or [ 123 I]iodide), fluorescent labels (including but nor limiting fluorescent proteins, like green fluorescent protein (GFP) or fluorophores, like fluorescein isothiocyanate (FITC)) or NMR labels (like gadolinium chelates).
• radioactive labels like [ 3 H]hydrogen [ 125 I]iodide or [ 123 I]iodide
• fluorescent labels including but nor limiting fluorescent proteins, like green fluorescent protein (GFP) or fluorophores, like fluorescein isothiocyanate (FITC)
• NMR labels like gadolinium chelates
• the present invention provides for a kit comprising the biologically active protein, the nucleic acid molecule encoding said biologically active protein, the vector comprising said nucleic acid molecule or the cell comprising said nucleic acid or said vector as described herein.
• the kit of the present invention further comprises, optionally (a) buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of medical, scientific or diagnostic assays and purposes.
• parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.
• the kit of the present invention may be advantageously used, inter alia, for carrying out the method of the invention and could be employed in a variety of applications referred herein, e.g., as diagnostic kits, as research tools or as medical tools. Additionally, the kit of the invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes.
• the manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
• Figure 1 Gene design for the Pro-Ala-Ser#l (PAS#1; SEQ ID NO: 18), Pro-Ala-Ser#2 (PAS#2; SEQ ID NO: 20), Pro-Ala-Ser#3 (PAS#3; SEQ ID NO: 22), (Pro-Ala- Ser#5 (PAS#5; SEQ ID NO: 26), Pro-Ala-Ser#lP2 (PAS#1P2; SEQ ID NO: 28 and Ser-Ala (piSA; SEQ ID NO: 2) polymer sequences.
• Figure 2 Cloning strategy for the Pro-Ala-Ser polymer sequences as fusion to IFNa2b and IL- Ira.
• the nucleotide sequence encodes for two Sapl restriction sites in reverse complementary orientation, which leads upon digest to protruding ends that are compatible with the synthetic gene cassettes shown in Figure 1 (indicated by bars). The recognition sequences are underlined.
• PAS(#l)400-IFNa2b and PAS(#l)600-IFNa2b are identical except that the PAS#1 polymer with 400 or 600 residues, i.e. 20 or 30 repetitive copies of the sequence shown in Figure IA, is encoded instead of PAS(# 1)200.
• the expression vectors for PAS(#2)200-IFNa2b and PAS(#3)200-IFNa2b carry a PAS#2 or PAS#3 polymer of 200 , i.e. 10 repetitive copies of the sequences shown in Figure IB and 1C, respectively.
• the expression vectors for PAS(#5)192-IFNa2b and PAS(#5)384-IFNa2b carry a PAS#5 polymer of 192 or 384 residues, i.e. 8 or 16 repetitive copies of the sequences shown in Figure ID.
• the expression vector for PAS(#lP2)140-IFNa2b carries a PAS#1P2 polymer of 140 residues, i.e.
• Expression vectors for PAS(#l)200-ILlra, PAS(#l)400-ILlra, PAS(#5)192- IL Ira and PAS(#5)384-ILlra are similar to the corresponding vectors for IFNa2b except for carrying the coding gene for IL- Ira instead of IFNa2b.
• Figure 3 Cloning strategy for the Pro-Ala-Ser and Ser-Ala polymer sequences according to Figure 1 as fusion to human neutrophil gelatinase-associated lipocalin, NGAL.
• NGAL-PAS(# 1)200 Plasmid map of pNGAL-PAS(# 1)200.
• the structural gene for NGAL- PAS(#l)200 (comprising the OmpA signal peptide, the modified NGAL, and PAS#1 with 200 residues, PAS(#l)200, as well as the Strep-tag II) is under transcriptional control of the tetracycline promoter/operator (tet p/o ) and ends with the lipoprotein terminator (ti pp ).
• the plasmid backbone i.e.
• Figure 4 Analysis of the purified recombinant IFNa2b, IL- Ira, and NGAL, as well as their polymer fusions by SDS-PAGE, followed by staining with Coomassie brilliant blue R-250.
• the recombinant proteins were produced in E. coli BL21 via periplasmic secretion and purified by means of the Strep-tag II using streptavidin affinity chromatography.
• the polymer fusions show significantly larger sizes than the calculated masses of 37.4 kDa for PAS(#l)200-IFNa2b, 37.4 kDa for PAS(#2)200-IFNa2b, 38.6 kDa for PAS(#3)200-lFNa2b, 36.7 kDa for PAS(#5)192-IFNa2b, and 31.7 kDa for PAS(#lP2)140-IFNa2b.
• This effect is again due to the Pro-Ala-Ser polymers with different lengths.
• (C) Calibration curve for the chromatograms from (A) and (B) using Superdex S200 10/300 GL.
• the logarithm of the molecular weight (MW) of marker proteins (RNase A, 13.7 kDa; carbonic anhydrase, 29.0 kDa; ovalbumin, 43.0 kDa; bovine serum albumin, 66.3 kDa; transferrin, 81.0 kDa; alcohol dehydrogenase, 150 kDa) was plotted vs. their elution volumes (black circles) and fitted by a straight line.
• IFNa2b 22.5 kDa (calculated: 20.9kDa); PAS(#l)200- IFNa2b: 176 kDa (calculated: 37.4 kDa); PAS(#l)400-IFNa2b: 346 kDa (calculated: 54.0 kDa); PAS(#l)600-IFNa2b: 522 kDa (calculated: 70.5 kDa); PAS(#5)192-IFNa2b: 162 kDa (calculated: 36.7 kDa); PAS(#5)384-IFNa2b: 280 kDa (calculated: 52.6 kDa).
• (E) Calibration curve for the chromatograms from (D) using the same Superdex S200 10/300 GL column.
• the logarithm of the molecular weight (MW) of marker proteins (RNase A, 13.7 kDa; carbonic anhydrase, 29.0 kDa; ovalbumin, 43.0 kDa; bovine serum albumin, 66.3 kDa; transferrin, 81.0 kDa; alcohol dehydrogenase, 150 kDa) was plotted vs. their elution volumes (black circles) and fitted by a straight line.
• (G) Calibration curve for the chromatograms from (F) using the same Superdex S200 10/300 GL column.
• the logarithm of the molecular weight (MW) of marker proteins (RNase A, 13.7 kDa; carbonic anhydrase, 29.0 kDa; ovalbumin, 43.0 kDa; bovine serum albumin, 66.3 kDa; transferrin, 81.0 kDa; alcohol dehydrogenase, 150 kDa) was plotted vs. their elution volumes (black circles) and fitted by a straight line.
• IL-lra 19.8 kDa (calculated: 18.8 kDa); PAS(#l)200-ILlra: 161 kDa (calculated: 35.3 kDa); PAS(#l)400-ILlra: 336 kDa (calculated: 51.9 kDa); PAS(#5)192-ILlra: 148 kDa (calculated: 34.6 kDa); PAS(#5)384-ILlra: 305 kDa (calculated: 50.5 kDa).
• NGAL 21.5 kDa (calculated: 21.5 kDa); NGAL- PAS(#l)100: 72.6 kDa (calculated: 29.8 kDa); NGAL-P AS(# 1)200: 106.4 kDa (calculated: 38.1 kDa); NGAL-piSAlOO: 54 kDa (calculated: 29.4 kDa).
