WO1992007935A1 - Proteines de fusion ciblees par clycosaminoglycane, leurs conception, construction et compositions - Google Patents

Proteines de fusion ciblees par clycosaminoglycane, leurs conception, construction et compositions Download PDF

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WO1992007935A1
WO1992007935A1 PCT/US1991/008105 US9108105W WO9207935A1 WO 1992007935 A1 WO1992007935 A1 WO 1992007935A1 US 9108105 W US9108105 W US 9108105W WO 9207935 A1 WO9207935 A1 WO 9207935A1
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sequence
residues
polypeptide
integer
sequences
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PCT/US1991/008105
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John A. Tainer
Leslie Kuhn
Maurice Boissinot
Cindy Fisher
Hans E. Parge
John H. Griffin
Guy T. Mullenbach
Robert A. Hallewell
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The Scripps Research Institute
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
    • C12N9/0089Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81Protease inhibitors
    • C07K14/8107Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811Serine protease (E.C. 3.4.21) inhibitors
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62DNA sequences coding for fusion proteins
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/01Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/20Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/31Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to
  • glycosaminoglycan-binding fusion proteins and methods for designing and constructing the fusion proteins. More particularly, the invention relates to methods and compositions for extending in vivo lifetimes of biologically active compounds and targeting them to specific cell surfaces or substrates.
  • Fusion proteins may be comprised of homologous or heterologous sources of polypeptides so long as the polypeptides being fused are not typically associated together. Particularly interesting are fusion proteins comprised of
  • polypeptides derived from independently folding structural regions (domains) of proteins that contain biological function.
  • fusion proteins have been formed using the techniques of genetic engineering. For example, a non-excretable protein can be fused to a ⁇ -lactamase moiety to give an excretable fused protein. At the genetic level, this fusion is
  • the fused protein is generated. See, e.g., Freifelder. Molecular
  • a DNA oligonucleotide can be prepared which codes for the hormone attached to a methionine group.
  • the synthetic oligonucleotide can be ligated to a cleaved vector adjacent to the lac Z gene for ⁇ -galactosidase in E. coli.
  • the enzyme region of the expressed protein can subsequently be removed by reaction with CNBr which cleaves the expressed protein at the methionine group.
  • the biologically active form of a peptide may be released by enzymatically removing the undesired protein fragment, e.g., with trypsin.
  • Many other variations can be envisioned. See, Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY pp.422-433 (1982).
  • SOD superoxide dismutase
  • HSOD human intracellular SOD
  • SOD enzymes have also been implicated in preventing alloxan diabetes [Grankvist et al., Nature, 294:158 (1981)] and in preventing metastasis of certain forms of cancer (EPO Application No. 0332464).
  • Extracellular SOD (EC-SOD) is the major SOD enzyme in extracellular fluids.
  • Heparin is a glycosaminoglycan (GAG) that
  • polysaccharide chains are covalently linked to
  • GAGs polypeptide backbones to form proteoglycans. Seven different groups of GAGs are distinguished by the types of sugar residues, the type of linkage between the sugar residues, and the number and location of sulfate groups. The presence of sulfate groups as well as carboxyl groups give GAGs a highly negative charge. The various forms of GAGs are distributed throughout the body in such areas as connective tissues, skin, cartilage, cornea, bone, blood vessels, lung, liver, cell surfaces, extracellular matrix and the like. In these areas, GAGs adopt an extended, random-coil conformation. GAGs are hydrophilic, forming hydrated gels at low concentrations. The negative charge of the chains attracts water as well as osmotically active cations. See, Lindahl et al., Annu. Rev Biochem., 47:385-417 (1978); and Chakrabarti et al., CRC Crit. Rev. Biochem., 8:225-313 (1980).
  • EC-SOD is shown to be heterogeneous with regard to heparin binding. Marklund et al., Proc. Natl.
  • EC-SOD anti-inflammatory agent
  • recombinant EC-SOD has been expressed successfully only in mammalian cell cultures making recombinant EC-SOD very expensive to produce. Tibell et al., Proc. Natl. Acad. Sci. USA 84:6634-8 (1987).
  • recombinant EC-SOD is formed as a heterogeneous mixture of SOD enzymes due to variations in carbohydrate content and extent of proteolysis.
  • a recombinant EC-SOD also has been described by Marklund et al. (WO 8701387).
  • HSOD Intracellular human SOD
  • HSOD Intracellular human SOD
  • Chemical approaches include conjugating HSOD to polyethylene glycol [White et al., Superoxide and Superoxide Dismutase in
  • Native HSOD is a CuZn dimer having a molecular weight of 32,000 Daltons.
  • recombinant HSOD analog differs from HSOD in that it is not N-acetylated.
  • BTG's HSOD shows pharmacological activity in preclinical studies that is indistinguishable from the natural protein.
  • Recombinant HSOD has also been expressed in yeast
  • the long-lived variants of proposed pharmaceutical agents will preferably be non- immunogenic, i.e., not trigger an immune response and therefore be suitable for repeated therapeutic use in a particular host animal.
  • the long-lived variants of proposed pharmaceutical agents will preferably include functionalities that minimize the costs and complexities associated with employing such variants, e.g., by facilitating their purification from reaction mixtures.
  • a class of glycosaminoglycan (GAG) -binding moieties have been identified in the present invention that can be operatively linked to a preselected protein to form a fusion protein and thereby increase the stability, plasma half-life and ease of
  • the present invention contemplates a fusion polypeptide having a minimum of two independently folding protein moieties operatively linked into a single polypeptide.
  • a first moiety is a
  • glycosaminoglycan (GAG)-binding moiety that provides a targeting function and introduces GAG-binding activity into the fusion protein.
  • the second moiety is a polypeptide having biological activity.
  • the present invention also affords a systematic method for identifying optimal configurations of fusion proteins having independently folding
  • the fusion proteins are amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids
  • amino acid residue sequences provide desired functionalities for the fused protein, and preferably contains a third amino acid residue sequence that serves to covalently link the first two sequences.
  • an amino acid residue sequence, which has superoxide dismutase activity, corresponding to that for HSOD is linked to a second sequence having glycosaminoglycan- binding activity.
  • a preferred linking sequence is Gly-Pro-Gly, which links the HSOD unit to the
  • compositions are also contemplated in which the compositions comprise a therapeutically effective amount of the fusion protein.
  • compositions comprise a therapeutically effective amount of the fusion protein.
  • pharmacologically active compound in an animal's bloodstream is extended by administering the heparin- binding fused protein to the animal.
  • the fused protein will comprise the heparin-binding moiety.
  • FIG. 1 illustrates the nucleotide sequence of a DNA segment that codes for a GAG-binding fusion protein, shown from left-to-right and in the direction of 5'-terminus to 3'-terminus using the single letter nucleotide base code.
  • the structural gene for the mature fusion protein begins at base 67 and ends at base 579, with the position number of the every tenth base residue in each row indicated above the row showing the sequence.
  • amino acid residue sequence for the fusion protein is indicated by the single letter code below the nucleotide base sequence, with the position number for the first residue in each row indicated to the left of the row showing the amino acid residue
  • the reading frame is indicated by placement of the deduced amino acid residue sequence below the nucleotide sequence such that the single letter that represents each amino acid is located below the first base in the corresponding codon.
  • the mature fusion protein amino acid residue sequence begins at residue 1 and ends at residue 171.
  • N-terminal A+ helix crosses the lower third of the picture, with the N-terminus, residue 5 ( ⁇ 1 -antitrypsin numbering), at far right and
  • residue 15 turns from the end of the helix, at center.
  • the H helix in the upper portion of the picture, starts at residue 269 (one residue to the right of the labeled residue, 270), and extends to residue 277 at upper left.
  • the positive charges on this helix are augmented by positive charges in residues 280-282, which have extended conformation.
  • the highly positive electrostatic potential (dots indicating a surface potential of ⁇ 3 kcal/mol) is generated by the many positive charges on these helices and constitutes the most favorable region on the PCI surface for binding of (negatively-charged) glycosaminoglycans. This arrangement of helices and their spacing is similar to the two-helix motif found for heparin binding in the crystallographic structure of platelet factor 4, a protein not homologous to PCI.
  • Figure 3 illustrates a model of the carboxy- terminal helices from the platelet factor 4 (PF4) dimer structure attached to the carboxy termini of the human superoxide dismutase (HSOD) dimer, comprising an optimal two-helix motif for glycosaminoglycan binding.
  • PF4 platelet factor 4
  • HSOD human superoxide dismutase
  • Residues 75-85 and 175-185 are the C-terminal helices of the two monomers in the PF4 crystal structure dimer.
  • the chains of the two dimers of HSOD are numbered 1-153 and 201-353.
  • FIG. 4 is block diagram of the system of the present invention.
  • Amino Acid Residue An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages.
  • the amino acid residues described herein are preferably in the "L” isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino acid residue is broadly defined to include the amino acids listed in the Table of
  • Base Pair a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double-stranded DNA molecule.
  • A adenine
  • T thymine
  • C cytosine
  • G guanine
  • U uracil
  • Base pairs are said to be "complementary" when their component bases pair up normally when a DNA or RNA molecule adopts a double- stranded configuration.
