HOBBA, Graham, Dean (2 Sylvia Court, Coromandel Valley, South Australia 5051, AU)
FRANCIS, Geoffrey, Leonard (6 Joyleen Court, Athelstone, South Australia 5076, AU)
HOBBA, Graham, Dean (2 Sylvia Court, Coromandel Valley, South Australia 5051, AU)
CLAIMS
1. A construct comprising: a) a peptide comprising the amino acid sequence LSTQ (SEQ ID NO: 19); and b) a polypeptide growth factor.
2. A construct according to claim 1, in which the construct is a recombinant polypeptide.
3. A construct according to claim 1 or claim 2, in which the construct exhibits growth factor activity.
4. A construct according to any one of claims 1 to 3 , in which the polypeptide growth factor is selected from the group consisting of insulin-like growth factor (IGF) , epidermal growth factor (EGF) , transforming growth factor- β (TGFβ) , fibroblast growth factor (FGF) , platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) .
5. A construct according to any one of claims 1 to 3, in which the polypeptide growth factor has an amino acid sequence which is homologous to the sequence of the human growth factor.
6. A construct according to any one of claims 1 to 5 , in which the growth factor is an IGF.
7. A construct according to any one of claims 1 to 6, in which the growth factor has an amino acid sequence which is homologous to the human IGF sequence.
8. A construct according to any one of claims 1 to 7, in which the growth factor is an IGF selected from the group consisting of IGF-I, IGF-II, [Arg]3IGF-I, [Arg] 6IGF- II, des (1-3) IGF-I, des (1-6) IGF-II, and analogues thereof.
8. A construct according to claim 7, in which the IGF is des (1-3) IGF-I.
9. A construct according to any one of claims 1 to 5, in which the growth factor is an EGF.
10. A construct according to any one of claims 1 to 9, in which (a) is an amino acid sequence selected from the group consisting of:
LSTQ (SEQ ID NO: 19)
VNLSTQ (SEQ ID NO: 23)
MFPAMPLSSLFLSTQ (SEQ ID NO: 24); MFPAMPLSSLFVNLSTQ (SEQ ID NO: 25);
MFPAMPLSSLFVNGLSTQ (SEQ ID NO: 26);
MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQLSTQ (SEQ
ID NO: 27) ;
MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNLSTQ (SEQ ID NO: 28) ; and
MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNGLSTQ (SEQ ID NO: 29) .
11. A construct according to any one of claims 1 to 8 , in which the construct is selected from the group consisting of:
MFPAMPLSSLFLSTQ-des (1-3) IGF-I (SEQ ID NO:30);
MFPAMPLSSLFVNLSTQ-des (1-3) IGF-I (SEQ ID N0:31);
MFPAMPLSSLFVNGLSTQ-des (1-3) IGF-I (SEQ ID NO:32); MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQLSTQ-des (1-
3) IGF-I (SEQ ID NO:33);
MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNLSTQ- des(l-3)IGF-I (SEQ ID NO:34); and
MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNGLSTQ- des(l-3)IGF-I (SEQ ID NO:35).
12. A construct according to any one of claims 1 to 11 in which (a) does not comprise the amino acid sequence FAHY (SEQ ID NO: 38) or GFAHY (SEQ ID NO: 39) .
13. A construct according to claim 1 which is metpGH(l- ll)VNLSTQdes (1-3) IGF-I.
14. An antibody specific for a construct according to any one of claims 1 to 13.
15. An isolated nucleic acid molecule encoding a construct according to any one of claims 1 to 13.
16. An isolated nucleic acid molecule according to claim
15. in which the nucleic acid sequence encoding the growth factor is of human origin.
17. An isolated nucleic acid molecule according to claim 15 or claim 16, which is a single-stranded DNA or double- stranded DNA molecule.
18. An isolated nucleic acid molecule according to claim any one of claims 15 to 17, which is a cDNA.
19. An isolated nucleic acid molecule according to claim 15 or claim 16, which is an RNA.
20. A vector or plasmid comprising a nucleic acid molecule according to any one of claims 15 to 19.
21. A vector according to claim 20, further comprising a second nucleic acid molecule encoding a desired polypeptide.
22. A host cell transformed by a vector or plasmid according to claim 20 or claim 21.
23. A host cell according to claim 22, in which the host cell is a prokaryotic cell or a eukaryotic cell.
24. A host cell according to claim 23, in which the prokaryotic cell is E. coli, a mammalian cell or a yeast.
25. A method for the production of a construct according to any one of claims 1 to 13, which process comprises:
(a) culturing a host cell according to any one of claims 22 to 24 under conditions favourable for cell growth and expression of the construct; and
(b) isolating the construct from the culture.