• Figure 6 Experimental secondary structure analysis of the purified recombinant IFNa2b, IL- Ira, NGAL, as well as their polymer fusions by circular dichroism (CD) spectroscopy. Spectra were recorded at room temperature in 50 mM K 2 SO 4 , 20 mM K-phosphate pH 7.5 and normalized to the molar ellipticity, ⁇ M , for each protein.
• CD circular dichroism
• the CD spectrum for IFNa2b shows the typical features of a predominant ⁇ -helix protein with two negative maxima around 208 nm and 220 nm (Sreerama in: Circular Dichroism - Principles and Applications (2000) Berova, Nakanishi and Woody (Eds.) Wiley, New York:601- 620 which indicates the correct folding of the bacterially produced human IFNa2b.
• Circular dichroism (CD) Circular dichroism spectra of the purified recombinant PAS(#2)200- IFNa2b, PAS(#3)200-IFNa2b and PAS(#lP2)140-IFNa2b, together with the one of IFNa2b.
• the spectra of the polymer fusion proteins reveal a dominant negative minimum around 205 nm, which is indicative of random coil conformation, and a shoulder around 220 nm, which results from the contribution of the correctly folded IFNa2b.
• the spectra of the four fusion proteins reveal a dominant negative minimum around 200 nm, which is indicative of random coil conformation.
• Serum stability of PAS(#l)200-IFNa2b (A) and PAS(#5)192-IFNa2b (B) was analyzed by incubation of the fusion protein at a concentration of 0.17 mg/ml in 83 % v/v mouse plasma (Rockland Immunochemicals, Gilbertsville, PA) at 37 0C for up to 48 h. Samples (6 ⁇ l) were taken at indicated time points and diluted with 54 ⁇ l SDS-PAGE electrophoresis buffer and 15 ⁇ l SDS-PAGE loading buffer containing ⁇ -mercaptoethanol.
• Figure 8 Pharmacokinetics of the purified recombinant IFNa2b and its PAS#1 polymer fusions with 200 or 400 residues.
• mice with body weights around 25 g received injections of ca. 125 ⁇ l of either IFNa2b, PAS(#l)200-IFNa2b, or PAS(#l)400-IFNa2b protein with a concentration of 1 mg/ml in PBS containing 1 niM EDTA to achieve a dose of 5 mg test protein per kg body weight (b.w.). Blood samples were taken as indicated. Aliquots of the cleared plasma samples were diluted 1 :5 with PBS. Aliquots of 10 ⁇ l of the diluted sample (corresponding to 1 ⁇ l plasma), were applied to a 12 % SDS-PAGE and blotted onto a nitrocellulose membrane.
• the recombinant proteins were detected by incubation with the mouse anti-human IFNa2b antibody 9D3 (Abeam, Cambridge, UK) followed by incubation with an anti -mouse IgG alkaline phosphatase conjugate (Sigma- Aldrich, St. Louis, MO) and developed in a chromogenic reaction.
• the other lanes show plasma samples for IFNa2b, PAS(#l)200-IFNa2b, and PAS(#l)400-IFNa2b at time points as indicated.
• Plasma samples from the same animals as investigated in Figure 8 were quantitatively assayed for IFNa2b, PAS(#l)200-IFNa2b or PAS(#l)400-IFNa2b concentrations using a sandwich ELISA. Therefore, the wells of a microtitre plate were coated with the anti-human IFNa antibody 9D3 (Abeam, Cambridge, UK) as capture antibody and dilution series of the plasma samples from animals of group A (injection of IFNa2b), group B (injection of PAS(#l)200-IFNa2b), and group C (injection of PAS(#l)400-IFNa2b) were applied.
• group A injection of IFNa2b
• group B injection of PAS(#l)200-IFNa2b
• group C injection of PAS(#l)400-IFNa2b
• Bound IFNa2b, PAS(#l)200-IFNa2b, and PAS(#l)400-IFNa2b were detected with a second anti-human IFNa2b antibody HRP conjugate (4E10-HRP; Abeam, Cambridge, UK), which recognizes a different epitope than the capture antibody, followed by chromogenic reaction.
• Concentrations of IFNa2b, PAS(#l)200-IFNa2b, and PAS(#l)400-IFNa2b were quantified by comparison with standard curves prepared with the same purified recombinant proteins applied at a known concentration.
• the unfused IFNa2b protein exhibited a very fast clearance with a half- life of 5.5 ⁇ Ix 10 "5 min.
• the elimination phase determined for PAS(#l)200-IFNa2b and PAS(#l)400-IFNa2b were significantly retarded, with half-lifes of 61.7 ⁇ 5.4 min and ca. 6 ⁇ 3 h, respectively, thus demonstrating a more than ten-fold and 60-fold prolonged circulation due to the Pro-Ala-Ser polymer fusion with 200 and 400 residues, respectively, compared with the unfused IFNa2b.
• Figure 10 Quantitative analysis of the pharmacokinetics of the purified recombinant IFNa2b PAS#1 polymer fusions with 200, 400, 600 residues and PAS#5 polymer fusions with 192 and 384 residues, respectively.
• PAS(#l)200-IFNa2b, PAS(#l)400-IFNa2b, PAS(#l)600- IFNa2b, PAS(#5)-IFNa2b or PAS(#5)384-IFNa2b protein with a concentration of 1 mg/ml in PBS containing 1 mM EDTA to achieve a dose of 7 mg test protein per kg body weight (b.w.).
• Blood samples were taken after 30 min, 240 min, 360 min, and 480 min. Plasma samples were quantitatively assayed for IFNa2b, PAS(#l)200-IFNa2b or PAS(#l)400-IFNa2b using a sandwich ELISA.
• Figurell Pharmacokinetics of the purified recombinant NGAL and its PAS#1 polymer fusions with 100 or 200 residues.
• NGAL NGAL-PAS(#l)100
• NGAL-P AS(# 1)200 protein with a concentration of 1 mg/ml in PBS to achieve a dose of 5 mg test protein per kg body weight (b.w.). Blood samples were taken as indicated. Aliquots of the cleared plasma samples were diluted 1 :5 with PBS. Three aliquots of 1.25 ⁇ l of the diluted sample (corresponding to 0.25 ⁇ l plasma) from animals each injected with one of the three different proteins were mixed and applied to a 12 % SDS-PAGE and blotted onto a nitrocellulose membrane.
• the recombinant proteins were detected by incubation with Strep Tactin® Alkaline Phosphatase conjugate (IBA, Gottingen, Germany), which recognizes the Strep-tag II, and developed in a chromogenic reaction.
• Strep Tactin® Alkaline Phosphatase conjugate IBA, Gottingen, Germany
• Figures HA and HB depict two time series with independent plasma samples of different animals from group A (injection of NGAL), group B (injection of NGAL-PAS(#l)100), and group C (injection of NGAL-P AS(# 1)200).
• the leftmost lanes in Figures HA and HB show the molecular size standard (with marker sizes on the left), the following lanes show mixtures of the three plasma samples containing NGAL, NGAL-PAS(#l)100, and NG AL-P AS(# 1)200 at time points indicated, and the rightmost lane shows a mixture of purified NGAL, NGAL-PAS(#l)100, and NG AL-P AS(# 1)200 (each 0.1 ⁇ g) as reference.
• the blots reveal the highest signals for all three protein samples at the earliest time point, i.e. after 5 min, with a rapid decay of the NGAL, which is no longer detectable after 30 min.
