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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • 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
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  • C07K2319/00—Fusion polypeptide
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/50—Fusion polypeptide containing protease site

Definitions

• the present invention relates to fusion polypeptides where two individual polypeptides or parts thereof are fused to form a single amino acid chain. Such fusion may arise from the expression of a single continuous coding sequence formed by recombinant DNA techniques.
• Fusion polypeptides are known, for example those where a polypeptide which is the ultimately desired product of the process is expressed with an N-terminal "leader sequence" which encourages or allows secretion of the polypeptide from the cell.
• leader sequence An example is disclosed in EP-A-116 201 (Chiron) .
• HSA Human serum albumin
• EP-A-147 198 discloses its expression in a transformed host, in this case yeast.
• EP-A-322 094 discloses N-terminal fragments of HSA, namely those consisting of residues 1-n where n is 369 to 419, which have therapeutic utility.
• the application also mentions the possibility of fusing the C-terminal residue of such molecules to other, unnamed, polypeptides.
• One aspect of the present invention provides a fusion polypeptide comprising, as at least part of the N-terminal portion thereof, an N-terminal portion of HSA or a variant thereof and, as at least part of the C-terminal portion thereof, another polypeptide except that, when the said N- terminal portion of HSA is the 1-n portion where n is 369 to 419 or a variant thereof then the said polypeptide is (a) the 585 to 1578 portion of human fibronectin or a variant thereof, (b) the 1 to 368 portion of CD4 or a variant thereof, (c) platelet derived growth factor, or a variant thereof, (d) transforming growth factor, or a variant thereof, (e) the 1-261 portion of mature human plasma fibronectin or a variant thereof, (f) the 278-578 portion of mature human plasma fibronectin or a variant thereof, (g) the 1-272 portion of mature human von illebrand's Factor or a variant thereof, or (h) alpha-1-antitryp
• the N-terminal portion of HSA is preferably the said 1-n portion, the 1-177 portion (up to and including the cysteine), the 1-200 portion (up to but excluding the cysteine) or a portion intermediate 1-177 and 1-200.
• HSA human serum albumin
• variants are intended to include (but not necessarily to be restricted to) minor artificial variations in sequence (such as molecules lacking one or a few residues, having conservative substitutions or minor insertions of residues, or having minor variations of amino acid structure).
• polypeptides which have 80%, preferably 85%, 90%, 95% or 99%, homology with HSA are deemed to be "variants”.
• variants are physiologically equivalent to HSA; that is to say, variants preferably share at least one pharmacological utility with HSA.
• any putative variant which is to be used pharmacologically should be non-immunogenic in the animal (especially human) being treated.
• Conservative substitutions are those where one or more amino acids are substituted for others having similar properties such that one skilled in the art of polypeptide chemistry would expect at least the secondary structure, and preferably the tertiary structure, of the polypeptide to be substantially unchanged.
• typical such substitutions include asparagine for glutamine, serine for asparagine and arginine for lysine.
• Variants may alternatively, or as well, lack up to ten (preferably only one or two) intermediate amino acid residues (ie not at the termini of the said N-terminal portion of HSA) in comparison with the corresponding portion of natural HSA; preferably any such omissions occur in the 100 to 369 portion of the molecule (relative to mature HSA itself) (if present).
• amino acids may be added, again in the 100 to 369 portion for preference (if present).
• physiologically functional equivalents also encompasses larger molecules comprising the said sequence plus a further sequence at the N-terminal (for example, pro-HSA, pre-pro-HSA and met-HSA) .
• the said "another polypeptide" in the fusion compounds of the invention cannot be the remaining portion of HSA, since otherwise the -whole polypeptide would be HSA, which would not then be a "fusion polypeptide”.
• the non-HSA portion is one of the said (a) to (h) entities.
• the 1 to 368 portion of CD4 represents the first four disulphide-linked immunoglobulin-like domains of the human T lymphocyte CD4 protein, the gene for and amino acid sequence of which are disclosed in D. Smith et al (1987) Science 328, 1704-1707. It is used to combat HIV infections.
• PDGF platelet-derived growth factor
• TGF- ⁇ transforming growth factors ⁇
• a cDNA sequence for the 1-261 portion of Fn was disclosed in EP-A-207 751 (obtained from plasmid pFH6 with endonuclease PvuII). This portion binds fibrin and can be used to direct fused compounds to blood clots.
• Variants of alpha-1-antitrypsin include those disclosed by Rosenburg et al (1984) Nature 312, 77-80.
• the present invention includes the Pittsburgh variant (Met ⁇ 5 ⁇ is mutated to Arg) and the variant where ro- ⁇ 57 and are mutated to alanine and arginine respectively. These compounds are useful in the treatment of septic shock and lung disorders.