26. A method according to claim 25, in which prior to step (b) the construct is subjected to dissolution and a refolding step to release a biologically active construct.
27. A method for culturing mammalian cells, the method comprising culturing the cells in a medium comprising a construct according to any one of claims 1 to 13.
28. A method according to claim 27, in which the mammalian cells encode a desired polypeptide, which is then isolated from the culture.
29. A method according to claim 27 or claim 28, in which the construct is at a concentration of between 10-100 ng/ml .
30. A method according to any one of claims 27 to 29, in which the medium does not contain serum.
31. A method according to any one of claims 27 to 30, in which the cells comprise an IGF receptor or an insulin receptor.
32. A method according to any one of claims 27 to 31, in which the medium comprises (a) an osmolality regulator;
(b) a buffer;
(c) an energy source; and one or more of
(d) glutamine; (d) at least one additional amino acid; and
(e) an inorganic, organic or recombinant iron source .
33. A method according to any one of claims 27 to 32, comprising culturing the cells in the absence of serum in a medium comprising, per litre of medium:
(a) about 10 ng-50 ng of a construct according to any one of claims 1 to 13 ;
(b) an osmolality regulator to maintain the osmolality of the medium within the range of about 200-350 mOsm;
(c) a buffer to maintain the pH of the medium within the range of about 6.5 to 7.5;
(d) about 1,000-10,000 mg of a monosaccharide;
(e) about 400-600 mg of L-glutamine; (f) about 10 -200 mg of at least one amino acid selected from the group consisting of L-alanine, L-arginine, L- asparagine, L-aspartic acid, L-cystine, L-cysteine, L- glutamic acid, glycine, L-histidine, L-isoleucine, L- leucine, L- lysine, L-methionine, L-phenylalanine, L- proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine;
(g) about 0.25-5 mg of an inorganic, organic or recombinant iron source; and (h) about 1-lOOOmg of albumin.
34. A method according to claim 25 or claim 26, in which the isolating of the construct or desired polypeptide comprises :
(a) homogenisation of the cultured host cell to release inclusion bodies;
(b) dissolution of the inclusion bodies;
(c) refolding of the construct; (d) cation exchange chromatography;
(e) reverse phase high performance liquid chromatography;
(f) size exclusion chromatography; and
(g) ultrafiltration.
35. A process according to any one of claims 25, 26 and 34, in which the isolated construct or desired polypeptide is subject to a freeze-drying step.
36. A composition comprising a construct according to any one of claims 1 to 13, together with a pharmaceutically, veterinarily or cell culture acceptable carrier.
37. A composition according to claim 36, which is a liquid composition, formulated in acetic acid or hydrochloric acid.
38. A liquid composition according to claim 37, formulated in 100 mM acetic acid.
39. A liquid composition according to claim 37, formulated in 10 mM hydrochloric acid.
40. A composition according to claim 36, which is in dry powder form.
41. A composition according to claim 40, which further comprises a bulking agent.
42. A cell culture system, comprising:
(a) a fermenter adapted for mammalian cell culture;
(b) a mammalian cell comprising a nucleic acid sequence encoding a desired polypeptide; and (c) a mammalian cell culture medium comprising a construct according to any one of claims 1 to 13.
43. A cell culture system comprising:
(a) a fermenter adapted for mammalian cell culture;
(b) a mammalian cell comprising a nucleic acid sequence encoding (i) a desired polypeptide and (ii)a construct according to any one of claims 1 to 13 ; and
(c) a mammalian cell culture medium.
44. A cell culture system according to claim 42 or claim 43, in which the cell culture medium comprises water, an osmolality regulator, a buffer, an energy source, at least one additional amino acid, and an inorganic or recombinant iron source .
45. A kit comprising a composition according to any one of claims 38 to 41 sealed in a vial, cartridge or container.
46. A method of treatment of a condition selected from the group consisting of protein accumulation deficiency condition or protein loss; chronic growth disorders, including growth hormone deficiency and somatomedin deficiency; disorders associated with insufficient growth or tissue wasting, including, but not limited to, cancer, cystic fibrosis, Duchenne muscular dystrophy, Becker dystrophy, autosomal recessive dystrophy, polymyositis and other myopathies; acute conditions associated with poor nitrogen status including, but not limited to, burns, skeletal trauma and infection; or to promote growth, improve nitrogen status and/or to treat catabolic disorders in infants or premature babies, in a subject in need of such treatment, comprising the step of administering an effective amount of a construct according to any one of claims 1 to 13 which exhibits insulin-like growth factor activity to a mammal in need of such treatment.