• both NGAL-PAS(#l)100 and NGAL- PAS(#l)200 are detectable for much longer periods, with a slightly stronger effect for the 200 residue fusion compared with the 100 residue fusion, indicating significantly prolonged circulation when compared to the unfused NGAL protein.
• proteolytic degradation for either protein sample.
• the polymer fusion moiety reveal high serum stability.
• none of the animals showed any signs of acute toxicity or inflammation, demonstrating high tolerance for the fusion proteins according to this invention.
• Figure 12 Quantitative analysis of the pharmacokinetics of the purified recombinant NGAL and its PAS#1 polymer fusion with 200 residues.
• Plasma samples from the same animals as investigated in Figure HA were assayed for NGAL or NGAL-P AS(# 1)200 concentrations using a sandwich ELISA. Therefore, the wells of a microtitre plate were coated with an anti- human Lipocalin-2/NGAL antibody (R&D Systems, Minneapolis, MN) as capture antibody and dilution series of the plasma samples from animals of group A (injection of NGAL) or group C (injection of NGAL-PAS(# 1)200) were applied. Bound NGAL and NGAL-P AS(# 1)200 were detected with StrepTactin® Alkaline Phosphatase conjugate, which recognizes the Strep-tag II, followed by chromogenic reaction.
• Concentrations of NGAL and NGAL- PAS(# 1)200 were quantified by comparison with a standard curve prepared with the same purified recombinant proteins applied at a known concentration.
• concentration values were plotted against time post intravenous injection and numerically fitted assuming a mono-exponential decay, whereby for better clarity only data points till 360 min are depicted.
• the unfused NGAL protein exhibited a very fast clearance with a half-life of 3.1 ⁇ 0.2 min. According to the principles of allometric scaling (Mahmood (2005) Interspecies Pharmacokinetic Scaling: Principles and Application of Allometric Scaling. Pine House Publishers, Rockville, Maryland) this value is in agreement with the half-life of 10 min described for the monomeric form of the natural NGAL in humans (Axelsson (1995) Scand J Clin Lab Invest 55:577-588), which indicates a mechanism of cellular uptake that may be unique to this particular protein.
• megalin a member of the low-density lipoprotein receptor, may act as a receptor for NGAL in kidney epithelial cells and mediate its uptake (Hvidberg (2005) FEBS Lett 579:773-777).
• the elimination phase determined for NGAL-P AS(# 1)200 was significantly slower, with a terminal half-life of 30.9 ⁇ 1.3 min, thus demonstrating a ten-fold prolonged circulation due to the Pro-Ala-Ser polymer fusion with 200 residues compared with the unfused NGAL.
• the retarding effect on the plasma half-life may be even more pronounced for a protein of interest that is not subject to a specific clearance mechanism as it is obviously the case for NGAL.
• Figure 13 Comparative activity analysis of the commercially available IntronA (Schering, Kenilworth, NJ), recombinant PAS(#l)200-IFNa2b, and a recombinant Fab fragment (serving as negative control) by IP-10 ELISA. 2x10 5 human peripheral blood mononuclear cells (PBMCs) were incubated with IntronA, PAS(# 1)200- IFNa2b or a Fab fragment, which was similarly prepared as PAS(#l)200-IFNa2b, at different concentrations. The specific activity of IntronA was 2.6x10 U/mg according to the data sheet of the manufacturer.
• IP-IO protein was quantified by the human IP-10 ELISA Set (BD OptEIATM, BD Biosciences Pharmingen, USA). IntronA and PAS(#l)200- IFNa2b induce the release of IP-IO in a concentration-dependent manner with similar effects. The unstimulated as well as the PBMCs treated with the Fab fragment did not show any significant IP-10 production.
• Figure 14 Theoretical prediction of secondary structure for the Pro-Ala-Ser and Ser-Ala polymer sequences according to the Chou-Fasman method (Chou and Fasman (1974) Biochemistry 13: 222-245).
• This illustration shows the output from the CHOFAS computer algorithm as implemented on the Sequence Comparison and Secondary Structure prediction server at the University of Virginia (URL: http://fasta.bioch.virginia.edu/fasta_www2).
• URL http://fasta.bioch.virginia.edu/fasta_www2
• the Chou-Fasman algorithm predicts ⁇ -helical secondary structure for 20 of 20 residues, i.e. 100 %. This is in clear contrast with the experimentally observed predominant ⁇ -sheet conformation for this polymer sequence as part of a fusion protein (see Figure 6).
• the Chou-Fasman algorithm predicts ⁇ -helical secondary structure for 12 of 20 residues, i.e. 60 %. This is in contrast with the experimentally observed predominant random coil conformation for this polymer sequence as part of a fusion protein (see Figure 6).
• Example 1 Gene synthesis for Pro-Ala-Ser and Ser-Ala amino acid polymers.
• PAS amino acid repeats consisting of Pro, Ala, and Ser residues
• PES amino acid repeats consisting of Pro, Ala, and Ser residues
• Gene fragments encoding a repetitive polymer sequence comprising Pro, Ala, and Ser residues (PAS#1 which corresponds to SEQ ID NO: 18, PAS#2 which corresponds to SEQ ID NO: 20, PAS#3 which corresponds to SEQ ID NO: 22, PAS#5 which corresponds to SEQ ID NO: 26, and PAS#1P2 which corresponds to SEQ ID NO: 28) or Ser and Ala (piSA which corresponds to SEQ ID NO: 2) were obtained by hybridisation and ligation of the two complementary oligodeoxynucleotides shown in Fig.
• Oligodeoxynucleotides were purchased from IBA (G ⁇ ttingen, Germany) and purified by preparative urea polyacrylamide gel electrophoresis.
• the amino acid sequences depicted in SEQ ID NOs 30, 32, 34, 36, 38 and 40 represent cloning versions of SEQ ID NOs 18, 20, 22, 26, 2 and 28, respectively, comprising an additional alanine.
• nucleic acid sequences depicted in SEQ ID NOs 29, 31, 33, 35, 37 and 39 comprise an additional egg codon for alanine, which becomes eliminated upon ligation via sticky ends.
• Enzymatic phosphorylation was performed by mixing 200 pmol of both oligodeoxynucleotides in 100 ⁇ l 50 mM Tris/HCl pH 7.6, 10 mM MgCl 2 , 5 mM DTT, ImM ATP and incubation for 30 min at 37 °C in the presence of 10 u polynucleotide kinase (MBI Fermentas, St.
• Example 2 Construction of expression vectors for PAS#1, PAS#2, PAS#3,PAS#5, and PAS#1P2 fusion proteins of interferon ⁇ -2b (IFNa2b)
• pASK-2xSapI harboring a nucleotide sequence with two Sapl restriction sites in reverse complementary orientation (Fig. 2A).
• This vector was cut with Sapl, dephosphorylated with shrimp alkaline phosphatase (USB, Cleveland, OH), and ligated with the synthetic DNA fragment (Fig. 2B).
• Resulting intermediate plasmids were designated pPAS(#l)200, pPAS(#2)200, pPAS(#3)200, pPAS(#5)192, and P PAS(#1P2)14O.
• plasmids were prepared and the sequences of the cloned synthetic nucleic acid inserts were confirmed by restriction analysis and automated double-stranded DNA sequencing (ABI- PrismTM310 Genetic analyzer, Perkin-Elmer Applied Biosystems, Rothstadt, Germany) using the BigDyeTM terminator kit as well as oligodeoxynucleotide primers that enabled sequencing from both sides.