• Variants of the non-HSA portion of the polypeptides of the invention include variations as discussed above in relation to the HSA portion, including those with conservative amino acid substitutions, and also homologues from other species.
• the fusion polypeptides of the invention may have N- ter inal amino acids which extend beyond the portion corresponding to the N-terminal portion of HSA.
• pre-, pro-, or pre- pro sequences may be added thereto, for example the yeast alpha-factor leader sequence.
• the fused leader portions of WO 90/01063 may be used.
• the polypeptide which is fused to the HSA portion may be a naturally-occurring polypeptide, a fragment thereof or a novel polypeptide, including a fusion polypeptide.
• a fragment of fibronectin is fused to the HSA portion via a 4 amino acid linker.
• the amino terminal portion of the HSA molecule is so structured as to favour particularly efficient translocation and export of the fusion compounds of the invention in eukaryotic cells.
• a second aspect of the invention provides a • transformed host having a nucleotide sequence so arranged as to express a fusion polypeptide as described above.
• “so arranged” we mean, for example, that the nucleotide sequence is in correct reading frame with an appropriate RNA polymerase binding site and translation start sequence and is under the control of a suitable promoter.
• the promoter may be homologous with or heterologous to the host.
• Downstream (3') regulatory sequences may be included if desired, as is known.
• the host is preferably yeast (for example Saccharomyces spp. , e.g. S. cerevisiae; Kluyveromyces spp., e.g. K.
• lactis Pichia spp.; or Schizosaccharomyces spp., e.g. S. pombe
• suitable host such as E. coli, B. subtilis, Asperqillus spp., mammalian cells, plant cells or insect cells.
• a third aspect of the invention provides a process for preparing a fusion polypeptide according to the first aspect of the invention by cultivation of a transformed host according to the second aspect of the invention, followed by separation of the fusion polypeptide in a useful form.
• a fourth aspect of the invention provides therapeutic methods of treatment of the human or other animal body comprising administration of such a fusion polypeptide.
• the fusion polypeptides of the present invention wherein the C-terminal portion is Fn 585-1578 can be used for wound healing applications as biosynthesised, especially where the hybrid human protein will be topically applied.
• the portion representing amino acids 585 to 1578 of human fibronectin can if desired be recovered from the fusion protein by preceding the first amino acid of the fibronectin portion by amino acids comprising a factor X cleavage site. After isolation of the fusion protein from culture supernatant, the desired molecule is released by factor X cleavage and purified by suitable chromatography (e.g. ion-exchange chromatography) .
• suitable chromatography e.g. ion-exchange chromatography
• Other sites providing for enzymatic or chemical cleavage can be provided, either by appropriate juxtaposition of the N-terminal and C-terminal portions or by the insertion therebetween of an appropriate linker.
• At least some of the fusion polypeptides of the invention also have an increased half-life in the blood and therefore have advantages and therapeutic utilities themselves, namely the therapeutic utility of the non-HSA portion of the molecule.
• the compound will normally be administered as a one-off dose or only a few doses over a short period, rather than over a long period, and therefore the compounds are less likely to cause an immune response.
• DNA sequences encoding portions of human serum albumin used in the construction of the following molecules are derived from the plas ids mH0B12 and pDBD2 (EP-A-322 094, Delta Biotechnology Ltd, relevant portions of which are reproduced below) or by synthesis of oligonucleotides equivalent to parts of this sequence.
• DNA sequences encoding portions of human fibronectin are derived from the plasmid pFHDELl, or by synthesis of oligonucleotides equivalent to parts of this sequence.
• Plasmid pFHDELl which contains the complete human cDNA encoding plasma fibronectin, was obtained by ligation of DNA derived from plasmids pFH6, 16, 54, 154 and 1 (EP-A-207 751; Delta Biotechnology Ltd) .
• This DNA represents an mRNA variant which does not contain the 'ED' sequence and had an 89-amino acid variant of the III-CS region (R.J. Owens, A.R. Kornblihtt and F.E. Baralle (1986) Oxford Surveys on Eukaryotic Genes _3 141- 160).
• the map of this vector is disclosed in Fig. 11 and the protein sequence of the mature polypeptide produced by expression of this cDNA is shown in Fig. 5.
• Oligonucleotides were synthesised on an Applied Biosystems 380B oligonucleotide synthesiser according to the manufacturer's recommendations (Applied Biosystems, Warrington, Cheshire, UK).
• An expression vector was constructed in which DNA encoding ' the HSA secretion signal and mature HSA up to and including the 387th amino acid, leucine, fused in frame to DNA encoding a segment of human fibronectin representing amino acids 585 to 1578 inclusive, was placed downstream of the hybrid promoter of EP-A-258 067 (Delta Biotechnology) , which is a highly efficient galactose- inducible promoter functional in Saccharomyces cerevisiae.