47. Use of a construct according to any one of claims 1 to 13 which exhibits insulin-like growth factor activity in the manufacture of a medicament for the treatment of a condition selected from the group consisting of protein accumulation deficiency condition or protein loss; chronic growth disorders, including growth hormone deficiency and somatomedin deficiency; disorders associated with insufficient growth or tissue wasting, including, but not limited to, cancer, cystic fibrosis, Duchenne muscular dystrophy, Becker dystrophy, autosomal recessive dystrophy, polymyositis and other myopathies; acute conditions associated with poor nitrogen status including, but not limited to, burns, skeletal trauma and infection; or for promoting growth, improving nitrogen status and/or treatment of catabolic disorders in infants or premature babies.
48. A peptide having the amino acid sequence LSTQ (SEQ ID NO: 19) .
49. Use of a peptide according to claim 48 in a leader sequence to increase expression of a recombinant growth factor.
50. Use of a peptide according to claim 49 as a cleavage site for an α-lytic protease.
51. A peptide leader sequence of up to 200 amino acids which sequence comprises LSTQ (SEQ ID NO: 19), in which the peptide leader sequence capable of increasing the expression of recombinant protein compared to recombinant protein expressed without a peptide leader sequence comprising LSTQ.
52. A fusion protein comprising: a) a peptide leader sequence of up to 200 amino acids comprising LSTQ (SEQ ID NO: 19); and b) a polypeptide growth factor.
53. A peptide leader sequence consisting essentially of LSTQ (SEQ ID N0:19), which is capable of increasing the expression of a recombinant protein compared to expression of the recombinant protein without a peptide leader sequence comprising LSTQ (SEQ ID NO: 19).
54. A method of increasing the yield of a recombinant protein, comprising the steps of: (a) providing a construct capable of expressing a fusion protein, in which the fusion protein comprises
(i) a peptide leader sequence of less than 200 amino acids comprising LSTQ (SEQ ID NO: 19); and
(ii) a polypeptide growth factor,- (b) transforming the construct into a suitable host, and (c) expressing said fusion protein.
55. A fusion protein comprising a peptide leader sequence of up to 200 amino acids containing LSTQ (SEQ ID NO: 19) and an amino acid sequence encoding a growth factor.
56. A construct comprising a first nucleic acid encoding a peptide leader sequence comprising the amino acid sequence LSTQ (SEQ ID NO:19), a second nucleic acid encoding a cleavage site and a third nucleic acid encoding a growth factor, in which said first, second and third nucleic acids are consecutive.
57 . MetpGH ( 1- 11 ) VNLSTQdes ( 1 -3 ) IGF- I . |
RECOMBINANT PROTEIN PRODUCTION
PRIORITY
This application claims priority from US provisional application No. 60/750359, the entire disclosure of which is incorporated herein by this cross-reference.
FIELD
This invention relates to methods of recombinant production of proteins, such as growth factors. In particular, the invention relates to improvements in methods for the production of growth factor proteins as fusion proteins . In some embodiments the protein is a growth factor such as IGF or EGF.
BACKGROUND
All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in Australia or in any other country.
Insulin-like growth factors (IGFs) are important mitogenic factors involved in cell survival, proliferation and metabolism, and locally produced IGFs regulate tissue growth and differentiation. IGF-I is a small protein which has been shown to stimulate the growth of a wide range of cells in culture.
Human, bovine and porcine IGF-I all share the
identical amino acid sequence, which is
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRL E MYCAPLKPAKSA (SEQ ID NO: 1) .