• the resulting plasmid haboring the ca. 200 residue polymer sequence served as an intermediate vector, which enabled the simple further subcloning of the polymer sequence insert.
• the coding gene for IFNa2b was amplified from the plasmid IRAMp995M1713Q (RZPD, Berlin, Germany) carrying the corresponding cDNA using the oligodeoxynucleotides 5'- TCTGTGGGCGCC AGCTCTTCTGCCTGTGATCTGCCTCAAACCCAC (SEQ ID NO: 59) and 5'-GAACCA AAGCTTATTCCTTACTTCTTAAAC (SEQ ID NO: 60) as primers.
• the first primer contains a Kasl restriction site at the 5 '-end, followed by a Sapl restriction site (underlined), whereas the second primer contains a Hindlll restriction site (underlined).
• the amplification product was purified and digested with Kasl and Hindlll and ligated with the accordingly cut vector pASK-IBA4 (IBA, G ⁇ ttingen, Germany). After transformation of E. coli XLl -Blue, plasmids were prepared and the sequences of the cloned synthetic nucleic acid inserts were confirmed by restriction analysis and automated double- stranded DNA sequencing.
• the plasmid coding for IFNa2b as fusion with a N-terminal Strep- tag II was designated pASK-IFNa2b (Fig. 2C).
• pASK-IFNa2b was cut with S ⁇ pl, dephosphorylated with shrimp alkaline phosphatase, and ligated with an excess of the gene fragment for the 200 residue polymer isolated from the intermediate plasmid pPAS(# 1)200 by restriction digest with S ⁇ pl (Fig. 2D).
• plasmids were prepared and the sizes of the polymer encoding insert were confirmed by restriction analysis.
• the plasmids coding for IFNa2b carrying a 200, 400 and 600 residue polymer sequence i.e. PAS(#l)200-IFNa2b, PAS(#l)400-IFNa2b, and PAS(#l)600-IFNa2b, were designated pASK-PAS(#l)200-IFNa2b (Fig. 2G), pASK- PAS(#l)400-IFNa2b, and pASK-PAS(#l)600-IFNa2b, respectively.
• PAS(#2)200-IFNa2b, PAS(#3)200-IFNa2b, PAS(#lP2)140-IFNa2b, PAS(#5)192-IFNa2b, and PAS(#5)384-IFNa2b were constructed in a similar manner using the appropriate corresponding gene cassette encoding each of the amino acid polymer sequences.
• Example 3 Construction of expression vectors for PAS#1 and PAS#5 fusion proteins of interleukin-l receptor antagonist (IL-lra)
• the coding gene for IL-lra (Carter (1990) Nature 344:633-638) was amplified from the plasmid IRANp969G0350D6ILlRN (RZPD, Berlin, Germany) with the cloned cDNA using the oligodeoxynucleotides 5'-ACGATCGGCGCCAGCTCTTCTGCCCGACCCTCTGGG AGAAAATCC (SEQ ID NO:61) and 5'- CTGGGC AAGCTTACTCGTCCTCCTGGA AGTAG (SEQ ID NO: 62) as primers.
• the first primer contains a K ⁇ sl restriction site at the 5 '-end, followed by a S ⁇ pl restriction site (underlined), whereas the second primer contains a HmdIII restriction site (underlined).
• the amplification product was purified and digested with K ⁇ sl and Hindlll and ligated with the accordingly cut vector pASK-IBA4 (IBA, G ⁇ ttingen, Germany). After transformation of E. coli XLl -Blue, plasmids were prepared and the sequences of the cloned synthetic nucleic acid inserts were confirmed by restriction analysis and automated double-stranded DNA sequencing.
• the plasmid coding for ILl ra as fusion with a N-terminal Strep-tag II was designated pASK-ILlra (Fig. 2E).
• pASK-ILlra was cut with S ⁇ pl, dephosphorylated with shrimp alkaline phosphatase, and ligated with an excess of the gene fragment for the 200 residue PAS#1 polymer or for the 192 residue PAS#5 polymer, respectivley, isolated from the corresponding intermediate plasmids pPAS(# 1)200 and pPAS(#5)192 by restriction digest with S ⁇ pl (Fig. 2F). After transformation of E. coli JM83 (Yanisch-Perron.
• Plasmids were prepared and the sizes of the polymer-encoding regions, which were inserted during ligation in one or several repeated copies, were determined by restriction analysis.
• PAS(#5)192-ILlra or PAS(#5)384-ILlra were designated pASK-PAS(#l)200-ILlra, pASK-PAS(#l)400-ILlra, pASK-PAS(#5)192-ILlra, and pASK-PAS(#5)384-ILlra respectively.
• Example 4 Construction of expression vectors for PAS#1 and piSA fusion proteins of neutrophil gelatinase-associated Iipocalin (NGAL).
• This vector dubbed pNGAL15- ⁇ co, was cut with EcoO109I, dephosphorylated with shrimp alkaline phosphatase (USB, Cleveland, OH), and ligated with the synthetic DNA fragment encoding PAS#1 or piSA (Fig. 3B).
• plasmids were prepared and the sequences of the cloned synthetic nucleic acid inserts were confirmed by restriction analysis and automated double-stranded DNA sequencing (ABI- PrismTM310 Genetic analyzer) using the BigDyeTM terminator kit as well as oligodeoxynucleotide primers that enabled sequencing from both sides.
• NGAL- PAS(#l)100 and NG AL-P AS(# 1)200 were named pNGAL-PAS(#l)100 and pNGAL- PAS(#l)200 (Fig. 3C), respectively.
• the plasmid coding for NGAL carrying a piSAlOO residue polymer sequence, NGAL-piSAlOO was named pNGAL-piSAlOO.
• Example 5 Bacterial production and purification of fusion proteins between IFNa2b and genetically encoded PAS#1, PAS#2, PAS#3, PAS#5, and PAS#1P2 polymers.
• IFNa2b (calculated mass: 20.9 kDa), PAS(#l)200-IFNa2b (calculated mass: 37.4 kDa), PAS(#l)400-IFNa2b (calculated mass: 54.0 kDa), PAS(#l)600-IFNa2b (calculated mass:
• PAS(#5)192-IFNa2b (calculated mass: 36.7 kDa)
• PAS(#5)384-IFNa2b calculated mass: 52.6 kDa
• E. coli BL21 Novagen, Madison, USA; Wood (1966) J MoI Biol 16:118-133 harboring the corresponding expression plasmids from Example 2 together with the folding helper plasmid pTUM4 (Schlapschy (2006) Protein Eng. Des. SeI.
• cells were harvested by centrifugation and resuspended during 10 min in ice-cold periplasmic fractionation buffer (500 mM sucrose, 1 mM EDTA, 100 mM Tris/HCl pH 8.0; 2 ml per L and OD 550 ). After adding 15 mM EDTA and 250 ⁇ g/ml lysozyme, the cell suspension was incubated for 20 min on ice, centrifuged several times, and the cleared supernatant containing the recombinant protein was recovered.
