Patent number 8642542 is assigned to Novozymes Biopharma DK A/S (Bagsvaerd, DK).
The following quote was obtained by the news editors from the background information supplied by the inventors: "Human serum albumin (HSA or HA), a protein of 585 ammo acids (SEQ ID NO: 26), is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. At present, HSA for clinical use is produced by extraction from human blood. The production of recombinant HA (rHA) in microorganisms has been disclosed in EP 330 451 and EP 361 991.
"The role of albumin as a carrier molecule and its inert nature are desirable properties for use as a stabiliser and transporter of polypeptides. The use of albumin as a component of a fusion protein for stabilising other proteins has been disclosed in WO 93/15199, WO 93/15200, and EP 413 622. The use of N-terminal fragments of HSA for fusions to polypeptides has also been disclosed (EP 399 666). Fusion to the said polypeptide is achieved by genetic manipulation, such that the DNA coding for HSA, or a fragment thereof, is joined to the DNA coding for the said polypeptide. A suitable host is then transformed or transfected with the fused nucleotide sequences, so arranged on a suitable plasmid as to express a fusion polypeptide. Nomura et al (1995) attempted to express human apolipoprotein E in S. cerevisiae as a fusion protein with HSA or fragments of HSA, using the HSA pre-sequence to direct secretion. Whilst fusion to full length HSA resulted in the secretion of low levels of the protein into the medium (maximum yield of 6.3 mg per litre), fusion to HSA (1-198) or HSA (1-390) did not result in secretion into the medium.
"Human growth hormone (reviewed by Strobl and Thomas, 1994) consists of a single polypeptide of 191 amino acids, internally cross-linked by two disulphide bonds. Two molecules of hGH receptor bind each molecule of hGH to facilitate signal transduction (Cunningham et al, 1991;
"In addition, hGH has been expressed intracellularly in yeast (
"In humans, hGH is secreted into the blood in pulses, and in the circulation has a half-life of less than 20 minutes (Haffner et al, 1994). Elimination of the hormone is primarily via metabolism in the liver and kidneys and is more rapid in adults than in children (Kearns et al, 1991). Treatment for hGH deficiency generally lasts for 6 to 24 months, during which hGH is administered either three times a week intramuscularly or on a daily basis subcutaneously. Such a regimen of frequent administration is necessary because of the short half-life of the molecule.
"Poznansky et al (1988) increased the half-life of porcine growth hormone by conjugation with either porcine or human serum albumin (HSA) to form relatively large conjugates of about 180 kD. Chemical reaction using the cross-linking reagent glutaraldehyde resulted in, on average, two molecules of albumin complexed with six molecules of growth hormone. The resulting 180 kD conjugate was found to have an extended half-life in the circulation of rats of 2 to 3 hours, compared to 5 minutes for unconjugated growth hormone. Activity assays showed that the conjugate retained full, and possibly increased activity in vitro, but was inactive in vivo."
In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventor's summary information for this patent: "The invention relates to proteins formed by the fusion of a molecule of albumin, or variants or fragments thereof, to a molecule of growth hormone or variants or fragments thereof, the fusion proteins having an increased circulatory half-life over unfused growth hormone. For convenience, we shall refer to human albumin (HA) and human growth hormone (hGH), but the albumin and growth hormones of other vertebrates are included also. Preferably, the fusion protein comprises HA, or a variant or fragment thereof, as the N-terminal portion, with hGH or a variant or fragment thereof as the C-terminal portion, so as to minimise any possible negative effects on receptor binding. Alternatively, a fusion protein comprising HA, or a variant or fragment thereof, as the C-terminal portion, with hGH or a variant or fragment thereof as the N-terminal portion, may also be capable of signal transduction. Generally, the polypeptide has only one HA-derived region and one GH-derived region.
"Additionally, the fusion proteins of the invention may include a linker peptide between the two fused portions to provide a greater physical separation between the two moieties and thus maximise the availability of the hGH portion to bind the hGH receptor. The linker peptide may consist of amino acids such that it is flexible or more rigid.
"The linker sequence may be cleavable by a protease or chemically to yield the growth hormone related moiety. Preferably, the protease is one which is produced naturally by the host, for example the S. cerevisiae protease kex2 or equivalent proteases. Hence, a further aspect of the invention provides a process for preparing growth hormone or a variant or fragment thereof by expressing a polynucleotide which encodes a polypeptide of the invention in a suitable host, cleaving the cleavable linker to yield the GH-type compound and recovering the GH-type compound from the host culture in a more pure form.
"We have discovered that the polypeptides of the invention are significantly more stable in solution than hGH. The latter rapidly becomes inactive when stored in solution at 4.degree. C. for over one month. Currently marketed hGH is sold as a freeze-dried powder.