The sequences of some mammalian IGF-Is and IGF-IIs are as follows:
IGF-I
Human GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMY CAPLKPAKSA (SEQ ID NO:1)
Cow GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMY CAPLKPAKSA (SEQ ID NO: 2)
I
I
Human AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYC ATPAKSE (SEQ ID NO: 7)
CO
£ 20 Pig AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVNRRSRGIVEECCFRSCDLALLETYC ATPAKSE (SEQ ID NO: 8) >
C
Cow AYRPSETLCGGELVDTLQFVCGDRGFYFSRPSSRINRRSRGIVEECCFRSCDLALLETYC ATPAKSE (SEQ ID NO: 9)
Sheep AYRPSETLCGGELVDTLQFVCGDRGFYFSRPSSRINRRSRGIVEECCFRSCDLALLETYC AAPAKSE (SEQ ID NO: 10)
25
Rat AYRPSETLCGGELVDTLQFVCSDRGFYFSRPSSRANRRSRGIVEECCFRSCDLALLETYC ATPAKSE (SEQ ID NO: 11)
IGF-I has a wide variety of potential applications, including its use to:
(1) treat growth hormone deficiencies in humans;
(2) suppress the loss of body protein in severe catabolic states, such as those resulting from burns, infection or other trauma, in humans and other mammals;
(3) increase growth rates, redistribute nutrients and enhance food conversion efficiency in farm animals; and (4) support the growth and survival of cells in mammalian culture. This includes commercial mammalian cell cultures wherein the cells are engineered to express polypeptides and peptides, including polypeptides and peptides to be used for therapeutic applications. IGF-I has also been widely used as a supplement in serum-free cell culture media. Analogues of IGF-I and IGF-II are described in US Patent No. 5,330,971, the entire disclosure of which is incorporated herein by this cross-reference. One particular IGF-I analogue disclosed therein, LONG 18 R 3 IGF-I (MetpGH (1-11) VNR 3 IGF-I) , is significantly more potent than human IGF-I in vitro. The enhanced potency is due to decreased binding of LONG 8 R 3 IGF- I to IGF binding proteins, which normally inhibit the biological actions of IGFs. LONG 0 R 3 IGF-I is currently used in the commercial manufacture of 7 therapeutic agents approved by the US Food and Drug administration.
Another analogue of IGF-I is human des (1-3) IGF-I (SEQ ID NO: 12) . This is a 67 amino acid analogue of IGF-I which lacks the N-terminal tripeptide Gly-Pro-Glu. Des(l- 3) IGF-I is more potent than IGF-I in vitro and in vivo.
This increased potency is due to reduced binding of human des (1-3) IGF-I to most of the IGF binding proteins. Des(l- 3) IGF-I binds to the type 1 IGF receptor with similar affinity to full length IGF-I. The expression of heterologous proteins using recombinant DNA technology in some cases requires a "fusion partner" to prevent degradation and to increase
expression in hosts such as E. coll. For IGF-I this has been achieved by inclusion of the first 11 amino acids of porcine growth hormone at the N-terminus as the fusion partner (Francis et al . , (1992), Journal of Molecular Endocrinology 8:213-223). Furthermore, the porcine growth hormone-derived fusion partner has been shown to promote the correct folding of the fusion protein into its preferred biologically active form (Milner et al . , (1995), Biochemical Journal 308:865-871). Methods for the recombinant production of IGF-I (SEQ ID NO: 13), IGF-II (SEQ ID NO: 12) and analogues thereof, such as LONG 0 R 3 IGF-I and des (1-3) IGF-I, include those methods described in Francis et al . , Milner et al . and US Patent No. 5,330,971. This patent discloses that expression yields during recombinant manufacture of IGFs are increased significantly if the IGF is expressed linked to a leader sequence, in which the leader sequence is a polypeptide having growth hormone activity or a fragment thereof. The polypeptide comprising the leader sequence is referred to as the fusion partner, and the IGF is referred to as the fusion protein. Preferably the leader sequence is homologous to the first 1-191 N-terminal amino acids of methionine porcine growth hormone (metpGH or MpGH) . This leader sequence is set out in SEQ ID NO: 14. The fusion protein may optionally further comprise a chemical or enzymatic cleavage site between the leader sequence and the IGF so as to allow the release of mature IGF or analogue following cleavage of the fusion protein. Without this leader sequence, yields of the IGF following cell culture are very low, and inadequate for manufacture of
IGF on a commercial scale.
US Patent No. 5,330,971 also discloses additional leader sequences, including: MFPAMPLSSLF- (SEQ ID NO: 15); MFPAMPLSSLFVN- (SEQ ID NO:16);
MFPAMPLSSLFVNFAHY- (SEQ ID NO: 17); and MFPAMPLSSLFVNGFAHY- (SEQ ID NO: 18).
- S -
These leader sequences contain an initiating methionine at their N-terminal end, followed by a sequence homologous to the first 10 amino acids of pGH. SEQ ID NOs 16, 17, and 18 also contain other amino acids, as indicated above. These are encoded by the Hpal restriction site.
The inventors have recognized a need in the art for improved methods for the commercial-scale production of proteins, such as IGFs and other growth factors, which provide protein yields which are higher than those which can be achieved with presently available techniques.