• ice-cold periplasmic fractionation buffer 500 mM sucrose, 1 mM EDTA, 100 mM Tris/HCl pH 8.0; 2 ml per L and OD 550 . After adding 15 mM EDTA and 250 ⁇ g/ml lysozyme, the cell suspension was incubated for 20 min on ice, centrifuged several times, and the cleared supernatant containing the recombinant protein was
• the IFNa2b variants were purified via the Strep-tag II fused to the N-terminus (Skerra (2000) Methods Enzymol 326:271-304) and via gel filtration using a Superdex S75 or S200 HiLoad 16/60 column (Amersham Biosciences, Uppsala, Sweden).
• PAS(#2)200-IFNa2b (calculated mass: 37.4 kDa)
• PAS(#3)200-IFNa2b calculated mass:
• 1 mM EDTA, 100 mM Tris/HCl pH 8.0 containing 50 ⁇ g lysozyme per ml was performed as described (Breustedt (2005) loc. cit.) and followed by purification via the Strep- tag II using streptavidin affinity chromatography (Skerra (2000) loc. cit.) with a high salt buffer (500 mM NaCl, 1 mM EDTA, 100 mM Tris/HCl, pH 8.0).
• the purified proteins were dialysed three times against PBS (115 mM NaCl, 4 mM KH 2 PO 4 , 16 mM Na 2 HPO 4 pH 7.4) and applied to a Q Sepharose FF 16/200 column (Amersham Biosciences, Uppsala, Sweden) using an Akta Purifier 10 system with a 50 ml superloop (Amersham Biosciences) and PBS as running buffer. The flow through containing the recombinant protein was collected and concentrated to ca.
• IL-lra (calculated mass: 19.8 kDa), PAS(#l)200-ILlra (calculated mass: 35.3 kDa), PAS(#l)400-ILlra (calculated mass: 51.9 kDa), PAS(#5)192-ILlra (calculated mass: 34.6 kDa), and PAS(#5)384-ILlra (calculated mass: 50.5 kDa) were produced in E.
• Example 7 Bacterial production and purification of fusion proteins between NGAL and genetically encoded PAS#1 and piSA polymers.
• the NGAL (calculated mass: 21.5 kDa) was produced in E. coli BL21 harboring the expression plasmid pNGAL15 using an 8 L bench top fermenter essentially as described in Example 4.
• the NGAL was purified via the Strep-tag II fused to the C-terminus (Skerra (2000) Methods Enzymol 326:271-304).
• Periplasmic extraction in the presence of 500 mM sucrose, 1 mM EDTA, 100 mM Tris/HCl pH 8.0 containing 50 ⁇ g lysozyme per ml was performed as described (Breustedt (2005) J Biol Chem 280:484-493) and followed by purification via the Strep-tag II using streptavidin affinity chromatography (Skerra (2000) loc. cit.) with a high salt buffer (500 mM NaCl, 1 mM EDTA, 100 mM Tris/HCl, pH 8.0).
• NGAL-PAS(#l)100 and NGAL-P AS(# 1)200 homogeneous protein preparations were obtained after the one step affinity chromatography (Fig. 4E) with yields of 0.1 mg L “1 OD “1 for NGAL, 0.5 mg L “1 OD “1 for NGAL-PAS(#l)100, and 0.8 mg L “1 OD “1 for NGAL- PAS(#l)200.
• NGAL-piSAlOO was further purified via gel filtration using a Superdex S75 HR 10/300 GL column (Amersham Biosciences, Uppsala, Sweden), yielding 0.01 mg L "1 OD “1 .
• NGAL, NGAL-PAS(#l)100, and NG AL-P AS(# 1)200 proteins were dialysed three times against PBS and applied to a Q Sepharose FF 16/200 column (Amersham Biosciences) using an Akta Purifier 10 system with a 50 ml superloop (Amersham Biosciences) and PBS as running buffer.
• the flow through containing the recombinant protein was collected and concentrated to ca. 1.5 mg/ml by ultrafiltration using Amicon Ultra centrifugal filter devices (10000 MWCO; 15 ml; Millipore, Billerica, MA).
• EndoTrap ® affinity columns (Profos AG, Regensburg, Germany) using PBS as running buffer.
• the final endotoxin content was between 5.17 and 21.9 EU/ml at a protein concentration of 1 mg/ml as determined using the Endosafe PTS Kit (Charles River Laboratories, L'Arbresle, France).
• Example 8 Measurement of the hydrodynamic volume for the recombinant fusion proteins between IFNa2b and genetically encoded PAS#1, PAS#2, PAS#3, PAS#5 or PAS#1P2 polymers of different length by analytical gel filtration.
• RNA samples 250 ⁇ l of a mixture of the following globular proteins (Sigma, Deisenhofen, Germany) were applied in PBS: RNase A (0.2 mg/ml), carbonic anhydrase (0.2 mg/ml), ovalbumin (0.5 mg/ml), bovine serum albumin (0.5 mg/ml), transferrin (0.2 mg/ml) and alcohol dehydrogenase (0.4 mg/ml).
• RNase A 0.2 mg/ml
• carbonic anhydrase 0.2 mg/ml
• ovalbumin 0.5 mg/ml
• bovine serum albumin 0.5 mg/ml
• transferrin 0.2 mg/ml
• alcohol dehydrogenase 0.4 mg/ml
• the fusion proteins with the PAS#1 polymers with 200, 400 and 600 residues and the PAS#5 polymers with 192 and 384 residues exhibited significantly larger sizes than corresponding globular proteins with the same molecular weight.
• the size increase for PAS(#l)200-IFNa2b, PAS(#l)400-IFNa2b and PAS(#l)600-IFNa2b was 8.4-fold, 16.5-fold and 24.9-fold, respectively, compared with the unfused IFNa2b protein. In contrast, the true mass was only larger by 1.8-fold, 2.6-fold and 3.4-fold.
• PAS(#5)192- IFNa2b and PAS(#5)384-IFNa2b The size increase for PAS(#5)192- IFNa2b and PAS(#5)384-IFNa2b was 7.7-fold and 13.3-fold, respectively, compared with the unfused IFNa2b ⁇ protein. In these cases the true mass was only by 1.8-fold and 2.5-fold larger.
• the fusion proteins with the PAS#2 and PAS#3 polymers with 200 residues exhibited significantly larger sizes than corresponding globular proteins with the same molecular weight.
• the size increase for PAS(#2)200-IFNa2b and PAS(#3)200-IFNa2b was 8- fold and 7-fold, respectively, compared with the unfused IFNa2b protein.
• the true mass was in both cases only larger by 1.8-fold.
• the fusion protein with the PAS#1P2 polymer with 140 residues exhibited also a larger size than the corresponding globular proteins with the same molecular weight.
• PAS(#lP2)140-IFNa2b was just 3- fold compared with the unfused IFNa2b protein, whereby the true mass was just 1.5 -fold larger.
• the size increase for PAS(#lP2)140-IFNa2b with a reduced number of proline residues (14 in PAS(#1P2)14O) was less pronounced, indicating a major influence of the Pro content on the random coil properties of the amino acid polymer sequences.
• RNA samples 250 ⁇ l of a mixture of the following globular proteins (Sigma, Deisenhofen, Germany) were applied in PBS: RNase A (0.2 mg/ml), carbonic anhydrase (0.2 mg/ml), ovalbumin (0.5 mg/ml), bovine serum albumin (0.5 mg/ml), transferrin (0.2 mg/ml) and alcohol dehydrogenase (0.4 mg/ml).