  • Complementary Nucleotide Sequence a sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to another single strand to specifically (non-randomly) hybridize to it with consequent hydrogen bonding.
  • nucleotide sequence is conserved with respect to a preselected (reference) sequence if it non-randomly hybridizes to an exact complement of the preselected sequence.
  • Duplex DNA a double-stranded nucleic acid molecule comprising two strands of substantially complementary polynucleotides held together by one or more hydrogen bonds between each of the complementary bases present in a base pair of the duplex. Because the nucleotides that form a base pair can be either a ribonucleotide base or a deoxyribonucleotide base, the phrase "duplex DNA” refers to either a DNA-DNA duplex comprising two DNA strands (ds DNA), or an RNA-DNA duplex comprising one DNA and one RNA strand.
  • Fusion Protein A protein comprised of at least two polypeptides. In some cases, a linking sequence is present to operatively link the two polypeptides into one continuous polypeptide (i.e., fusion
  • At least one, and preferably two, of the polypeptides comprising a fusion protein is
  • the two polypeptides linked in a fusion protein are typically derived from two
  • a fusion protein comprises two linked polypeptides not normally found linked in nature.
  • Gene a nucleic acid whose nucleotide sequence codes for a RNA, DNA or polypeptide molecule. Genes may be uninterrupted sequences of nucleotides or they may include such intervening segments as introns, promoter regions, splicing sites and repetitive sequences. A gene can be either RNA or DNA.
  • Hybridization the pairing of complementary nucleotide sequences (strands of nucleic acid) to form a duplex, heteroduplex, or complex containing more than two single-stranded nucleic acids, by
  • Hybridization is a
  • Linking Sequence an amino acid residue sequence comprising zero to seven amino acid residues.
  • a linking sequence serves to chemically link two
  • Nucleotide a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate group, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of base and sugar is a nucleoside.
  • nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose, it is referred to as a nucleotide.
  • nucleotides is typically referred to herein as a "base sequence” or “nucleotide sequence”, and their
  • Nucleotide Analog a purine or pyrimidine nucleotide that differs structurally from an A, T, G, C, or U base, but is sufficiently similar to
  • Inosine (I) is a nucleotide analog that can hydrogen bond with any of the other nucleotides. A, T, G, C, or U. In addition, methylated bases are known that can participate in nucleic acid hybridization.
  • Polynucleotide a polymer of single or double stranded nucleotides.
  • polynucleotide and its grammatical equivalents will include the full range of nucleic acids.
  • a polynucleotide will typically refer to a nucleic acid molecule comprised of a linear strand of two or more deoxyribonucleotides and/or ribonucleotides. The exact size will depend on many factors, which in turn depends on the ultimate conditions of use, as is well-known in the art.
  • the polynucleotides of the present invention include primers, probes, RNA/DNA segments, oligonucleotides or "oligos" (relatively short polynucleotides), genes, vectors, plasmids, and the like.
  • Polypeptide or Peptide or Protein a linear series of at least two amino acid residues in which adjacent residues are connected by peptide bonds between the alpha-amino group of one residue and the alpha- carboxy group of an adjacent residue.
  • Recombinant DNA (rDNA) molecule a DNA molecule produced by operatively linking a nucleic acid
  • a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature. rDNAs not having a common biological origin, i.e., evolutionarily different, are said to be "heterologous”.
  • Vector a DNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment.
  • a DNA segment e.g., gene or polynucleotide
  • Vectors capable of directing the expression of genes encoding for one or more proteins are referred to herein as "expression vectors”.
  • a glycosaminoglycan (GAG)-binding fusion protein i.e., a GAG-targeted fusion protein, is a protein comprising two functional elements defined by two independently folding polypeptides that are
  • the first functional element is a GAG-binding moiety comprised of a first polypeptide that independently folds into a functional three-dimensional protein structural domain having GAG-binding activity.
  • the second functional element is a biologically active moiety comprised of a second polypeptide that independently folds into a functional three-dimensional protein structural domain having a preselected biological activity.
  • GAGs Glycosaminoglycans
  • GAG-binding moiety for use in the present invention is a
  • polypeptide sequence that has an affinity for binding with a GAG, and is therefore useful in a fusion protein of the present invention to target the fusion protein to the vicinity of a GAG molecule.
  • a GAG-binding moiety for use in a fusion protein includes the following structural parameters as defined by the teachings of the present invention:
  • the GAG-binding moiety is a polypeptide of 6-20 amino acid residues in length
  • a helix-promoting residue is one of the following: leucine, alanine, glutamic acid, phenylalanine, threonine, isoleucine, serine, tyrosine, valine, asparagine, lysine, arginine, and aminoisobutyric acid;
  • polypeptide comprising the GAG- binding moiety exhibits amphipathic character when modeled as an ⁇ -helix
  • the GAG-binding moiety contains no more than two helix-breaking residues (e.g., glycine or proline).
  • a general formula for a GAG-binding moiety for use in the present invention is a polypeptide
  • X g [+] h X i [+] j -X k [+] l X m [+] n X o where [+] and X are amino acid residues (designated in single letter code), [+] is R or K; X is L, A, E, F, T, I, S, Y, V, N, K, R or aminoisobutyric acid; g is an integer from 0-9, h is an integer from 1-3, i is an integer from 1-5, j is an integer from 1-3, k is an integer from 1-7, 1 is an integer from 0-7, m is an integer from 0-7, n is an integer from 0-2, and o is an integer from 0-2; and with the proviso that
  • g+h+i+j+k+l+m+n+o is equal to or less than 20.
  • X can be H, Q, M, C, W, D, G or P and X contains zero to two of the helix-breaking residues selected from the group consisting of G and P.
  • Polypeptide sequences having an amino acid residue sequence according to the above formula represents a GAG-binding moiety for use in the present invention and can be designed de novo or can be identified from known protein sequences.
  • the polypeptide sequence is selected from a protein having known GAG-binding activity. Proteins of known sequence that have known GAG-binding activity have been described extensively in the literature and are summarized in Table 1.
  • Neural cell adhesion protein Mouse Neural cell adhesion protein Rat
  • Preferred GAG-binding moieties are polypeptides including the sequences having the formula: [+] 2 X 2 [+] 2 where [+] and X are as described above. Particularly preferred in this embodiment is the polypeptide having the sequence YKKIIKKLLES, which is derived from the platelet factor 4 (PF4) C-terminal helix.
  • PF4 platelet factor 4
  • GAG-binding moiety is the polypeptide including the formula: [+]X 3 [+]X 2 [+] 3 , wherein the [+] and X are as defined above.
  • this embodiment is the polypeptide having the sequence HRHHPREMKKRVEDL, derived from the amino terminus of protein C inhibitor and corresponding to the A+ helix as described herein.
  • GAG-binding moiety is the polypeptide including the formula: [+]X 2 [+] 2 X[+], wherein the [+] and X are as defined above.
  • preferred polypeptide according to this embodiment is an internal sequence corresponding to a section of C- terminal end of the D helix of antithrombm III having the sequence KLNCRLYRKANK.
  • polypeptide according to the above formula is the internal H helix of protein C inhibitor having the sequence EKTLRKWLK.
  • GAG-binding moiety for use in the present invention is a polypeptide including the formula: [+] 2 X 5 [+]X 3 [+] where [+] and X are as defined above.
  • a preferred polypeptide according to this embodiment is a section of the N-terminal end of the internal A helix of antithrombin III having the sequence RRVWELSKANSR.
  • GAG-binding moiety for use in a fusion protein is the PCI A+ helix identified above and utilized in the HSOD-A+ fusion protein described in Example 5.
  • GAG-binding moiety for use in the present invention is a polypeptide having the sequence
  • a linker or linking means for use in the present invention to connect a glycosaminoglycan (GAG)-binding moiety to a biologically active moiety in the fusion protein of the present invention has a structure that depends on the amino acid sequence of the two moieties being linked. Considerations for selection of a linker are discussed in detail herein and in the discussion of identifying linkers using SEARCHWILD.
  • a linking means operatively links two polypeptide portions of a fusion protein through peptide bonds and in one embodiment is comprised of zero or more amino acid residues i.e., a linker sequence, and is
  • a fusion protein typically less than 20 amino acid residues, preferably less than seven residues, and more preferably is three residues.
  • a fusion protein can simply have a peptide bond as the operative linkage (linking means) between two polypeptide domains of the fusion protein, it is more typical that the linking means in a fusion protein is one or more residues in a linker sequence for operatively connecting the protein's independently folding polypeptide domains.
  • parvalbumin B (PDB code 3cpv).
  • linker sequence REACG corresponding to residues 113-117 of p-hydroxybenzoate hydroxylase ternary complex (PDB lphh); the linker VE
  • linkers can be included in a fusion protein of the present invention involving a reverse turn of 4 residues in length.
  • Reverse turns are preferred because they are surface-exposed, well- defined structures stabilized by internal hydrogen bonds between residues within the turn [Richardson et al.. Adv. Prot. Chem., 34:168-364 (1981)] and because the preferred residue types in the four positions of type I and type II turns, the most common reverse turns, are known. Wilmot et al.. J. Mol. Biol.,
  • polypeptide sequences NDSG, NSSG, NSRG, and NSDG.