• the codon for the 1578th amino acid of human fibronectin was directly followed by a stop codon (TAA) and then the S. cerevisiae phosphoglycerate kinase (PGK) gene transcription terminator.
• TAA stop codon
• PGK phosphoglycerate kinase
• a similar vector is constructed so as to enable secretion by S. cerevisiae of a hybrid molecule representing the N-terminal 195 amino acids of HSA C- terminally fused to amino acids 585 to 1578 of human fibronectin.
• Figure 1 depicts the amino acid sequence currently thought to be the most representative of natural HSA, with (boxed) the alternative C-termini of HSA(l-n);
• Figure 2 depicts the DNA sequence coding for mature HSA, wherein the sequence included in Linker 3 is underlined;
• FIG. 3 illustrates, diagrammatically, the construction of mH0B16
• Figure 4 illustrates, diagrammatically, the construction of pH0B31
• Figure 5 (on 6 sheets) illustrates the mature protein sequence encoded by the Fn plasmid pFHDELl
• Figure 6 illustrates Linker 5, showing the eight constituent oligonucleotides
• Figure 7 shows schematically the construction of plasmid pDBDF2
• Figure 8 shows schematically the construction of plasmid pDBDF5
• Figure 9 shows schematically the construction of plasmid pDBDF9
• Figure 10 shows schematically the construction of plasmid DBDF12, using plasmid pFHDELl.
• FIG. 11 shows a map of plasmid pFHDELl.
• EXAMPLE 1 HSA 1-387 FUSED TO Fn 585-1578
• the human serum albumin coding sequence used in the construction of the following molecules is derived from the plasmid M13mpl9.7 (EP-A-201 239, Delta Biotech- nology Ltd. ) or by synthesis of oligonucleotides equivalent to parts of this sequence. Oligonucleotides were synthesised using phosphoramidite chemistry on an Applied Biosystems 380B oligonucleotide synthesizer according to the manufacturer's recommendations (AB Inc., Warrington, Cheshire, England) .
• Linker A An oligonucleotide was synthesised (Linker A) which represented a part of the known HSA coding sequence (Figure 2) from the PstI site (1235-1240, Figure 2) to the codon for valine 381 wherein that codon was changed from GTG to GTC:
• Linker 1 was ligated into the vector M13mpl9 (Norrander et al, 1983) which had been digested with PstI and Hindi and the ligation mixture was used to transfect E.coli strain XLl-Blue (Stratagene Cloning Systems, San Diego, CA) . Recombinant clones were identified by their failure to evolve a blue colour on medium containing the chromogenic indicator X-gal (5-bromo-4-chloro-3-indolyl- ⁇ - D-galactoside) in the present of IPTG (isopropylthio- ⁇ - galactoside) .
• M13mpl9.7 consists of the coding region of mature HSA in M13mpl9 (Norrander et al, 1983) such that the codon for the first amino acid of HSA, GAT, overlaps a unique Xhol site thus:
• the ligation mix was then used to transfect E.coli XL1- Blue and template DNA was prepared from several plaques and then analysed by DNA sequencing to identify a clone, pDBDl ( Figure 4), with the correct sequence.
• a 1.1 kb HindiII to PstI fragment representing the 5' end of the HSA coding region and one half of the inserted oligonucleotide linker was isolated from pDBDl by agarose gel electrophoresis. This fragment was then ligated with double stranded mHOB12 previously digested with HindiII and PstI and the ligation mix was then used to transfect E.coli XLl-Blue.
• Single stranded template DNA was prepared from mature bacteriophage particles of several plaques.
• the DNA was made double stranded in vitro by extension from annealed sequencing primer with the Klenow fragment of DNA polymerase I in the presence of deoxynucleoside triphosphates. Restriction enzyme analysis of this DNA permitted the identification of a clone with the correct configuration, mHOB15 ( Figure 4).
• Linker 3 represents from the codon for the 382nd amino acid of mature HSA (glutamate, GAA) to the codon for lysine 389 which is followed by a stop codon (TAA) and a HindiII site and then a BamHI cohesive end:
• oligonucleotides 585 to 640, Fig. 2 were prepared by synthesising six oligonucleotides (Linker 5, Fig. 6).
• the oligonucleotides 2, 3, 4, 6, 7 and 8 were phosphorylated using T4 polynucleotide kinase and then the oligonucleotides were annealed under standard conditions in pairs, i.e. 1+8, 2+7, 3+6 and 4+5.
• the annealed oligonucleotides were then mixed together and ligated with mHOB12 which had previously been digested with the restriction enzymes Hindi and EcoRI.
• pDBDFl a clone in which a linker of the expected sequence had been correctly inserted into the vector was designated pDBDFl (Fig. 7).