• RNase A 0.2 mg/ml
• carbonic anhydrase 0.2 mg/ml
• ovalbumin 0.5 mg/ml
• bovine serum albumin 0.5 mg/ml
• transferrin 0.2 mg/ml
• alcohol dehydrogenase 0.4 mg/ml
• the fusion proteins with the PAS#1 polymers with 200 and 400 residues and the PAS#5 polymers with 192 and 384 residues exhibited significantly larger sizes than corresponding globular proteins with the same molecular weight.
• the size increase for PAS(#l)200-ILlra and PAS(#l)400-ILlra was 8-fold and 17-fold, respectively, compared with the unfused IL-lra protein. In contrast, the true mass was only larger by 1.8-fold and 2.6- fold.
• the size increase for PAS(#5)192-ILlra and PAS(#5)384-ILlra was 7-fold and 15-fold, respectively, compared with the unfused IL-lra protein. In these cases the true mass was only by 1.7-fold and 2.5-fold larger.
• Example 10 Measurement of the hydrodynamic volume for the recombinant fusion proteins between NGAL and genetically encoded PAS#1 and piSA polymers by analytical gel filtration.
• aprotinin 0.5 mg/ml
• ribonuclease 0.4 mg/ml
• myoglobin 0.2 mg/ml
• carbonic anhydrase 0.2 mg/ml
• ovalbumin 0.5 mg/ml
• bovine serum albumin 0.5 mg/ml
• transferrin 0.2 mg/ml
• cytochrome c (0.2 mg/ml), carbonic anhydrase (0.2 mg/ml), ovalbumin (0.5 mg/ml), bovine serum albumin (0.5 mg/ml), transferrin (0.2 mg/ml) and alcohol dehydrogenase (0.4 mg/ml) for the Superdex S200 10/300 GL run.
• the fusion protein with the piSA polymer with 100 residues showed a less significant size increase compared with corresponding globular proteins having the same molecular weight.
• the size increase for NGAL-piSA100 was just 2.5-fold compared with the unfused NGAL protein whereby the true mass was by 1.4 fold bigger.
• fusion with the 100 residue Pro-Ala-Ser polymer leads to a significantly larger increase in the hydrodynamic volume than with the 100 residue Ala-Ser polymer.
• Example 11 Detection of random coil conformation for the genetically encoded PAS#1 polymer fused to IFNa2b by circular dichroism spectroscopy.
• ⁇ Obs denotes the measured ellipticity, c the protein concentration [mol/1], d the path length of the quartz cuvette [cm].
• the ⁇ M values were plotted against the wavelength using Kaleidagraph (Synergy Software, Reading, PA).
• the circular dichroism (CD) spectrum for the recombinant IFNa2b is in accordance with previously published data for this ⁇ -helix bundle protein (Radhakrishnan (1996) Structure 4:1453-1463), whereas the spectra for PAS(#l)200-IFNa2b, PAS(#l)400-IFNa2b, and PAS(#l)600-IFNa2b reveal significant contributions of random coil conformation (Fig. 6A).
• Example 12 Detection of random coil conformation for the genetically encoded PAS#5 polymer fused to IFNa2b by circular dichroism spectroscopy.
• Pro-Ala-Ser sequence as part of the recombinant fusion protein appears to be present as a random coil polymer under physiological buffer conditions.
• Example 13 Detection of random coil conformation for the genetically encoded PAS#2, PAS#3 and PAS#1P2 polymer fused to IFNa2b by circular dichroism spectroscopy.
• the Pro-Ala-Ser sequence as part of the recombinant fusion protein appears to be present as a random coil polymer under physiological buffer conditions.
• the CD signal for random coil is significantly reduced, indicating a dependency of the random coil character upon the Pro content in the amino acid polymer sequences.
• Example 14 Detection of random coil conformation for the genetically encoded PAS#1 and PAS#5 polymer fused to IL-lra by circular dichroism spectroscopy.
• Example 16 Detection of ⁇ -sheet conformation for the genetically encoded piSA polymer fused to NGAL by circular dichroism spectroscopy.
• Example 17 Quantitative analysis of the secondary structure of IFNa2b, NGAL, and their polymer fusions
• the fraction of unstructured conformation comprising random coil and turns for the whole protein clearly increases with the length of the PAS(#1) and PAS(#5) polymers fused to IFNa2b (see bottom row of the Table shown above, which summarizes the results of the CD spectra deconvolution with the program CDNN).
• a generally similar but less pronounced effect can be seen for NGAL- PAS(#l)100 and NGAL-P AS(# 1)200.
• the amount of turns and random coil is even lower than in the recombinant NGAL 5 whereas the amount of anti-parallel ⁇ -sheet increases from 38.3 % in NGAL to 50.0 % in NGAL-piSA100.
• the piSAlOO polymer comprising only Ser and Ala residues assumes a ⁇ -sheet structure rather than a random coil, which is reflected by the less significant increase in the hydrodynamic volume as measured in Example 10.
• Example 18 Test of serum stability of PAS(#l)200-IFNa2b and PAS(#5)192-IFNa2b
• Serum stability of PAS(#l)200-IFNa2b and PAS(#5)192-IFNa2b was analyzed by mixing of 10 ⁇ l test protein at a concentration of 1 mg/ml and 50 ⁇ l mouse plasma (Rockland Immunochemicals, Gilbertsville, PA), resulting in a test protein concentration of 0.17 mg/ml and a plasma concentration of 83 % (v/v). The samples were incubated at 37 0 C for 24 h or 48 h.
• the membrane was incubated for 10 min in 20 ml PBST containing 2 ⁇ g/ml egg- white avidin to mask endogenous protein-bound biotin groups and then 20 ⁇ l of the StrepTactin® Alkaline Phosphatase conjugate (IBA, Gottingen, Germany) were directly added (at a dilution of 1 :1000).
• the chromogenic reaction was performed (without shaking) by adding 10 ml of AP buffer with 5 ⁇ l nitroblue tetrazolium (NBT, Biomol, Hamburg, Germany; 75 mg/ml in 70 % w/v DMF) and 30 ⁇ l 5-bromo-4-chloro-3-indolyl- phosphate p-toluidine salt (BCIP, Roth, Düsseldorf, Germany; 50 mg/ml in DMF) until the bands appeared. The reaction was stopped by washing with water and air-drying of the membrane.
• Example 19 Detection of prolonged plasma half-life for the recombinant fusion proteins between IFNa2b and a genetically encoded PAS#1 polymer in vivo.
• Adult BALB/c mice (Harlan-Winckelmann, Borchen, Germany) were injected intravenously according to the following table:
• the total volume of intravenously administered test item was calculated according to the individual body weight recorded on the day of administration (e.g. an animal with 25 g body weight (b.w.) received 125 ⁇ l of 1 mg/ml test item). Blood sampling was performed 30 min, 120 min, and 360 min after injection according to the following table:
• 10 ⁇ l aliquots of the cleared plasma samples were diluted with 90 ⁇ l PBS. 10 ⁇ l thereof (corresponding to 1 ⁇ l plasma) were diluted with 6 ⁇ l PBS and mixed with 4 ⁇ l SDS-PAGE loading buffer (250 mM Tris/HCl pH 8.0, 7.5 % w/v SDS, 25 % v/v glycerol, 0.25 mg/ml bromophenol blue) containing 12.5 % v/v 2-mercaptoethanol. After 5 min heating at 95 °C, these samples were subjected to 10 % SDS-PAGE.