  • a preferred linker includes the amino acid residues Gly-Pro-Gly.
  • An important consideration in the linker is to provide a short extension away from one polypeptide structure into another polypeptide structure that confers a sharp turn such that the two polypeptides can lie against one another.
  • the three residues Gly-Pro-Gly provide an appropriate length for such a turn between the two structural elements provided by many such polypeptide moieties of a fusion protein.
  • Glycine has a high degree of conformational flexibility and thus allows the two structural elements which are joined to interact in an optimal way.
  • the rigid, kink-forming residue proline has high propensity to form turns.
  • the Gly-Pro-Gly structure forms turns with rotational flexibility at the ends.
  • both glycine and proline have small sidechains and are less likely to cause packing problems between the structural elements of the two polypeptide moieties.
  • Gly-Pro-Gly is particularly preferred as a linker and is utilized in the HSOD-A+ fusion protein
  • Bioly active polypeptide moieties for use in a fusion protein of this invention can be derived from any number of proteins of known primary amino acid residue sequence that provide therapeutic
  • At least four classes of proteins of known structure have potential therapeutic applications that can benefit from having glycosaminoglycan-binding properties either to
  • proteins include the following: 1) serine proteases; 2) protease inhibitors including serine protease inhibitors, which are also called serpins; 3) antioxidant enzymes; and 4) receptors and immunoglobulins.
  • Tissue-plasminogen activator (tPA), urokinase, and single-chain urokinase-like plasminogen activator (scuPA) [Haber et al., Science, 243:51-56 (1989)] are representative proteases in the serine protease class of proteins. These proteases are used for the
  • tPA and urokinase can benefit from the addition of a glycosaminoglycan-binding domain to their structure.
  • protease consists of a protease and a binding domain, the latter of which promotes binding to heparin.
  • the proteases Upon binding to heparin, however, the proteases naturally undergo a cleavage resulting in a separation of the protease domain from the binding domain.
  • tPa protease domain is more active in dissolving blood clots than the full-length form.
  • the tPA protease domain although more active, lacks heparin-binding capacity.
  • the construction of an expression vector in which a glycosaminoglycan-binding domain is incorporated into the serine protease domain of these molecules can correct this deficiency.
  • the resulting fusion protein can then have improved stability and clot targeting capacity compared to a tPA protease domain alone.
  • Protease inhibitors categorized as anti- proteases, include alpha-1-antitrypsin, acid-stable proteinase inhibitor and human secretory leukocyte protease inhibitor.
  • serpin is synonymous with serine protease inhibitors. The prototypical serine protease inhibitor or serpin is
  • alpha-1-antitrypsin This protein is the principal natural inhibitor of the protease, leukocyte elastase, which is known to cause major tissue damage in lung inflammatory diseases like emphysema. Elastase is also known to be closely associated with various glycosaminoglycans found in tissue. Travis et al., Am. J. Medicine, 84 (sup. 6A): 37-42 (1988). Heparan sulfate, a glycosaminoglycan, is a major component of the alveolar interstitial tissue where the protease damage occurs. Crystal et al., In: Pulmonary Diseases and Disorders. 2nd ed., Fishman, Ed., McGraw-Hill Book Company (1987). Alpha-1-antitrypsin does not
  • alpha-1-antitrypsin with a glycosaminoglycan-binding moiety should be more readily targeted to the site of action in the alveolar interstitial tissue and have an increased half-life.
  • SLPI human secretory leukocyte protease inhibitor
  • SLPI serine protease inhibitor in that it can reversibly inhibit a broad range of proteases involved in tissue damage during inflammation and also has a unique ability to gain access to proteolytic
  • SLPI sequestered microenvironments that are inaccessible to other fluid-phase inhibitors.
  • SLPI has a low molecular weight (11,700 D) , it is expected to be rapidly cleared by kidney filtration.
  • the addition of a glycosaminoglycan-binding peptide to SLPI can increase its half-life and can result in direct cell surface targeting.
  • antioxidants which function as anti-inflammatory agents.
  • the medically important antioxidant enzymes of known structures are superoxide dismutase, catalase and glutathione peroxidase. These enzymes are involved in the prevention of
  • immunoglobulins Targeting of immunoglobulins is normally an intrinsic function of these proteins. Antibodies to circulatory antigens and antibodies that have a catalytic function, however, can benefit from a heparin-binding moiety. Since immunoglobulins have a beta-barrel fold like that of SOD, they will behave like SOD upon fusion with a glycosaminoglycan-binding domain. Catalytic antibodies that are more stable and have enhanced tissue-targeting ability can provide a more efficient therapeutic agent. Alternatively, for industrial or purification processes, a
  • glycosaminoglycan-binding domain added either to a catalytic as well as a standard variable domain antibody or to a single-chain antibody can enable the antibody to be immobilized via the glycosaminoglycan- binding domain on a solid support such as a
  • heparin-sepharose column Recovery of the free catalyst or antibody can be easily accomplished by a gentle salt gradient elution as described for HSOD-A+.
  • many receptors are known to have an immunoglobulin-like structure.
  • the CD4 soluble receptor and especially the VI domain are contemplated as a potential drug against HIV. Ashkenazi et al., Proc. Natl. Acad. Sci., 87:7175-7154 (1990). These molecules can have enhanced tissue targeting and stability via glycosaminoglycan binding.
  • proteins can benefit from an increased circulatory half-life and tissue targeting through an attached glycosaminoglycan-binding function.
  • Solubilized domains from medically important receptors represent a major potential application.
  • transmembrane domain of the full-length molecule have been removed without affecting the activity.
  • glycosaminoglycan-binding peptide should not be
  • glycosaminoglycan-binding peptide can prove to be a general strategy for the surface targeting of soluble forms of receptors.
  • subtilisin carlsberg subtilisin carlsberg (subtilopeptidase *a)
  • subtilisin BPN* (e.c.3.4.21.14)
  • subtilisin carlsberg e.c.3.4.21.14
  • n-acetyl eglin-c 1sic subtilisin /bpn(prime)
  • streptomyces subtilisin inhibitor e.c.3.4.21.14
  • subtilisin novo e.c.3.4.21.14 complex with chymotrypsin inhibitor 2 (CI-2)
  • 3sgb proteinase b from streptomyces griseus
  • beta-trypsin orthorhombic at p*h5.0
  • beta-trypsin (e.c.3.4.21.4) complex with
  • alpha-lytic protease e.c.3.4.21.12
  • alpha chymotrypsin a tosylated (e.c.3.4.21.1)
  • 4cha alpha-chymotrypsin (e.c.3.4.21.1)
  • alpha chymotrypsin a (e.c.3.4.21.1)
  • alpha chymotrypsin a (e.c.3.4.21.1) complex
  • PEBA phenyethane boronic acid
  • alpha-chymotrypsin e.c.3.4.21.1
  • turkey ovomucoid third domain OMTKY3
  • subtilisin carlsberg e.c.3.4.21.14
  • hne human neutrophil elastase (hne) (e.c.3.4.21.37)
  • 1ntp modified beta trypsin (monoisopropylphosphoryl inhibited) (e.c.3.4.21.4) (neutron data) 1p01 : alpha-lytic protease (e.c.3.4.21.12) complex with boc-*ala-*pro-*valine boronic acid
  • alpha-lytic protease (e.c.3.4.21.12) complex with methoxysuccinyl-*ala-*ala-*pro-*alanine boronic acid
  • alpha-lytic protease (e.c.3.4.21.12) complex with methoxysuccinyl-*ala-*ala-*pro-*valine boronic acid
  • alpha-lytic protease (e.c.3.4.21.12) complex with methoxysuccinyl-*ala-*ala-*pro-*isoleucine boronic acid
  • alpha-lytic protease (e.c.3.4.21.12) complex with methoxysuccinyl-*ala-*ala-*pro-*norleucine boronic acid
  • trypsin orthorhombic, 2.4 m ammonium sulfate
  • 3ptn trypsin (trigonal, 2.4 m ammonium sulfate)
  • 3rp2 rat mast cell protease ii (rmcpii)
  • beta-trypsin (e.c.3.4.21.4) complex with
  • actinidin sulfhydryl proteinase
  • Acid Proteinases and Their Inhibitors 4ape acid proteinase (e.c.3.4.23.10), endothiapepsin 2app : acid proteinase (e.c.3.4.23.7),penicillopepsin 2apr : acid proteinase (rhizopuspepsm) (e.c.3.4.23.6) 3apr : acid proteinase (rhizopuspepsm) (e.c.3.4.23.6) complex with reduced peptide inhibitor
  • pepsin e.c.3.4.23.1
  • pepsin e.c.3.4.23.1
  • pepsin e.c.3.4.23.1
  • 2fb4 immunoglobulin fab 1fbj : ig*a fab fragment (j539) (galactan-binding) 1fc1 : fc fragment (iggl class)
  • antioxidant enzymes of the superoxide dismutase (SOD) class are particularly preferred.
  • cauliflower SOD [Steffens et al., Biol. Chem. Hoppe- Seyler, 367:1007-1016 (1986)]; cabbage SOD [Steffens et al., Physiol. Chem., 367:1007-1016 (1986)]; maize SOD [Cannon et al., Proc. Natl. Acad. Sci. USA,
  • SOD fusion proteins are particularly preferred and an exemplary embodiment using HSOD has been prepared in Example 6.