• This plasmid was then digested with PstI and EcoRI and -the approx. 0-24kb fragment was purified and then ligated with the 1.29kb BamHI-PstI fragment of pDBD2 (Fig. 7) and BamHI + EcoRI digested pUC19 (Yanisch-Perron, et al. , 1985) to form pDBDF2 (Fig. 7).
• pFHDELl A plasmid containing a DNA sequence encoding full length human fibronectin, pFHDELl, was digested with EcoRI and Xhol and a 0.77kb EcoRI-XhoI fragment (Fig. 8) was isolated and then ligated with EcoRI and Sail digested Ml3 mpl ⁇ (Norrander et al., 1983) to form pDBDF3 (Fig. 8).
• Linker 6 The following oligonucleotide linker (Linker 6) was synthesised, representing from the PstI site at 4784-4791 of the fibronectin sequence of EP-A-207 751 to the codon for tyrosine 1578 (Fig. 5) which is followed by a stop codon (TAA) , a HindiII site and then a BamHI cohesive end: Linker 6
• EP-A-258 067 includes the promoter of EP-A-258 067 to direct the expression of the HSA secretion signal fused to DNA encoding amino acids 1- 387 of mature HSA, in turn fused directly and in frame with DNA encoding amino acids 585-1578 of human fibronectin, after which translation would terminate at the stop codon TAA. This .is then followed by the S.cerevisiae PGK gene transcription terminator.
• the plasmid also contains sequences which permit selection and maintenance in Escherichia coli and S.cerevisiae (EP-A-258 067) .
• This plasmid was introduced into S.cerevisiae S150-2B (leu2-3 leu2-112 ura3-52 trpl-289 his3- 1) by standard procedures (Begg ⁇ , 1978). Transformants were subsequently analysed and found to produce the HSA-fibronectin fusion protein.
• the first domain of human serum albumin (amino acids 1-195) is fused to amino acids 585- 1578 of human fibronectin.
• the plasmid pDBD2 was digested with BamHI and Bglll and the 0.79kb fragment was purified and then ligated with BamHI-digested M13mpl9 to fo ' rm pDBDF6 (Fig. 6).
• a mutagenic primer was used as a mutagenic primer to create a Xhol site in pDBDF6 by in vitro mutagenesis using a kit supplied by Amersham International PLC. This site was created by changing base number 696 of HSA from a T to a G (Fig. 2). The plasmid thus formed was designated pDBDF7 (Fig. 9). The following linker was then synthesised to represent from this newly created Xhol site to the codon for lysine 195 of HSA (AAA) and then from the codon for isoleucine 585 of fibronectin to the ends of oligonucleotides 1 and 8 shown in Fig. 6.
• This linker was ligated with the annealed oligonucleotides shown in Fig. 3, i.e. 2+7, 3+6 and 4+5 together with Xhol and EcoRI digested pDBDF7 to form pDBDF ⁇ (Fig. 9). Note that in order to recreate the original HSA DNA sequence, and hence amino acid sequence, insertion of linker 7 and the other oligonucleotides into pDBDF7 does not recreate the Xhol site.
• pDBDF9 The 0.83kb BamHI-StuI fragment of pDBDF8 was purified and then was ligated with the 0.68kb EcoRI-BamHI fragment of pDBDF2 and the 2.22kb StuI-EcoRI fragment of pFHDELl into Bglll-digested pKV50 to form pDBDF9 (Fig. 9).
• This plasmid is similar to pDBDF5 except that it specifies only residues 1-195 of HSA rather than 1-387 as in pDBDF5.
• the plasmid When introduced into S.cerevisiae S150-2B as above, the plasmid directed the expression and secretion of a hybrid molecule composed of residues 1-195 of HSA fused to residues 585-1578 of fibronectin.
• This linker was then ligated with the annealed oligonucleotides shown in Fig. 6, i.e. 2+7, 3+6 and 4+5 into Hin i andEcoRI digested mH0B12, to form pDBDFlO (Fig. 7).
• the plasmid was then digested with PstI and EcoRI and the roughly 0.24kb fragment was purified and then ligated with the 1.29kb BamHI-PstI fragment of pDBD2 and BamHI and EcoRI digested pUC19 to form pDBDFll (Fig. 10) .
• the 1.5kb BamHI-StuI fragment of pDBDFll was then ligated with the 0.68kb EcoRI-BamHI fragment of pDBDF4 and the 2.22kb StuI-EcoRI fragment of pFHDELl into Bglll-diges ed pKV50 to form pDBDF12 (Fig. 10).
• This plasmid was then introduced into S.cerevisiae S150-2B.
• the purified secreted fusion protein was treated with Factor X to liberate the fibronectin fragment representing residues 585-1578 of the native molecule.

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