• SDS-PAGE loading buffer 250 mM Tris/HCl pH 8.0, 7.5 % w/v SDS, 25 % v/v glycerol, 0.25 mg/ml bromophenol blue
• the membrane was placed in a dish and washed 3 times for 20 min with 10 ml PBST (PBS containing 0.1 % v/v Tween 20). Then the membrane was incubated for 10 min in 20 ml PBST containing 20 ⁇ l of the mouse anti-human IFNa2b antibody 9D3 (Abeam, Cambridge, UK; at a dilution of 1 :1000).
• PBST PBS containing 0.1 % v/v Tween 20
• the membrane was incubated for 10 min in 20 ml PBST containing 20 ⁇ l of the mouse anti-human IFNa2b antibody 9D3 (Abeam, Cambridge, UK; at a dilution of 1 :1000).
• the membrane was washed 3 times for 20 min with 10 ml PBST and then incubated with a anti-mouse IgG Alkaline Phosphatase conjugate (Sigma- Aldrich, St. Louis, MO) for 60 min.
• a anti-mouse IgG Alkaline Phosphatase conjugate Sigma- Aldrich, St. Louis, MO
• the chromogenic reaction was performed (without shaking) by adding 10 ml of AP buffer with 5 ⁇ l nitroblue tetrazolium (NBT, Biomol, Hamburg, Germany; 75 mg/ml in 70 % w/v DMF) and 30 ⁇ l 5- bromo-4-chloro-3-indolyl-phosphate p-toluidine salt (BCIP, Roth, Düsseldorf, Germany; 50 mg/ml in DMF) until the bands appeared. The reaction was stopped by washing with water and air-drying of the membrane.
• Fig. 8 shows samples containing IFNa2b, PAS(#l)200-IFNa2b, and PAS(#l)400-IFNa2b from equivalent time points. While IFN a2b is no longer detectable after 120 min, PAS(#l)200-IFNa2b and PAS(#l)400-IFNa2b can be detected for periods up to 360 min. These data indicate that the plasma half-life of IFNa2b is significantly prolonged when fused with the Pro-Ala-Ser polymers.
• the wells of a 96 well microtitre plate were coated overnight at 4 °C with 100 ⁇ l of a 5 ⁇ g/ml solution of the mouse anti-human IFNa2b antibody 9D3 (Abeam, Cambridge, UK) in 5 % (w/v) NaHCO 3 pH 9.3. After removal of the coating solution the wells were blocked with 200 ⁇ l of 2 % (w/v) BSA in PBS for 1 h and washed three times with PBST.
• the plasma samples of animals no. 1/2 (IFNa2b), no. 3/4 (PAS(#l)200-IFNa2b), and no.
• ⁇ ⁇ /2 is the plasma half-life
• C 0 is the total blood concentration at time point zero, which was set to a fixed value of 78 ⁇ g/ml under the assumption of an average animal weight of 25 g and a typical blood to body weight ratio for mouse of 0.064.
• Fig. 9 depicts the kinetics of blood clearance in vivo. While the recombinant IFNa2b shows a rapid clearance from blood with a half-life of just ca. 5.5 min, the PAS(#l)200-IFNa2b and PAS(#l)400-IFNa2b fusion proteins have a more than 10-fold and 60-fold extended half- life of ca. 61 min and 6 h respectively. These data are in agreement with the Western blot analysis shown above and prove that the in vivo plasma half-life of IFNa2b is significantly prolonged due to fusion with the Pro-Ala-Ser polymers, whereby the half-life becomes longer with increasing length of the amino acid polymer.
• Example 20 Detection of prolonged plasma half-life for the recombinant fusion proteins between IFNa2b and the genetically encoded PAS#1 and PAS#5 polymers in vivo.
• mice (Charles River Laboratories, L'Arbresle, France) were injected intravenously according to the following table:
• the total volume of intravenously administered test item was calculated according to the individual body weight recorded on the day of administration (e.g. an animal with 18 g body weight (b.w.) received 125 ⁇ l of 1 mg/ml test item). Blood sampling was performed 30 min, 120 min, 240 min, and 480 min after injection according to the following table:
• the wells of a 96 well microtitre plate were coated overnight at 4 0 C with 100 ⁇ l of a 5 ⁇ g/ml solution of the mouse anti-human IFNa2b antibody 9D3 (Abeam, Cambridge, UK) in 5 % (w/v) NaHCO 3 pH 9.3. After removal of the coating solution the wells were blocked with 200 ⁇ l of 2 % (w/v) BSA in PBS for 1 h and washed three times with PBST.
• the plasma samples of animals no. 1/2 (PAS(#l)200-IFNa2b), no. 3/4 (PAS(#l)400-IFNa2b), no.
• the chromogenic reaction was started by adding 100 ⁇ l of 1 mg/ml ABTS peroxidase substrate solution in the recommended buffer (Roche Diagnostics, Mannheim, Germany) and, after incubation for 20 min at 25 °C, the absorbance at 405 nm was measured.
• Concentrations of PAS(#l)200-IFNa2b, PAS(#l)400- IFNa2b, PAS(#l)600-IFNa2b, PAS(#5)192-IFNa2b, and PAS(#5)384-IFNa2b in the plasma samples were quantified by comparison with standard curves, which were determined for dilution series for the corresponding purified recombinant proteins at defined concentrations in PBST containing 0.25 % (v/v) dummy mouse plasma.
• the concentration values, c(t) were determined for each time point from the ELISA measurements and plotted against time post intravenous injection, t. These data were numerically fitted using KaleidaGraph software assuming a mono-exponential decay according to the equation
• ⁇ i /2 is the plasma half-life
• C 0 is the total blood concentration at time point zero, which should have a value of ca. 116 ⁇ g/ml under the assumption of an average animal weight of 18 g and a typical blood to body weight ratio for mouse of 0.064.
• Fig. 10 depicts the kinetics of blood clearance in vivo.
• the half-life of PAS(#l)200-IFNa2b is ca. 66 min, which is in good agreement with the half-life of 61 min for PAS(#l)200-IFNa2b in Example 19, although a lower dose of 5 mg/kg b.w. compared to 7 mg/kg b.w. was used here. Thus, variation of the mouse line and the dose had no significant influence on the pharmacokinetics.
• the PAS(#l)400-IFNa2b and PAS(#l)600-IFNa2b fusion proteins have a more than 60-fold and 70-fold extended half-life of ca.
• the PAS(#5)192-IFNa2b and PAS(#5)384-IFNa2b fusion proteins have a more than 7-fold and 58-fold extended half-life of ca. 40 min and 321 min, respectively.
• Example 21 Detection of prolonged plasma half-life for the recombinant fusion proteins between NGAL and the genetically encoded PAS#1 polymer in vivo.
• the total volume of intravenously administered test item was calculated according to the individual body weight recorded on the day of administration (e.g. animal no. 104 with 21O g body weight (b.w.) received 1050 ⁇ l of 1 mg/ml NGAL). Blood sampling was performed 5 min, 10 min, 30 min, 60 min, 120 min, 240 min, 360 min, and 1440 min after injection according to the following table:
• a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) by means of a semi-dry blotting apparatus the membrane was placed in a dish and washed 3 times for 20 min with 10 ml PBST (PBS containing 0.1 % v/v Tween 20). Then the membrane was incubated for 10 min in 20 ml PBST containing 2 ⁇ g/ml egg-white avidin to mask endogenous protein-bound biotin groups and 20 ⁇ l of the StrepTactin® Alkaline Phosphatase conjugate (IBA GmbH, G ⁇ rtingen, Germany) were directly added (at a dilution of 1 :1000) and incubation was continued for 60 min.