  • a SOD-containing-fusion protein is also referred to as a SOD-GAG-binding protein.
  • This embodiment, designated HS0D-A+ fusion protein has an amino acid residue sequence for the mature, expressed protein as shown in Figure 1 from residue 1 to residue 171.
  • linker sequences and GAG-binding moieties identified herein as preferred are also contemplated.
  • a fusion protein has a polypeptide sequence that corresponds, and preferably is identical, to the formula A+-L-HSOD, where A+ is at the amino terminus and HSOD is at the carboxy terminus, A+ corresponds to the PCI A+ GAG- binding helix having the formula HRHHPREMKKRVED, HSOD is a polypeptide having an amino acid residue sequence that corresponds, and preferably is identical, to the sequence in Figure 1 from residue 1 to residue 153, and L represents an operative linkage between A+ and HSOD in the form of either a peptide bond, i.e., no intervening amino acid residues, or one of the linker polypeptides YYK or SMD.
  • a fusion protein has a polypeptide sequence that corresponds, and preferably is identical, to the formula HSOD-L-PF4+, where HSOD is at the amino terminus and PF4+ is at the carboxy terminus, HSOD has a sequence as defined above, PF4+ is a GAG-binding helix having the formula YKKIIKKLLES, and L is either a peptide bond or is one of the linker polypeptides DEDG or IGVMP.
  • Another embodiment contemplates a fusion protein having a polypeptide sequence that corresponds, and preferably is identical, to the formula PF4+-L-HSOD, where PF4+ is at the amino terminus and HSOD is at the carboxy terminus, PF4+ is the polypeptide defined above, HSOD has a sequence as defined above, and L is one of the linker polypeptides REACG, VE or VMAS.
  • fusion proteins that contain a single GAG-binding moiety operatively linked to a single independently folding protein domain (i.e., a biologically active polypeptide moiety), where the GAG-binding moiety is linked at either its carboxy or amino terminus.
  • a fusion protein is also contemplated where more than one GAG-binding moiety is linked, for example, one at the carboxy and one at the amino terminus of the biologically active polypeptide moiety.
  • a fusion protein having a polypeptide sequence that corresponds, and preferably is identical, to the formula A+-L 1 -HSOD-L 2 - PF4+, where A+ is at the amino terminus and PF4+ is at the carboxy terminus, A+ corresponds to a polypeptide of the formula HRHHPREMKKRVED, PF4+ is the polypeptide as defined above, HSOD has a sequence as defined above L 1 is either a peptide bond or is one of the linker polypeptides YYKK or SMD and L 2 is either a peptide bond or is one of the linker polypeptides DEDG or IGVMP.
  • GAG-binding affinity is optimized by choosing linkers that encourage the GAG-binding helices to adopt the arrangement found in PF4 dimers (where each monomer contributes one helix) and in PCI; namely, two amphipathic, positively charged ⁇ -helices that lie roughly in a plane, that are aligned side-by-side, and that have parallel or anti-parallel axes separated by 10-14 angstroms.
  • the PF4 dimer helices have been superimposed by molecular graphics on the HSOD dimer, such that the two fold symmetry axes of the HSOD and PF4 dimers are coincident and the N-termini of the PF4 helices are as close to the C-termini of the HSOD monomers as
  • a linker of at least five residues in length is preferred.
  • the extra two to three residues for the PF4 linker relative to the A+ linker are required because the PF4 helix is one turn shorter than the A+ helix-containing peptide.
  • the fusion protein may contain two GAG-binding moieties, one attached at each end of the biologically active polypeptide.
  • a fusion protein can contain more than one GAG-binding moiety in tandem at a terminus of a biologically active polypeptide, for example, according to the general formula: -Y-L-Z n - or -Z n -L-Y-, where Y is a biologically active
  • L is a linking means
  • Z is a GAG-binding moiety
  • n is an integer of about 1-5, preferably about 2.
  • multiple GAG-binding moieties can be positioned according to the formula: -(Z-L) n -Y- or -Y-(L-Z) n -, where L, Z and Y are as defined above and n is an integer from 1 to 5, and preferably is 1, 2 or 3.
  • Y is HSOD, preferably
  • L is methionine (M)
  • n is 1, 2 or 3.
  • a GAG-binding protein comprises a polypeptide including the formula -Y-L-Z-, where Y and Z are amino acid residue sequences, L is a linking means, Y comprises a polypeptide having biological activity as described herein, and Z is a GAG-binding moiety according to the general formula described above, with the proviso that when L is -GPG-, Z is not -LWERQ-; and the proviso that when L is -PLY-, z is not -YKKII-.
  • the GAG-binding protein comprises a polypeptide including the formula -Z-L-Y-, where Z, L, and Y are as described above, with the provisio that when L is -HVG-, Z is not -RVEDL-.
  • Another embodiment contemplates inclusion of multiple GAG-binding moieties associated with a biologically active polypeptide moiety according to the formula -U b -(Z-L) a -Y- or -Y-(L-Z) a -U b - where Z, L ar?d Y are as defined before, U is an amino acid, a is an integer from 1 to 10, and b is an integer from 0 to 1.
  • fusion protein where L is methionine, Z is a polypeptide according to the sequence -RVPRESGKKRKRKRLKPS-, Y is a polypeptide having an amino acid residue sequence that corresponds to the sequence shown in Figure 1 from residue 1 to residue 171, z is 1, 2, or 3 and b is 1.
  • Fusion proteins comprising a preselected biologically active polypeptide moiety operatively linked to a glycosaminoglycan (GAG)-binding moiety are particularly useful due to the properties that the GAG-binding moiety imparts on the fusion protein.
  • GAG glycosaminoglycan
  • Therapeutic proteins administered to the blood are cleared from the blood. Addition of a GAG-binding moiety imparts a targeting function that directs the fusion protein to GAGs in the blood vessel wall and into the tissues rather than into the general
  • the targeting function takes the fusion protein away from free circulation, thereby increasing the fusion protein's effective half-life and
  • addition of a GAG- binding moiety imparts a means to more readily isolate a fusion protein from the expression medium in which it was synthesized or the fluid in which it is
  • the preparation of a fusion protein of this invention involves a combination of molecular
  • design considerations are resolved in the present invention by computer modeling methods that determine the regions of independently folding protein domains and particularly that design suitable linkers to combine two or more biologically active polypeptide moieties to form the fusion protein.
  • Example 1 A detailed description of the modeling of the protein C inhibitor is provided in Example 1. The methods generally involve a series of computer graphics and computer modeling manipulations based on the primary amino acid residue sequence of the
  • polypeptide to be modeled is exemplary of the methods used to solve protein structures in general.
  • glycosaminoglycan-binding fusion protein involves the following steps:
  • a GAG-binding moiety is selected according to the formula presented earlier.
  • a biologically active protein moiety is at least an independently folding protein domain that contains by its structure an identifiable biological activity, when assayed by standard biochemical methods for the presence of the identifiable biological assay.
  • a biologically active protein moiety is a complete protein, although there is no requirement that the protein be complete. For example. Fab fragments of immunoglobulins, or the single chain antigen binding protein described by Bird et al.
  • polypeptides The final amino acid residue sequence of the fusion protein is defined by the sum of the three parts, namely first polypeptide, linker and second polypeptide operatively linked into a single polypeptide.
  • Modeling a polypeptide can be accomplished by a variety of methods. Preferred are the homology modeling
  • SEARCHWILD scans a database of protein sequences for all occurrences of a specified sequence pattern. This pattern may include "linker" sequences (of a specified range of lengths) for which no sequence preference is specified. SEARCHWILD can be used to identify sequences forming natural (and thus
  • SEARCHWILD will identify all sequences of the protein structural database that are similar to the C- and N-terminal sequences separated by a linker of 0 or more residues. In doing so, SEARCHWILD successfully identifies linkers that provide favorable structures for linking structural units in a fusion protein.
  • An exemplary and preferred protein structure database is the Protein Data Bank available from Brookhaven National
  • SEARCHWILD is attached hereto as Appendix 1 to provide detailed description of the logic for completing a SEARCHWILD computer analysis.
  • SEARCHWILD can be run on any computer using a
  • UNIX operating system such as a SUN SPARCstation 1 or SLC, a SUN 3 or 4, a Convex 1 or 240, or a Stardent GS 1000 or Titan.
  • the executable SEARCHWILD code (the compiled and linked code in Appendix 1) is run on a (Unix operating system) computer by typing the
  • the command line includes symbols which mean the following: "pdbsearchwild" invokes the program
  • Execution of the described command initiates the program that passes parameters into SEARCHWILD, sorts the matches found in the sequence database (for example, the sequences corresponding to structural coordinates in the Protein Data Bank (PDB)), and lists the sequence matches found by the search in order from most similar to least similar to the input sequences. On each line containing a c-terminal and N-terminal sequence match is the sequence of the identified linker between them.
  • sequence database for example, the sequences corresponding to structural coordinates in the Protein Data Bank (PDB)
  • the SEARCHWILD parameters required at the command line include certain default values which are
  • the first parameter is the carboxy-terminal 7 residues of the polypeptide to precede the linker.