SUMMARY
We have now found that if a growth factor such as
IGF or epidermal growth factor (EGF) is expressed so that , it is linked to a leader sequence which comprises the amino acid sequence LSTQ (SEQ ID NO: 19), yields of recombinant protein are increased compared to yields obtained using prior art methods. We have also found that the amino acid sequence LSTQ acts as a cleavage site for α-lytic proteases, which are described in Haggett et al (1994), Arch. Biochem. Phys . 314 (1) 132-141. As far as we are aware there has not been any previous disclosure of a peptide having this sequence, or any suggestion that this sequence might be useful in the recombinant production of proteins, either to provide a protease cleavage site or to increase the yield of a protein.
In its general aspects the invention provides: 1. A peptide of amino acid sequence LSTQ (SEQ ID NO: 19) .
2. Use of a peptide of the amino acid LSTQ (SEQ ID NO: 19) in a leader sequence to increase expression of a recombinant growth factor. 3. Use of a peptide of the amino acid LSTQ (SEQ ID NO: 19) as a cleavage site for an α-lytic protease. 4. A peptide leader sequence of up to 200 amino acids
which comprises LSTQ (SEQ ID NO: 19), in which the peptide leader sequence is capable of increasing the expression of recombinant protein compared to recombinant protein expressed without a peptide leader sequence comprising LSTQ.
5. A fusion protein comprising: a) a peptide leader sequence of up to 200 amino acids comprising LSTQ (SEQ ID NO: 19); and b) a polypeptide growth factor. 6. A peptide leader sequence consisting essentially of LSTQ (SEQ ID NO: 19), which is capable of increasing the expression of a recombinant protein compared to expression of the recombinant protein without a peptide leader sequence comprising LSTQ (SEQ ID NO: 19) . 7. A method of increasing the yield of a recombinant protein, comprising the steps of:
(a) providing a construct capable of expressing a fusion protein, in which the fusion protein comprises
(i) a peptide leader sequence of less than 200 amino acids comprising LSTQ (SEQ ID N0:19); and (ii) a polypeptide growth factor;
(b) transforming the construct into a suitable host, and
(c) expressing said fusion protein. (d) A fusion protein comprising a peptide leader sequence of up to 200 amino acids containing LSTQ (SEQ ID NO: 19) and an amino acid sequence encoding a growth factor.
8. A construct comprising a first nucleic acid encoding a peptide leader sequence comprising the amino acid sequence LSTQ (SEQ ID N0:19), a second nucleic acid encoding a cleavage site and a third nucleic acid encoding a growth factor, in which said first, second and third nucleic acids are consecutive . Accordingly, in a first aspect the invention provides a polypeptide fusion protein or construct (hereafter referred to as "the construct") comprising:
a) a peptide or polypeptide leader sequence comprising the amino acid sequence LSTQ (SEQ ID NO: 19); and b) a polypeptide growth factor. In some embodiments, the growth factor is selected from the group consisting of insulin-like growth factor, epidermal growth factor (EGF) , transforming growth factor (TGFβ) , including TGFβl, TGFβ2, and TGFβ3 , fibroblast growth factor (FGF) , platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) . The person skilled in the art would expect on the basis of the results reported in this specification that the beneficial effects of LSTQ observed for IGF and EGF would also apply to the other growth factors listed above. In some embodiments, the amino acid sequence for the growth factor is homologous to that of the human growth factor or a biologically active fragment thereof.
In some embodiments, the construct of the invention exhibits growth factor activity. In some embodiments, the polypeptide growth factor is an IGF which stimulates protein synthesis in rat L6 myoblasts . Methods for assaying this activity are widely known in the art, and are described in G. L. Francis et al . Biochem. J. 233, 207-213, (1986), the entire disclosure of which is incorporated herein by this cross-reference.
In other embodiments, the IGF is a biologically active fragment, functional analogue or derivative of IGF- I or IGF-II. In some embodiments, the IGF is selected from the group consisting of: [Arg] 3 IGF-I:
GPRTLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLE MYCAPLKPAKSA (SEQ ID NO: 20); [Arg] 6 IGF-II: AYRPSRTLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLET YCATPAKSE (SEQ ID NO: 21); des (1-3) IGF-I: TLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYC
APLKPAKSA (SEQ ID NO: 12); and des (1-6) IGF-II:
TLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATP A KSE (SEQ ID NO: 22) In some embodiments the IGF is des (1-3) IGF-I (SEQ ID NO: 12) .
In some embodiments the leader sequence consists essentially of 4 to approximately 200 amino acids. In some embodiments, in addition to LSTQ the leader sequence comprises a polypeptide, or a fragment thereof, which has growth hormone activity. In one embodiment the leader sequence comprises a peptide sequence which is homologous to the first n to 191 N-terminal amino acids of methionine porcine growth hormone (metpGH or MpGH) , or an N-terminal fragment thereof, in which n is an integer between 1 and 190. In some embodiments n is i, 2, 3, 4, 5, 6, 7, 8, 9,
10 , 11 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24 , 25 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38 , 39 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 5 522,, 5 533, 54 or 55.