• PBST PBS containing 0.1 % v/v Tween 20
• the chromogenic reaction was performed (without shaking) by adding 10 ml of AP buffer with 5 ⁇ l nitroblue tetrazolium (NBT, Biomol, Hamburg, Germany; 75 mg/ml in 70 % w/v DMF) and 30 ⁇ l 5- bromo-4-chloro-3-indolyl-phosphate p-toluidine salt (BCIP, Roth, Düsseldorf, Germany; 50 mg/ml in DMF) until the bands appeared. The reaction was stopped by washing with water and air-drying of the membrane.
• Fig. 11 shows two series of mixed samples containing NGAL, NGAL-PAS(#l)100, and NGAL-P AS(# 1)200 from equivalent time points. While NGAL is no longer detectable after 10 min, NGAL-PAS(#l)100 and NGAL-P AS(# 1)200 can be detected for periods up to 120 min. These data indicate that the plasma half-life of NGAL is significantly prolonged when fused with the Pro-Ala-Ser polymer.
• the wells of a 96 well microtitre plate were coated overnight at 4 °C with 50 ⁇ l of a 5 ⁇ g/ml solution of an anti-human Lipocalin-2/NGAL antibody (R&D Systems, Minneapolis, MN) in PBS. After washing three times with PBST the wells were blocked with 200 ⁇ l of 3 % (w/v) BSA in PBST for 2 h and washed again three times with PBST.
• the plasma samples of animals 104/105 (NGAL) and 116/117 (NG AL-P AS(# 1)200) were applied in dilution series in PBST containing 2.5 % (v/v) rat plasma from untreated animals (Elevage Janvier, Le Genest ST. Isle, France; Aurigon Life Science, Tutzing, Germany) and incubated for 1.5 h.
• the wells were then washed three times with PBST and incubated for 1 h with 50 ⁇ l of a 1 :1000 dilution of StrepTactin® Alkaline Phosphatase conjugate.
• ⁇ ⁇ a is the plasma half-life parameter
• C 0 is the total blood concentration at time point zero, which should have a value of ca. 80 ⁇ g/ml under the assumption of an average animal weight of 210 g and a typical blood to body weight ratio for rat of 0.064.
• Fig. 12 depicts the kinetics of blood clearance in vivo. While the recombinant NGAL shows a rapid clearance from blood with a half-life of just ca. 3 min, the NGAL-P AS(# 1)200 fusion protein has a ten-fold extended half-life of ca. 31 min. These data are in agreement with the Western blot analysis shown above and prove that the in vivo plasma half-life of NGAL is significantly prolonged due to fusion with the Pro-Ala-Ser polymer.
• Example 22 Comparison of the activity of the commercially available IntronA and the recombinant PAS(#l)200-IFNa2b by IP-10 release assay with human PBMCs.
• the concentration of the released IP-10 (CXCLlO; interferon gamma inducible 10 kDa protein) in the supernatant upon induction by interferon alpha was determined by using the human IP-10 ELISA Set (BD OptEIATM, BD Biosciences Pharmingen, USA).
• Fig. 13 depicts the activity of the three test proteins. While the recombinant PAS(# 1)200- IFNa2b shows at higher concentrations comparable activities as IntronA, the latter is more active at lower concentrations, yielding on average a similar activity profile. Unstimulated PBMCs as well as PBMCs stimulated with the Fab fragment did not release significant amounts of IP-IO. As endotoxin, which might also induce the release of IP-IO, was removed in the preparations both of PAS(#l)200-IFNa2b and of the Fab fragment as described in Example 5, the activity of PAS(#l)200-IFNa2b can be clearly attributed to the IFNa2b moiety of the fusion protein. Thus, the Pro-Ala-Ser polymer does not interfere with the biological activity of IFNa2b.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Biochemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Molecular Biology (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Gastroenterology & Hepatology (AREA)
• Toxicology (AREA)
• Diabetes (AREA)
• Immunology (AREA)
• Hematology (AREA)
• Heart & Thoracic Surgery (AREA)
• Endocrinology (AREA)
• Urology & Nephrology (AREA)
• Cardiology (AREA)
• Epidemiology (AREA)
• Rheumatology (AREA)
• Vascular Medicine (AREA)
• Communicable Diseases (AREA)
• Obesity (AREA)
• Emergency Medicine (AREA)
• Transplantation (AREA)
• Oncology (AREA)
• Pain & Pain Management (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (17)

Applications Claiming Priority (4)

Related Child Applications (3)

Publications (1)

Family

ID=39776993

Family Applications (1)

Country Status (19)

Cited By (180)

Families Citing this family (38)

Citations (1)

Family Cites Families (8)

• 2008
  • 2008-06-20 DE DE602008005596T patent/DE602008005596D1/de active Active
  • 2008-06-20 JP JP2010512606A patent/JP5351889B2/ja active Active
  • 2008-06-20 WO PCT/EP2008/005020 patent/WO2008155134A1/en not_active Ceased
  • 2008-06-20 AU AU2008266448A patent/AU2008266448B2/en active Active
  • 2008-06-20 SI SI200830978T patent/SI2369005T1/sl unknown
  • 2008-06-20 PT PT08773567T patent/PT2173890E/pt unknown
  • 2008-06-20 PL PL08773567T patent/PL2173890T3/pl unknown
  • 2008-06-20 NZ NZ580670A patent/NZ580670A/en unknown
  • 2008-06-20 DK DK08773567.6T patent/DK2173890T3/da active
  • 2008-06-20 DK DK11158295.3T patent/DK2369005T3/da active
  • 2008-06-20 PL PL11158295T patent/PL2369005T3/pl unknown
  • 2008-06-20 AT AT08773567T patent/ATE502114T1/de active
  • 2008-06-20 EA EA201000040A patent/EA021222B8/ru active IP Right Grant
  • 2008-06-20 EP EP08773567A patent/EP2173890B1/en active Active
  • 2008-06-20 EP EP11158295A patent/EP2369005B1/en active Active
  • 2008-06-20 KR KR1020097025293A patent/KR101631323B1/ko active Active
  • 2008-06-20 CA CA2691386A patent/CA2691386C/en active Active
  • 2008-06-20 HR HR20110368T patent/HRP20110368T1/hr unknown
  • 2008-06-20 CN CN200880019017.4A patent/CN101970678B/zh active Active
  • 2008-06-20 SI SI200830250T patent/SI2173890T1/sl unknown
  • 2008-06-20 KR KR1020157020384A patent/KR101701080B1/ko active Active
  • 2008-06-20 ES ES11158295T patent/ES2422007T3/es active Active
  • 2008-06-20 ES ES08773567T patent/ES2362386T3/es active Active
  • 2008-06-20 US US12/665,565 patent/US8563521B2/en active Active
• 2011
  • 2011-05-27 CY CY20111100514T patent/CY1111491T1/el unknown
• 2013
  • 2013-08-09 US US13/963,953 patent/US9260494B2/en active Active

Patent Citations (1)

Non-Patent Citations (2)