  • the second parameter is the amino-terminal 7 residues of the polypeptide to follow the linker.
  • the third parameter identifies the minimum linker length (in residues) between the two polypeptides to be linked, with a minimum value of zero, and is referred to as "minlinkerlen” in pdbsearchwild.
  • the fourth parameter is the maximum linker length between the two polypeptide regions, is specified as 7 residues and is referred to as "maxlinkerlen” in pdsearchwild.
  • the choice of 7 residues for the lengths of the amino and carboxy termini and for the linker length in the described SEARCHWILD program was made because 7 residues is sufficient to form any of the preferred types of protein structure for a linker in the present invention, namely reverse turns, helical turns, and open turns or loops having internal hydrogen bonds.
  • the fifth parameter in SEARCHWILD is used to measure the similarity between the input sequence and the database sequence, and gives a value for each substitution of one residue type for another. Higher matrix values indicate more similar residues.
  • the preferred matrix best.matrix (E.D. Getzoff and J.A. Tainer), is a weighted combination of 7 individual matrices
  • the last parameter, matrix tolerance, is a value equaling the smallest value in the amino acid
  • substitution matrix for a substitution of one residue by another
  • this is set to some value greater than the smallest value in the matrix (to prevent all sequences in the database from being printed out with their scores, since clearly most sequences are not similar) and less than the value at which statistically significant scores are produced (as described below; thus at least all the significant matches will be printed out).
  • matrix tolerance is a residue-selection criterion. This parameter is referred to as "mat_tol" in pdbsearchwild, and an appropriate value is zero for most choices of input sequence when using best.matrix.
  • sequence database file to be searched by SEARCHWILD referred to as "pdbseq.asc"
  • PDB Brookhaven Protein Data Bank
  • amino acid equivalence matrices can be used with SEARCHWILD in place of best.matrix described herein, so long as the matrix provides for residue substitutions.
  • Typical factors involved in designing a rational substitution matrix include the following: hydrophobicity, evolutionary occurrence, sidechain charge and polarity, turn, strand or helix preference characteristics, size and the like.
  • Schirmer (supra) methodology is applied to best.matrix rather than the matrix of relative substitution frequencies described by Schulz and Schirmer (since the level of statistical significance depends on the values in the substitution matrix).
  • This methodology determines the mean and standard deviation of the distribution of scores for the sequence matches produced by searchwild.
  • a best.matrix score greater than three standard deviations above the mean score shows significant relatedness at a confidence level of more than 99.7%. This is a restrictive criteria since it gives a frequency of 0.005 for all 5-residue peptides and 0.0014 for all 13-residue peptides occurring in 2222 known protein sequences.
  • matchextractpdb (incorporating pdbresrange and pdbchain programs), extracts from the protein database (PDB) the three- dimensional coordinates of the linker residues
  • the selected sequence represents a potential linker sequence that must be evaluated by structural appropriateness criteria in order to be positively selected for use as a linker in a fusion protein.
  • the identified linkers are evaluated for structural appropriateness of the identified sequence in the context of the two polypeptide moieties to be linked.
  • SEARCHWILD linker sequences identified by SEARCHWILD have structures that are highly dependent on adjacent structures (an undesirable feature)
  • packing and hydrogen bonding within the linker structure in the PDB are evaluated using the tiny probe program of E.D. Getzoff (Chapter 8, Ph.D. Thesis, Duke University, 1982).
  • Preferred structures for linker residues to be included in a fusion protein of present invention are reverse turns, open turns, helical turns, and short loops having local hydrogen bonds and packing
  • linkers are selected in which the linker structure generates a favorable globular fold between the protein and the GAG-binding moiety as measured by: 1) exposing the GAG-binding sidechains at the solvent- accessible surface of the fusion protein; 2) producing buried surface (as measured by MS with a 1.4 ⁇ probe) between the protein and the GAG-binding moiety without producing undue cavities" or interpenetrations; and 3) absence of steric collisions that cannot be resolved by single bond rotations.
  • polypeptide moiety to a glycosaminoglycan-binding moiety as follows:
  • Representative modeling methods for obtaining a structural model include the homology modeling approach described by Summers et al. rj. Mol. Biol., 210:785-811 (1989)], and the related approach exemplified herein at Example 1.
  • FIG. 4 A system of the present invention for identifying linker sequences is shown in Figure 4.
  • the system comprises an input device 11 such as a keyboard for entering commands and data, a ROM or RAM (read-only- memory or random access memory) 13 with a stored program (SEARCHWILD), a computer processor 15
  • an input device 11 such as a keyboard for entering commands and data
  • ROM or RAM read-only- memory or random access memory
  • SEARCHWILD stored program
  • RAM random-access-memory
  • auxiliary storage device 17 for storing entered data and predetermined sequence data.
  • the system may include a CRT
  • the invention also contemplates a method of determining an amino acid residue sequence suitable for linking selected molecules, the method comprising the steps of:
  • the invention contemplates a system for determining an amino-acid residue sequence
  • amino acid residue sequence of a protein or polypeptide is directly related via the genetic code.to the deoxyribonucleic acid (DNA) sequence of the structural gene that codes for the protein.
  • DNA deoxyribonucleic acid
  • a structural gene can be defined in terms of the amino acid residue sequence, i.e., protein or polypeptide, for which it codes.
  • a DNA sequence (i.e., DNA segment) of the present invention comprises a structural gene. Usually, the DNA sequence is present as an uninterrupted linear series of codons where each codon codes for an amino acid residue, i.e., the DNA sequence contains no introns.
  • any desired target fragment such as a nucleic acid having an intervening sequence, a promoter, a
  • a DNA segment of this invention defines a
  • the DNA segment includes a nucleotide base sequence according to the sequence in Figure 1 from nucleotide base 535 to base 579.
  • the DNA segment is no more than about 5,000 and preferably no more than 2,500 nucleotides (bases) in length.
  • a DNA segment of the present invention can easily be synthesized by chemical techniques, for example, via the phosphotriester method of Matteucci et al. [J. Am. Chem. Soc., 103:3185 (1981)] or using
  • duplex DNA molecules typically are duplex DNA molecules having cohesive termini, i.e., "overhanging" single-stranded portions that extend beyond the double-stranded portion of the molecule.
  • cohesive termini i.e., "overhanging" single-stranded portions that extend beyond the double-stranded portion of the molecule.
  • the presence of cohesive termini on the DNA molecules of the present invention is generally preferred.
  • oligonucleotides in the form of a "cassette", i.e., having convenient restriction enzyme site-defined cohesive termini, can easily be prepared by ligating smaller oligonucleotides.
  • single-stranded oligonucleotides of between 40-75 nucleotide bases in length are prepared with
  • ds DNA double stranded
  • RNA ribonucleic acid
  • the present invention further contemplates a recombinant DNA (rDNA) that includes a DNA segment of the present invention operatively linked to a vector for replication and/or expression.
  • rDNA recombinant DNA
  • a preferred rDNA is characterized as being capable of directly
  • expressing in a compatible host, a GAG-binding fusion protein of the present invention.
  • directly expressing is meant that the mature polypeptide chain of the expressed fusion protein is formed by
  • An exemplary and preferred rDNA of the present invention is the rDNA molecule pPHSODI q HPCI4 described in Example 6.
  • a rDNA molecule of the present invention can be produced by operatively linking a vector to a DNA segment of the present invention.
  • vector refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are operatively linked are referred to herein as
  • expression vector and can be expressed in a suitable host cell.
  • GAG-binding fusion protein encoding DNA segment of the present invention is operatively linked depends upon the functional properties desired, e.g., protein expression, and upon the host cell to be transformed. These limitations are inherent in the art of constructing recombinant DNA molecules.
  • invention is at least capable of directing the
  • replication and preferably also expression, of a gene operatively linked to the vector.
  • a vector contemplated by the present invention includes a procaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recomb
  • Typical bacterial drug resistance genes are those that confer resistance to ampicillin, tetracycline, or kanamycin.
  • Those vectors that include a procaryotic replicon may also include a procaryotic promoter capable of directing the expression (transcription and
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Bacterial expression systems, and choice and use of vectors in those systems is described in detail in "Gene Expression Technology", [Meth.
  • Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Bio-Rad
  • Expression vectors compatible with eucaryotic cells can also be used to form the recombinant DNA molecules of the present invention.
  • Eucaryotic cell expression vectors are well-known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired gene. Typical of such vectors are pSVL and pKSV-10
  • the eucaryotic cell expression vectors used to construct the recombinant DNA molecules of the present invention include a selectable phenotypic marker that is effective in a eucaryotic cell, such as a drug resistance selection marker or selective marker based on nutrient
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene.
  • retroviral expression vector refers to a DNA molecule that includes a promoter sequence derived from the long terminal repeat (LTR) region of a retrovirus genome.
  • the expression vector is typically a retroviral expression vector that is preferably replication-incompetent in eucaryotic cells.
  • retroviral vectors The construction and use of retroviral vectors has been described by Sorge et al., Mol. Cell. Biol., 4:1730-37 (1984).