"Suitably, the fusion polypeptides are produced as recombinant molecules by secretion from yeast, a microorganism such as a bacterium, human cell line or a yeast. Preferably, the polypeptide is secreted from the host. We have found that, by fusing the hGH coding sequence to the HA coding sequence, either to the 5' end or 3' end, it is possible to secrete the fusion protein from yeast without the requirement for a yeast-derived pro sequence. This was surprising, as other workers have found that a yeast derived pro sequence was needed for efficient secretion of hGH in yeast. For example, Hiramitsu et al (1990, 1991) found that the N-terminal portion of the pro sequence in the Mucor pusillus rennin pre-pro leader was important. Other authors, using the MF.alpha.-1 signal, have always included the MF.alpha.-1 pro sequence when secreting hGH. The pro sequences were believed to assist in the folding of the hGH by acting as an intramolecular chaperone. The present invention shows that HA or fragments of HA can perform a similar function.
"Hence, a particular embodiment of the invention comprises a DNA construct encoding a signal sequence effective for directing secretion in yeast, particularly a yeast-derived signal sequence (especially one which is homologous to the yeast host), and the fused molecule of the first aspect of the invention, there being no yeast-derived pro sequence between the signal and the mature polypeptide.
"The Saccharomyces cerevisiae invertase signal is a preferred example of a yeast-derived signal.
"Conjugates of the kind prepared by Poznansky et al (1988), in which separately-prepared polypeptides are joined by chemical cross-linking, are not contemplated.
"The albumin or hGH may be a variant of normal HSA/rHA (termed hereinafter 'HA') or hGH, respectively. By 'variants' we include insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially alter one or more of the oncotic, useful ligand-binding and non-immunogenic properties of albumin or, in the case of hGH, its non-immunogenicity and ability to bind and activate the hGH receptor. In particular, we include naturally-occurring polymorphic variants of human albumin and fragments of human albumin, for example those fragments disclosed in EP 322 094 (namely HA (1-n), where n is 369 to 419). The albumin or growth hormone may be from any vertebrate, especially any mammal, for example human, cow, sheep, pig, hen or salmon. The albumin and GH parts of the fusion may be from differing animals.
"By 'conservative substitutions' is intended swaps within groups such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The variant will usually have at least 75% (preferably at least 80%, 90%, 95% or 99%) sequence identity with a length of normal HA or hGH which is the same length as the variant and which is more identical thereto than any other length of normal HA or hGH, once the allowance is made for deletions and insertions as is customary in this art. Generally speaking, an HA variant will be at least 100 amino acids long, preferably at least 150 amino acids long. The HA variant may consist of or comprise at least one whole domain of HA, for example domains 1 (1-194), 2 (195-387), 3 (388-585), 1+2 (1-387), 2+3 (195-585) or 1+3 (1-194,+388-585). Each domain is itself made up of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with flexible inter-subdomain linker regions comprising residues Lys106 to Glu199, Glu292 to Val315 and Glu492 to Ala511. Preferably, the HA part of the fusion comprises at least one subdomain or domain of HA or conservative modifications thereof. If the fusion is based on subdomains, some or all of the adjacent linker is preferably used to link to the hGH moiety. The hGH variant should have GH activity, and will generally have at least 10 amino acids, (although some authors have found activity with only 4 residues), preferably at least 20, preferably at least 50, 100, 150, 180 or 191, amino acids long, and preferably retains its cysteines for both internal disulphide bonds.
"The fused molecules of the invention generally have a molecular weight of less than 100 kD, for example less than 90 kD or 70 kD. They are therefore much smaller than the 180 kD conjugates of Poznansky et al (referred to above), which were inactive in vivo. They will normally have a molecular weight of at least 20 kD, usually at least 30 kD or 50 kD. Most fall within the molecular weight range 60-90 kD.
"A second main aspect of the invention provides a yeast transformed to express a fusion protein of the invention.
"In addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. Especially if the polypeptide is secreted, the medium will thus contain the polypeptide, with the cells, or without the cells if they have been filtered or centrifuged away.
"Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris, filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
"The desired protein is produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid.
"The yeasts are transformed with a coding sequence for the desired protein in any of the usual ways, for example electroporation. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
"Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce the desired polypeptide. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975)
"Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems,
"A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
"Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3'-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities.
"The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
"Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including
"A desirable way to modify the DNA in accordance with the invention, if, for example, HA variants are to be prepared, is to use the polymerase chain reaction as disclosed by
"Exemplary genera of yeast contemplated to be useful in the practice of the present invention as hosts for expressing the fusion proteins are Pichia (Hansenula), Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like. Preferred genera are those selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora. Examples of Saccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii. Examples of Kluyveromyces spp. are K. fragilis, K. lactis and K. marxianus. A suitable Torulaspora species is T. delbrueckii. Examples of Pichia (Hansenula) spp. are P. angusta (formerly H. polymorpha), P. anomala (formerly H. anomala) and P. pastoris.
"Methods for the transformation of S. cerevisiae are taught generally in EP 251 744, EP 258 067 and WO 90/01063, all of which are incorporated herein by reference.