Alternatively in other embodiments in addition to LSTQ (SEQ ID NO: 19) the leader sequence comprises a polypeptide which is useful in the expression of one or more other growth factors. Examples of other fusion partners used in the expression of growth factors include (a) Uhlen et al . (1992) Curr. Opin.
Biotechnol. ;3 (4) :363-9. The ZZ domain of staphylococcal protein A with IGF-I to improve the solubility of hIGF-I during refolding. (b) Su et al (2006) Protein Pept Lett .; 13 (8) : 785-92. SUMO was fused to hEGF to improve expression and refolding.
(c) Andrades et al (2001) Growth Factors .; 18 (4) : 261-75. A hexa-his tag and a collagen binding domain which contained an enzyme sensitive cleavage site was fused to bFGF to improve purification and targeting of the molecule .
(d) Wilkinson et al (2004) Protein Expr. Purif.; 35 (2) :334-43. Salmon IGF-II was fused with thioredoxin to improve expression levels and refolding levels of this protein. Further examples of growth factor leader sequences may be found on the NCBI Entrez protein database. Entries for the following may be found at the web site http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?CMD=search&D B=protein)
Human FGF accession number 5442453 Human VEGF accession number 181971 Human TGF-alpha accession number 4507461 Human TGF-B2 accession number 4507463 Human PDGF accession number 338209
In other embodiments, the peptide leader sequence in (a) is an amino acid sequence selected from the group consisting of: LSTQ (SEQ ID NO: 19) ; VNLSTQ (SEQ ID NO:23);
• MFPAMPLSSLFLSTQ (SEQ ID NO: 24); MFPAMPLSSLFVNLSTQ (SEQ ID NO: 25); MFPAMPLSSLFVNGLSTQ (SEQ ID NO: 26);
MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQLSTQ (SEQ ID NO: 27) ;
MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNLSTQ (SEQ ID NO: 28) ; and
MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNGLSTQ (SEQ ID NO: 29) . However, it will be clearly understood that it is not essential that the LSTQ portion of the peptide leader sequence is linked consecutively to the growth factor. Thus there may be additional amino acids in the leader sequence on either the N-terminal or the C-terminal side of the LSTQ sequence (SEQ ID NO: 19), or both.
The construct may optionally further comprise a chemical or enzymatic cleavage site between the leader
sequence and the growth factor so as to allow the release of mature growth factor following cleavage of the construct, and /or one or more amino acids encoded by a restriction enzyme recognition site. For example in one embodiment the construct comprises the amino acid sequence VN, which is encoded by the Hpal restriction enzyme recognition site.
In some embodiments, the construct is selected from the group consisting of: MFPAMPLSSLFLSTQ-des (1-3) IGF-I (SEQ ID NO:30);
MFPAMPLSSLFVNLSTQ-deS (1-3) IGF-I (SEQ ID N0:31); MFPAMPLSSLFVNGLSTQ-des (1-3) IGF-I (SEQ ID NO:32); MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQLSTQ~des(l- 3) IGF-I (SEQ ID NO: 33); MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNLSTQ- des (1-3) IGF-I (SEQ ID NO: 34); and
MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNGLSTQ- des (1-3) IGF-I (SEQ ID NO:35).
In some embodiments, the construct is: MFPAMPLSSLFVNLSTQTLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIV DECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO: 36) .
In one embodiment the construct is metpGH(l- 11)VNLSTQdes (1-3) IGF-I (SEQ ID NO:37).
In another embodiment the polypeptide is EGF or an analogue or derivative thereof which retains at least one biological activity of EGF (for the amino acid sequence of EGF, see Bell et al . ; Nucleic Acids Res. 14 (21), 8427- ■ 8446 (1986) ; for a review of EGF activities and methods for their assay, see Jost et al . Eur J Dermatol. 2000 10 (7) :505-10) .
In a further embodiment, the peptide leader sequence in (a) does not comprise either of the amino acid sequences FAHY (SEQ ID NO:38) or GFAHY (SEQ ID NO:39).
In some embodiments, the construct is substantially isolated. For example, it may be substantially isolated from a cell culture medium following recombinant production of the construct .