  • virus-based expression systems can be used, as is well-known, including systems based on SV-40, Epstein-Barr, Vaccinia, and the like. See, for example, "Gene Expression Technology", (Supra), at pp.485-569.
  • yeast a variety of vector are known in the art, in particular the vector, pCl/1 described by Brake et al., Proc. Natl. Acad.
  • the ribosome-binding site in E. coli includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotides upstream from the initiation codon (the Shine-Dalgarno sequence). See, Shine et al., Nature, 254:34 (1975). Methods for including a ribosome- binding site in mRNAs corresponding to the expressed proteins are described by Maniatis, et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY pp. 412-417 (1982). Ribosome binding sites can be modified to produce optimum configuration relative to the structural gene for maximal expression of the structural gene. [Hallewell et al., Nucl. Acid Res., 13:2017-2034 (1985)].
  • the vectors employed herein will contain restriction sites in all three reading frames of the DNA sequences.
  • other vectors will be suitable in which synthetic linkers are inserted to allow the fusion protein gene to be inserted in-frame. Synthetic linkers containing a variety of restriction sites are commercially available from a number of sources including
  • RNA sequences including the removable fragments and/or the linking sequences may also be prepared by direct synthesis techniques. Also contemplated by the present invention are RNA
  • the nucleic acids are combined with linear DNA molecules in an admixture thereof and a ligase will be added to effect ligation of the components.
  • a ligase Any ligase available commercially is contemplated to perform the ligation reaction effectively using methods and conditions well-known to those skilled in the art.
  • a preferred ligase is T4 DNA ligase.
  • Volume exclusion agents may also be used to accelerate the ligation reaction. However, such agents may cause excessive intramolecular
  • the recombinant DNA molecules of the present invention are introduced into host cells via a
  • the host cell can be either procaryotic or eucaryotic. Bacterial cells are preferred
  • procaryotic host cells typically are a strain of E. coli such as, for example, the MC1061 or JM109 strains.
  • Preferred eucaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
  • Preferred eucaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61 and NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658.
  • One preferred means of effecting transformation is electroporation.
  • Transformation of appropriate host cells with a recombinant DNA molecule of the present invention is accomplished by well-known methods that typically depend on the type of vector used. With regard to transformation of procaryotic host cells, see, for example, Cohen et al. [Proc. Natl. Acad. Sci. USA, 69:2110 (1972)] and Maniatis et al. [Molecular
  • rDNA recombinant DNA
  • cells resulting from the introduction of an rDNA of the present invention can be cloned to produce monoclonal colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J. Mol. Biol., 98:503 (1975) or Berent et al., Biotech., 3:208 (1985).
  • expression vector produce a polypeptide displaying a characteristic antigenicity.
  • Samples of a culture containing cells suspected of being transformed are harvested and assayed for a subject polypeptide using antibodies specific for that polypeptide antigen, such as those produced by an appropriate hybridoma.
  • telomere sequence a suitable plasmid, e.g., pLG. Since the plasmid lacks a promoter and the Shine-Dalgarno sequence, no ⁇ -galactosidase is synthesized. However, when a portable promoter fragment is properly
  • plasmids are used to construct a fusion protein having ⁇ -galactosidase activity.
  • Plasmids having optimally placed promoter fragments are thereby recognized. These plasmids can then be used to reconstitute the fusion protein gene which is expressed at high levels.
  • cultures of the cells are contemplated as within the present invention.
  • the cultures include monoclonal (clonally homogeneous) cultures, or
  • a "serum-free" medium is preferably used.
  • the present method entails culturing a nutrient medium containing host cells transformed with a recombinant DNA molecule of the present invention that is capable of expressing a gene encoding a subject polypeptide.
  • the culture is maintained for a time period sufficient for the transformed cells to express the subject polypeptide.
  • the expressed polypeptide is then recovered from the culture.
  • the plasmid selected will have additional cloning sites which allow one to score for insertion of the gene assembly. See,
  • Bacterial cultures transformed with the plasmids are grown for a few hours to increase plasmid copy number, e.g., to more than 1000 copies per cell.
  • Induction may be performed in some cases by elevated temperature and in other cases by addition of an inactivating agent to a represser. Very large increases in cloned fusion proteins can potentially be obtained in this way.
  • Methods for recovering an expressed polypeptide from a culture include fractionation of the polypeptide-containing portion of the culture using well-known biochemical techniques. For instance, the methods of gel filtration, gel chromatography, ultrafiltration, electrophoresis, ion exchange, affinity chromatography, and the like, can be used to isolate the expressed proteins found in the culture. In addition, immunochemical methods, such as immunoaffinity, immunoabsorption, and the like, can be performed using well-known methods.
  • a preferred method for isolating a fusion protein in this invention is by affinity chromatography.
  • Isolation and purification of an expressed fusion protein containing a GAG-binding domain can be
  • a preferred affinity chromatography column in this invention is heparin immobilized to Affi-gel as shown in Example 7. After the lysate is applied to the column, the GAG- binding domain of the fusion protein binds to the heparin. After washing the column to remove non-bound proteins, the fusion protein can be specifically eluted with an increasing ionic strength salt
  • fractions containing the purified fusion protein are collected and tested for activity in an appropriate assay, preferably in a gel activity assay.
  • the fractions containing the highest activity of the fusion proteins are thereafter pooled.
  • Affinity chromatography purification of fusion proteins by these means can result in greater than 95% purity.
  • micromolar means microliter
  • ug means microgram
  • compositions of the present invention contain a physiologically tolerable carrier together with a GAG-binding fusion protein, as described herein, dissolved or dispersed therein as an active ingredient.
  • a physiologically tolerable carrier together with a GAG-binding fusion protein, as described herein, dissolved or dispersed therein as an active ingredient.
  • therapeutic composition is not immunogenic when administered to a mammal or human patient for
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • compositions are prepared as
  • injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or
  • suspensions in liquid prior to use can also be prepared.
  • the preparation can also be emulsified.
  • the active ingredient can be mixed with
  • excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations
  • composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • the therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts
  • salts formed with the free carboxy1 groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as
  • Physiologically tolerable carriers are well-known in the art.
  • Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are
  • glycerin vegetable oils such as cottonseed oil, and water-oil emulsions.
  • Methods for reducing tissue damage caused by oxygen free radical (superoxide) in vivo or in vitro are contemplated by the present invention, using a HSOD-GAG-binding fusion protein.
  • SOD SOD-oxide anion
  • tendonitis tendovaginitis, bursitis, epicondylitis, periarthritis
  • tendonitis tendovaginitis, bursitis, epicondylitis, periarthritis
  • tissue-targeted SOD should help alleviate the toxic secondary effect of anti- cancer radio and chemotherapy.
  • Drug (antibiotic and anticancer) induced nephritis also can be reduced by a more potent SOD.
  • the present invention contemplates a method of in vivo scavenging superoxide radicals in a mammal that comprises administering a therapeutically effective amount of a physiologically tolerable composition containing a ⁇ SOD-GAG-binding fusion protein to a mammal in a predetermined amount calculated to achieve the desired effect.
  • the HSOD-GAG- binding fusion protein is administered in an amount sufficient to deliver 1 to 50 milligrams (mg),
  • a preferred dosage can alternatively be stated as an amount sufficient to achieve a plasma concentration of from about 0.1 ug/ml to about 100 ug/ml, preferably from about 1.0 ug/ml to about 50 ug/ml, more preferably at least about 2 ug/ml and usually 5 to 10 ug/ml.
  • GAG-binding fusion proteins having superoxide dismutase (SOD) activity for use in a therapeutic composition typically have about 200 to 5000 units (U) of enzyme activity per mg of protein.
  • Enzyme assays for SOD activity are well-known, and a preferred assay to standardize the SOD activity in a fusion protein is that described by McCord et al., J.Biol.Chem.,
  • a dosage of about 1 to 20 mg, preferably about 4 to 8 mg is administered intra-articularly per week per human adult. In certain cases, as much as 20 mg can be administered per kilogram (kg) of patient body weight.
  • a dosage of 5 mg per kg of body weight is preferred to be administered intravenously.
  • the therapeutic compositions containing a GAG- binding fusion protein are conventionally administered intravenously, or intra-articularly (ia) in the case of arthritis, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as a unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in
  • diluent i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient
  • Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration.
  • PCI plasma serpin protein C inhibitor
  • PCI residues 20-391 ( ⁇ 1 AT numbering) was built by sidechain substitution with the molecular editor Moledt (Biosym Technologies, Inc.), using the x-ray structure of ⁇ 1 AT available in the Brookhaven Protein Data Bank [Bernstein et al., J. Mol. Biol., 112:535-42 (1977)]; entry 6API as a template and following the original sidechain torsion angles. Sidechain collisions were corrected using new torsion angles from a rotamer library [Ponder et al., J. Mol. Biol., 193:775-91 (1987)], and small
  • the peptide bond between residue 19 (the C- terminus of the amino-terminal segment) and residue 20 (the N-terminus of residues 20-391 of PCI) was made by optimally orienting the N-terminal segment, then making minimal changes in the backbone torsion angles of residues 19 and 20 and their nearest neighbors (using Moledt) in order to align the carbonyl C of residue 19 with the amino N of residue 20.