"Suitable promoters for S. cerevisiae include those associated with the
"Convenient regulatable promoters for use in Schizosaccharomyces pombe are the thiamine-repressible promoter from the nmt gene as described by Maundrell (1990) J. Biol. Chem. 265, 10857-10864 and the glucose-repressible fbp1 gene promoter as described by Hoffman & Winston (1990) Genetics 124, 807-816.
"Methods of transforming Pichia for expression of foreign genes are taught in, for example, Cregg et al (1993), and various Phillips patents (eg U.S. Pat. No. 4,857,467, incorporated herein by reference), and Pichia expression kits are commercially available from
"Gleeson et al (1986) J. Gen. Microbiol. 132, 3459-3465 include information on Hansenula vectors and transformation, suitable promoters being MOX1 and FMD1; whilst EP 361 991, Fleer et al (1991) and other publications from
"The transcription termination signal is preferably the 3' flanking sequence of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, ie may correspond to the promoter. Alternatively, they may be different in which case the termination signal of the S. cerevisiae ADH1 gene is preferred.
"The desired fusion protein may be initially expressed with a secretion leader sequence, which may be any leader effective in the yeast chosen. Leaders useful in S. cerevisiae include that from the mating factor .alpha. polypeptide (MF.alpha.-1) and the hybrid leaders of EP-A-387 319. Such leaders (or signals) are cleaved by the yeast before the mature albumin is released into the surrounding medium. Further such leaders include those of S. cerevisiae invertase (SUC2) disclosed in JP 62-096086 (granted as 91/036516), acid phosphatase (PHO5), the pre-sequence of MF.alpha.-1, .beta.-glucanase (
"The fusion protein of the invention or a formulation thereof may be administered by any conventional method including parenteral (eg subcutaneous or intramuscular) injection or intravenous infusion. The treatment may consist of a single dose or a plurality of doses over a period of time.
"Whilst it is possible for a fusion protein of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be 'acceptable' in the sense of being compatible with the fusion protein and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free. The formulation should be non-immunogenic; vaccine-type formulations involving adjuvants are not contemplated.
"The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the fusion protein with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
"Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders.
"Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
"The fusion proteins of the invention may be used in the treatment of any condition in which growth hormone is indicated, for example isolated growth hormone deficiency, panhypopituitarism, following cranial irradiation (eg in the treatment of leukaemia or brain tumours), Turner's syndrome, Down's syndrome, intrauterine growth retardation, idiopathic growth deficiency, chronic renal failure, achondroplasia, female infertility and various catabolic disorders. They may also be used in the stimulation of growth, and/or enhancement of lean meat proportion, in farm animals such as cows, sheep, goats and pigs.
"The fusion protein may be administered together with insulin-like growth factor I (IGF-I).
"The dosage can be calculated on the basis of the potency of the fusion protein relative to the potency of hGH, whilst taking into account the prolonged serum half-life of the fusion proteins compared to that of native hGH. Growth hormone is typically administered at 0.3 to 30.0 IU/kg/week, for example 0.9 to 12.0 IU/kg/week, given in three or seven divided doses for a year or more. In a fusion protein consisting of full length HA fused to full length GH, an equivalent dose in terms of units would represent a greater weight of agent but the dosage frequency can be reduced, for example to twice a week, once a week or less.
"Preferred examples of the invention will now be described by way of example and with reference to the accompanying figures, in which:
"FIG. 1 shows the human growth hormone cDNA sequence (SEQ ID NO: 22), encoding mature hGH (SEQ ID NO: 23);
"FIG. 2 shows a restriction enzyme map of pHGH1;
"FIG. 3 shows a restriction enzyme map of pBST(+) and the DNA sequence of the polylinker (SEQ ID NO: 24);
"FIG. 4 shows the construction of pHGH12;
"FIG. 5 shows the construction of pHGH16;
"FIG. 6A-B show the HSA cDNA sequence (SEQ ID NO: 25), more particularly the region encoding the mature protein (SEQ ID NO: 26);
"FIG. 7 shows the construction of pHGH14;
"FIG. 8 shows the construction of pHGH38;
"FIG. 9 shows the construction of pHGH31;
"FIG. 10 shows the construction of pHGH58 or pHGH59 (Example 7);
"FIG. 11 is a scheme for constructing fusions having spacers (Example 7); and
"FIG. 12 shows the results of a pharmacokinetic study showing the clearance of .sup.125I-labelled rHA-hGH compared to that of hGH following iv injection in rats. Data are from two rats in each group, and include total radioactivity and radioactivity which could be precipitated by TCA, ie that associated with protein rather than as free .sup.125I. The calculated clearance half-life for hGH was approximately 6 minutes, compared to approximately 60 minutes for the rHA-hGH fusion protein. See Example 3. .circle-solid.--hGH (total counts) .box-solid.--hGH (TCA precipitated counts) --rHA-hGH (total counts) .tangle-solidup.--rHA-hGH (TCA precipitated counts)."
URL and more information on this patent, see: Ballance,
Keywords for this news article include: Esterases, Treatment, Polymerase, Amino Acids,
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