In a second aspect the invention provides an antibody against a construct according to the invention. It will be clearly understood that specific polyclonal or monoclonal antibodies against the constructs of the invention may readily be raised using methods routine in the art. These antibodies may be directed to an epitope comprising the tetrapeptide LSTQ (SEQ ID NO: 19), and may be used in assays such as immunoassays for the construct of the invention. Any immunoassay format may be used, including but not limited to radioimmunoassays, enzyme- linked immunosorbent assay (ELISA) , chemiluminescence assays, scintillation proximity assays, immunohistochemistry, immunoblotting, for example Western blotting, and immunofluorescence. In a third aspect the invention provides an isolated nucleic acid molecule which encodes a construct according to the invention. The nucleic acid may comprise nucleotide sequences of human origin or synthetic origin, and may be single-stranded or double-stranded DNA. The nucleic acid may have codons for optimized expression in a desired host organism, such as E. coli. In some embodiments the nucleic acid molecule is cDNA or RNA.
In a fourth aspect the invention provides a vector comprising a nucleic acid molecule according to the invention. In some embodiments the vector may comprise a first nucleic acid molecule encoding the construct and a second nucleic acid molecule encoding a desired polypeptide. For example, the desired polypeptide may be a therapeutically useful protein. The vector may be of any suitable type, and may be an expression vector or a plasmid. For example, the plasmid pGHXSC.4 may be used; this plasmid comprises the pGH coding region.
In a fifth aspect the invention provides a host cell transformed by a vector according to the invention. In some embodiments the host cell is a prokaryotic cell. One useful host cell is E. coli, for example JE?. coli strain JMlOl. Alternatively the host cell may be a eukaryotic
cell, such as a mammalian cell. In some embodiments, the mammalian cell is a CHO cell, Per.Cδ™ cell, HEK293 cell, Vero cell, or MDCK cell, or any fibroblast cell line. Alternatively the host cell may be a yeast, such as Saccharomyces cereviseae or Pichia pastoris.
In a sixth aspect, the invention provides a process for the production of a construct according to the invention, which process comprises:
(a) culturing a host cell according to the invention under conditions favourable for cell growth and expression of the construct; and
(b) isolating the construct from the culture.
In some embodiments the invention provides a process for the production of a construct according to the invention, comprising the steps of:
(a) introducing a vector into a unicellular organism, in which the vector can express a nucleic acid molecule encoding a construct according to the invention;
(b) culturing the unicellular organism; (c) expressing the construct encoded by the nucleic acid molecule; and
(d) isolating the construct from the culture. In some embodiments, the unicellular organism is a prokaryotic organism. The prokaryotic organism may, for example, be a bacterial strain, such as a strain of E. coli. The E. coli strain JMlOl is one suitable unicellular organism.
The process may further include the step of cleaving the construct at a cleavage site between the leader sequence and the growth factor sequence to release the growth factor polypeptide, which may then be isolated. It is known from US Patent No. 5,330,971 that cleavage to remove the growth hormone-derived leader sequence may not be required to achieve adequate biological activity if this leader sequence is small. For example, where the leader sequence is approximately 10 amino acids, then cleavage of the leader sequence may not be required. The
person skilled in the art will readily be able to determine whether cleavage is required in any specific leader sequence-growth factor combination, using routine methods . In a seventh aspect the invention provides a method for culturing mammalian cells, comprising culturing the cells under cell growing conditions in a cell culture medium comprising a construct according to the invention in which the growth factor is IGF. The medium suitably also comprises
(a) an osmolality regulator;
(b) a buffer;
(c) an energy source;
(d) at least one additional amino acid; and (e) an inorganic, organic or recombinant iron source .
In some embodiments, the polypeptide is at a concentration of between 10-100 ng/ml . For example the polypeptide may be at a concentration of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng/ml. In some embodiments, the medium does not contain serum. In other embodiments, each component of the medium is obtained from a source other than directly from an animal source. The medium may further comprise one or more of non-ferrous metals, vitamins, transition metals, carrier proteins such as albumin or cofactors.
The cells may comprise an IGF receptor.
Alternatively the cells may comprise an insulin receptor. In some embodiments, the mammalian cell is a CHO cell, Per.C6™ cell, HEK293 cell, Vero cell, MDCK cell, or fibroblast cell line.
In one embodiment, the invention provides a method for culturing mammalian cells, the method comprising culturing and growing the cells in the absence of serum, in a medium comprising, per litre of medium:
(a) about 10 ng-50 ng of a construct according to the invention;
(b) an osmolality regulator to maintain the osmolality of the medium within the range of about 200-350 mOsm;
(c) a buffer to maintain the pH of the medium within the range of about 6.5 to 7.5;
(d) about 1,000-10,000 mg of a monosaccharide;
(e) about 400-600 mg of L-glutamine;
(f) about 10 -200 mg of at least one amino acid selected from the group consisting of L-alanine, L- arginine, L-asparagine, L-aspartic acid, L-cystine, L- glutamic acid, glycine, L-histidine, L-isoleucine, L- leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine; and (g) about 0.25-5 mg of an inorganic or recombinant iron source .