  • Plausible models of PCI including the N-terminus were energy- minimized (using the methods described above) to alleviate unfavorable residue contacts and to improve the conformations of the residue 19-20 turn and the N- terminal segment.
  • sequences of surface ⁇ -helices are often amphipathic with a period of ⁇ 3.6 residues.
  • glycosaminoglycans GAGs
  • Electrostatic potentials were calculated at all points on the solvent-accessible surfaces of the energy-minimized PCI models using programs ESPOT and ESSURF [Getzoff et al., Nature, 306:287-90 (1983);
  • PCI exhibited an overall electrostatic dipole, with a highly positive region including the H helix opposed by a weakly negative region centered on Asp 121.
  • electrostatic potential surfaces In the electrostatic potential surfaces
  • the models including the A+ helix had a single, highly positive ( ⁇ 3 kcal mol -1 ) surface region centered on Arg 10 and Lys 274, which protrude from the A+ and H helices.
  • Other central positive residues were Lys 14 and Lys 270 in model I and Arg 6, Lys 277 and Lys 280 in model II.
  • the positive region in both models formed a single face of the protein, has an area (1365 ⁇ 2 in model I and 1705 ⁇ 2 in model II) consistent with other protein interfaces [Janin et al., J. Mol.
  • Residual APC activity was determined by the rate of change in absorbance at 405 nm, compared to controls without added PCI. Pseudo- first order rate constants were calculated from initial slopes in plots of the natural log (In) of APC activity versus time and k 2 values were obtained based on the concentration of PCI and are shown in Table 3.
  • PCI (4 ⁇ g) was incubated for 60 minutes at 22C with the anti-PCI antibodies API39 (48 ⁇ g), API60 (48 ⁇ g), or buffer, in 200 ⁇ l of 0.01 M Tris and 0.14 M NaCl at pH 7.4. The sample was adjusted to 0.1 M NaCl in a final volume of 400 ⁇ l and loaded onto a 0.6 ml column of heparin- agarose (Sigma). Using an FPLC liquid chromatography gradient programmer (Pharmacia), PCI was eluted (0.1 ml fractions) with a linear gradient from 0.1 to 0.6 M NaCl. The elution profiles were determined using an ELISA assay for PCI antigen as described by Espafia et al., Thromb. Res. 55:671-82 (1989).
  • an anti-PCI monoclonal antibody neutralizes heparin stimulation of APC inhibition by PCI [Meijers et al. Blood, 72:1401-3 (1988)], and by ELISA and peptide competition assays binds specifically a peptide corresponding to the A+ helix.
  • Antibody API39 prevented PCI from binding to a heparin-agarose column.
  • a control antibody that binds to PCI but not to peptides from the A+ or H helix regions does not affect heparin stimulation nor prevent PCI from binding to a heparin-agarose column.
  • the strikingly positive helix pairs that forms the heparin recognition surface of PCI identified by the studies in Example 1-3 is similar to the twin helical motif thought to bind heparin in dimers of platelet factor 4, a nonhomologous protein whose structure has recently been determined. St. Charles et al., J. Biol. Chem., 264: 2092-2099 (1989).
  • GAG recognition in ATIII may be a variation on this common theme, involving positive residues in both the D helix [Carrell et al.. Thrombosis and Haemostasis 1987, Verstraete et al., eds., Leuven University, pp.1-15 (1987)], and the N-terminal region.
  • a fusion protein was constructed to contain the heparin binding region of PCI, namely the A+
  • S0D-A+ contains three subunits: a first region comprised of a polypeptide having the amino acid residue sequence of HSOD, a second region comprised of a polypeptide linker to connect the first and third regions, and a third region comprised of a polypeptide having the amino acid residue sequence of the A+ helix of PCI.
  • the amino acid residue sequence of SOD-A+ is shown in Figure 1, including the first SOD region defined by residues 1-153, the second linker region defined by residues 154-156, and the third A+ region defined by residues 157-171.
  • S0D-A+ was done in the pPHSODlacI vector from Chiron Corporation (Emeryville, CA). This vector contains the Sall-EcoRI fragment from pBR322, coding for the ⁇ -lactamase and the origin of replication.
  • the lad gene was
  • the HSOD protein encoded by the synthetic HSOD gene differs from wild type HSOD in that it contains alanine and serine in place of the cysteines at amino acid residue positions 6 and 111, respectively . All experiments were carried out using E. coli MC1061 (araD139, delta (araleu)7696, delta (lac) 174, galU, galK, hsdR, strA) [Huynh et al., DNA Cloning, vol.1.
  • the HSOD be produced in yeast to obtain amino terminal acetylation like wild type HSOD protein found in humans.
  • yeast expression system for HSOD is described in Hallewell et al., J. Biol. Chem., 264:5260-5268 (1989) and also in
  • Applied Biosystems DNA synthesizer model 380B To add the Gly-Pro-Gly linker and the A+-helix to the carboxy terminus of HSOD, two oligonucleotides corresponding to the HSOD, sequence from the BamHI site of the synthetic gene to the end of the amino acid coding sequence were designed.
  • the coding strand was
  • the complementary strand was extended by a glycine
  • GCC anticodon
  • the Xmal site in the linker sequence allows further modifications of the linker sequence if needed.
  • Oligonucleotide HUCLI corresponds to the sequence of oligonucleotides 488 to 523.
  • Oligonucleotide HUCLIZ is the complement of nucleotides 492 to 528.
  • Oligonucleotide PCIHEPBI corresponds to the nucleotides sequence 524 to 583.
  • Oligonucleotide PCIHEPBZ is the complement of
  • oligonucleotides where hybridized pair wise, HUCLI with HUCLIZ and PCIHEPBI with PCIHEPBZ, 10 ⁇ g of each in 100 ml water for 1 minute at 90C followed by cooling down to room temperature for 5 minutes.
  • the hybridized oligos, HUCLI with HUCLIZ and PCIHEPBI with PCIHEPBZ, were ligated with T4 DNA ligase (New England Biolabs) according to the manufacturer's instructions.
  • T4 DNA ligase New England Biolabs
  • the resulting BamHI-Sall cassette was substituted for the BamHI-SalI fragment of the HSOD synthetic gene.
  • the BamHI-Sall cassette was ligated into the
  • Clones having a larger insert after Ncol-Sall digestion and including a sequence shown in Figure 1 from nucleotide base 1 to base 588 were selected and designated as containing the plasmid pPHSODI q HPCI4.
  • the plasmid pPHSODI q HPCI4 has been deposited with the American Type Culture Collection (ATCC; Bethesda, MD) in the form of a transformed E. coli containing the plasmid on November 1, 1990, by the depositor Chiron Corporation (Emeryville, CA) and has been assigned a deposit accession number that is available from the ATCC.
  • ATCC American Type Culture Collection
  • Chiron Corporation Emeryville, CA
  • Alternate expression vectors capable of producing HSOD-A+ fusion protein can be prepared from the deposited plasmid material using methodologies well- known. For general methods of molecular biology, see “Gene Expression Technologies” in Meth. Enzymol.
  • An exemplary alternate expression system can be prepared as follows.
  • the approximately 30 base pair (bp) Ncol-Pstl polylinker is first isolated from the pPROK-1 vector available from Clontech Laboratories (Palo Alto, CA).
  • the SalI site of pKK233-2 available from Clontech is disabled by first digesting pKK233-2 with SalI, filling in the cohesive SalI termini, then religating the resulting biunt ends to form a circular pKK233-2 plasmid with a disabled Sall.
  • pKK233-2 is digested with Ncol and Pstl, and the 30 bp Ncol-Pstl polylinker is ligated into pKK233-2 to provide a
  • Ncol-Sall site Deposited pPHSODI q HPCI4 is digested with Ncol and Sall to remove the HSOD-A+ fusion protein encoding gene cassette, and the cassette is inserted into the Ncol and SalI site of the above- modified pKK233-2 vector. Thereafter, the pKK233-2 vector having the HS0D-A+ protein encoding gene can be introduced into a suitable lacl q strain of E. coli
  • IPTG isopropylthio- ⁇ -D-galactoside
  • the periplasmic fraction of the bacterial cells was extracted by a modification of the osmotic shock procedure of Koshland et al., Cell, 20:749-760 (1980). The cells were centrifuged down into two one liter bottles (3.5k rpm for 15 minutes in a Beckman J-6B centrifuge maintained at 4C). Each pellet was
  • the periplasmic fraction was estimated to contain 5 mg per ml of HS0D-A+ as determined by coomassie blue staining of SDS-polyacrylamide gel [Laemmli, UK,
  • HSOD-A+ was then further isolated from the periplasmic fraction first purified by heparin
  • periplasmic fraction was loaded onto a 40 ml Affi-Gel heparin column.
  • the column was eluted at a flow rate of 1 ml per minute with 200 ml of a 0.2 M Tris pH 7.0 buffer generating a linear gradient from 0.03 M to 0.4 M NaCl.
  • Fractions of 5 ml were collected and tested by SDS-polyacrylamide gel electrophoresis.
  • HSOD-A+ eluted in fractions number 18 to 28 corresponding to elution buffer containing around 0.2 M salt. After that purification step, HSOD-A+ was estimated to be more than 95% pure and fully active based on the above gel activity assay.
  • heparin binding property of HSOD-A+ was demonstrated in vitro by using a heparin binding assay that measures retention of HSOD-A+ on the heparin column described above.
  • co- elution was conducted and compared using equivalent amounts of crude HS0D-A+ and of recombinant purified HSOD made in yeast [Hallewell et al., Biotechnology, 5:363-366 (1987)].
  • the HSOD was all eluted before the gradient reached 0.1 M salt while SOD-A+ eluted at about 0.2 M, indicating that the addition of a GAG- binding moiety to HSOD significantly increased the GAG-binding capacity of the SOD-A+ fusion protein.
  • mice were injected with 2 mg of HSOD-A+ or with recombinant HSOD [Hallewell et al., Biotech., 5:363- 366 (1987)] for control.
  • the proteins were
  • the half-life was estimated by the SOD gel activity assay as described above.
  • the recombinant HSOD have a half-life of less than 13 minutes, most likely between 7 and 10 minutes.
  • the HSOD-A+ half-life can be estimated to be around 15 minutes.

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Abstract

L'invention décrit des procédés de conception et de construction de protéines de fusion, c'est-à-dire des protéines comprenant au moins deux unités structurelles distinctes, dont chacune produit la fonctionnalité désirée. Selon un aspect préféré de l'invention, on conçoit et on exprime des molécules d'ADN recombiné codant des protéines possédant des polypeptides fusionnés de liaison au glycosaminoglycane et de dismutase de superoxyde (SOD). Les protéines de fusion obtenues conservent les activités du polypeptide de liaison au glycosaminoglycane (GAG) et des enzymes de dismutase de superoxyde (SOD). La durée de SOD intracellulaire humain dans le flux sanguin peut se prolonger par la fixation du groupe de liaison au glycosaminoglycane et cette dernière fonction cible également l'enzyme sur des surfaces cellulaires.
PCT/US1991/008105 1990-11-01 1991-11-01 Proteines de fusion ciblees par clycosaminoglycane, leurs conception, construction et compositions WO1992007935A1 (fr)

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EP0723398A1 (fr) * 1993-10-15 1996-07-31 Duke University Superoxyde-dismutase et ses mimetiques
US5866402A (en) * 1995-05-05 1999-02-02 Chiron Corporation Chimeric MCP and DAF proteins with cell surface localizing domain
WO1999023215A2 (fr) * 1997-10-31 1999-05-14 University Of Florida Matieres et procedes permettant de prevenir les atteintes cellulaires chez l'homme et l'animal
US5994339A (en) * 1993-10-15 1999-11-30 University Of Alabama At Birmingham Research Foundation Oxidant scavengers
US6103714A (en) * 1994-09-20 2000-08-15 Duke University Oxidoreductase activity of manganic porphyrins
EP1158046A1 (fr) * 1994-04-11 2001-11-28 Human Genome Sciences, Inc. Superoxide Dismutase-4
US6479477B1 (en) 1998-04-24 2002-11-12 Duke University Substituted porphyrins
US6544975B1 (en) 1999-01-25 2003-04-08 National Jewish Medical And Research Center Substituted porphyrins
US6583132B1 (en) 1993-10-15 2003-06-24 Duke University Oxidant scavengers
EP1456239A2 (fr) * 2001-07-31 2004-09-15 Wayne State University Proteines hybrides a domaine se liant a l'heparine de neureguline pour cibler des proteoglycanes de sulfate d'heparane
WO2005054285A1 (fr) * 2003-12-04 2005-06-16 Protaffin Biotechnologie Ag Proteines de liaison aux gag
US6916799B2 (en) 1997-11-03 2005-07-12 Duke University Substituted porphyrins
WO2007038942A1 (fr) * 2005-09-21 2007-04-12 7Tm Pharma A/S Agonistes selectifs du recepteur y4 pour applications therapeutiques
WO2007038943A1 (fr) * 2005-09-21 2007-04-12 7Tm Pharma A/S Agonistes selectifs du recepteur y2 pour applications therapeutiques
US7485721B2 (en) 2002-06-07 2009-02-03 Duke University Substituted porphyrins
WO2010071190A1 (fr) * 2008-12-19 2010-06-24 国立大学法人 新潟大学 Érythropoïétine à affinité pour l'héparine
US8470808B2 (en) 1999-01-25 2013-06-25 Jon D. Piganelli Oxidant scavengers for treatment of type I diabetes or type II diabetes
WO2021014220A1 (fr) * 2019-07-23 2021-01-28 Csts Health Care Inc. Administration médiée par les plaquettes de composés thérapeutiques
CN114703154A (zh) * 2022-03-30 2022-07-05 云南大学 一种多肽、含有其的蛋白及应用
US11382895B2 (en) 2008-05-23 2022-07-12 National Jewish Health Methods for treating injury associated with exposure to an alkylating species

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US5994339A (en) * 1993-10-15 1999-11-30 University Of Alabama At Birmingham Research Foundation Oxidant scavengers
EP0723398A1 (fr) * 1993-10-15 1996-07-31 Duke University Superoxyde-dismutase et ses mimetiques
US6583132B1 (en) 1993-10-15 2003-06-24 Duke University Oxidant scavengers
EP1158046A1 (fr) * 1994-04-11 2001-11-28 Human Genome Sciences, Inc. Superoxide Dismutase-4
US6635252B2 (en) 1994-04-11 2003-10-21 Human Genome Sciences, Inc. Antibodies to superoxide dismutase-4
US6103714A (en) * 1994-09-20 2000-08-15 Duke University Oxidoreductase activity of manganic porphyrins
US5866402A (en) * 1995-05-05 1999-02-02 Chiron Corporation Chimeric MCP and DAF proteins with cell surface localizing domain
WO1999023215A2 (fr) * 1997-10-31 1999-05-14 University Of Florida Matieres et procedes permettant de prevenir les atteintes cellulaires chez l'homme et l'animal
WO1999023215A3 (fr) * 1997-10-31 1999-07-15 Univ Florida Matieres et procedes permettant de prevenir les atteintes cellulaires chez l'homme et l'animal
US6916799B2 (en) 1997-11-03 2005-07-12 Duke University Substituted porphyrins
US6479477B1 (en) 1998-04-24 2002-11-12 Duke University Substituted porphyrins
US8546562B2 (en) 1999-01-25 2013-10-01 James D. Crapo Substituted porphyrins
US9289434B2 (en) 1999-01-25 2016-03-22 Aeolus Sciences, Inc. Substituted porphyrins
US8946202B2 (en) 1999-01-25 2015-02-03 Aeolus Sciences, Inc. Substituted porphyrins
US7189707B2 (en) 1999-01-25 2007-03-13 National Jewish Medical Research Center Substituted porphyrins
US8470808B2 (en) 1999-01-25 2013-06-25 Jon D. Piganelli Oxidant scavengers for treatment of type I diabetes or type II diabetes
US8217026B2 (en) 1999-01-25 2012-07-10 Aeolus Sciences, Inc. Substituted porphyrins
US6544975B1 (en) 1999-01-25 2003-04-08 National Jewish Medical And Research Center Substituted porphyrins
US7820644B2 (en) 1999-01-25 2010-10-26 Aelous Pharmaceuticals, Inc. Substituted porphyrins
EP1456239A4 (fr) * 2001-07-31 2005-04-27 Univ Wayne State Proteines hybrides a domaine se liant a l'heparine de neureguline pour cibler des proteoglycanes de sulfate d'heparane
EP1456239A2 (fr) * 2001-07-31 2004-09-15 Wayne State University Proteines hybrides a domaine se liant a l'heparine de neureguline pour cibler des proteoglycanes de sulfate d'heparane
US7527794B2 (en) 2001-07-31 2009-05-05 Wayne State University Hybrid proteins with neuregulin heparin-binding domain for targeting to heparan sulfate proteoglycans
AU2002322762B2 (en) * 2001-07-31 2008-10-16 Wayne State University Hybrid proteins with neuregulin heparin-binding domain for targeting to heparan sulfate proteoglycans
US7485721B2 (en) 2002-06-07 2009-02-03 Duke University Substituted porphyrins
JP2007536906A (ja) * 2003-12-04 2007-12-20 プロタフィン・ビオテヒノロギー・アクチェンゲゼルシャフト Gag結合蛋白質
WO2005054285A1 (fr) * 2003-12-04 2005-06-16 Protaffin Biotechnologie Ag Proteines de liaison aux gag
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WO2007038942A1 (fr) * 2005-09-21 2007-04-12 7Tm Pharma A/S Agonistes selectifs du recepteur y4 pour applications therapeutiques
JP2009508885A (ja) * 2005-09-21 2009-03-05 7ティーエム ファーマ エイ/エス 治療的介入のためのy4選択性レセプターアゴニスト
US11382895B2 (en) 2008-05-23 2022-07-12 National Jewish Health Methods for treating injury associated with exposure to an alkylating species
JP5799409B2 (ja) * 2008-12-19 2015-10-28 国立大学法人 新潟大学 ヘパリン親和性エリスロポエチン
WO2010071190A1 (fr) * 2008-12-19 2010-06-24 国立大学法人 新潟大学 Érythropoïétine à affinité pour l'héparine
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