In an eighth aspect the invention provides a composition comprising a construct according to the invention, together with a pharmaceutically, veterinarily or cell culture acceptable carrier. In some embodiments the composition is in dry powder form, and may further comprise a bulking agent. In other embodiments the composition comprises a polypeptide according to the invention formulated in acid. In some embodiments the acid is acetic acid or hydrochloric acid. In other embodiments, the polypeptide is formulated in 100 mM acetic acid or 10 mM hydrochloric acid.
In a ninth aspect the invention provides a cell culture system, comprising (a) a fermenter adapted for mammalian cell culture;
(b) a mammalian cell comprising a nucleic acid sequence encoding a desired polypeptide; and
(c) a mammalian cell culture medium comprising a construct according to the invention. For example, the mammalian host cell may be engineered to express both a desired protein such as a protein therapeutic agent and a construct according to the
invention, which stimulates the proliferation of the mammalian host cell and increases yields of the desired protein. Thus in some embodiments the invention provides a cell culture system comprising: (a) a fermenter adapted for mammalian cell culture;
(b) a mammalian cell comprising a nucleic acid sequence encoding (i) a desired polypeptide and (ii)a construct according to the invention; and
(c) a mammalian cell culture medium. In some embodiments the construct comprises an IGF sequence and exhibits growth factor activity, which stimulates the proliferation of the mammalian host cell, increases cell survival, prolongs cell viability, and/or increases yield of the protein. In some embodiments the cell culture medium comprises water, an osmolality regulator, a buffer, an energy source, at least one additional amino acid and an inorganic, organic or recombinant iron source.
In some embodiments, the host cell is a CHO cell, the desired polypeptide is an anti-inflammatory protein, and the construct is:
MFPAMPLSSLFWLSTQTLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIV DECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO: 32)
In a tenth aspect the invention provides a kit comprising a liquid composition or dry powder composition comprising a construct according to the invention sealed in a vial, cartridge or container. The kit may be labelled by, or accompanied with, instructions for use in mammalian cell culture. In some embodiments, the instructions specify that the contents are not suitable for human therapeutic use .
In a eleventh aspect the invention provides a composition for the treatment of a protein accumulation deficiency condition or protein loss in a mammal, comprising an effective amount of a construct according to the invention, together with a pharmaceutically acceptable diluent, carrier or excipient, in which the construct
exhibits insulin-like growth factor activity.
In some embodiments, the composition comprises the construct in an amount sufficient to provide a dose of approximately 0.01 to 10, preferably 0.1 to 1 mg/kg body weight/day. The construct may be present in a unit dosage form in amounts from approximately 0.02 to 2000 mg. Slow- release pellet implants, as provided in conventional practice, are the preferred method of administration to animals . In a twelfth aspect the invention provides a method of treatment of a condition selected from the group ■ consisting of protein accumulation deficiency condition or protein loss; chronic growth disorders, including growth hormone deficiency and somatomedin deficiency; disorders associated with insufficient growth or tissue wasting, including, but not limited to, cancer, cystic fibrosis, Duchenne muscular dystrophy, Becker dystrophy, autosomal recessive dystrophy, polymyositis and other myopathies; acute conditions associated with poor nitrogen status including, but not limited to, burns, skeletal trauma and infection; or to promote growth, improve nitrogen status and/or to treat catabolic disorders in infants or premature babies, in a subject in need of such treatment, comprising the step of administering an effective amount of a construct according to the invention which exhibits insulin-like growth factor activity to a mammal in need of such treatment .
In a thirteenth aspect the invention provides the use of a construct according to the invention which exhibits insulin-like growth factor activity in the manufacture of a medicament for the treatment of a condition selected from the group consisting of protein accumulation deficiency condition or protein loss; chronic growth disorders, including growth hormone deficiency and somatomedin deficiency; disorders associated with insufficient growth or tissue wasting, including, but not limited to, cancer, cystic fibrosis,
Duchenne muscular dystrophy, Becker dystrophy, autosomal recessive dystrophy, polymyositis and other myopathies; acute conditions associated with poor nitrogen status including, but not limited to, burns, skeletal trauma and infection; or for promoting growth, improving nitrogen status and/or treatment of catabolic disorders in infants or premature babies .
The mammal may be a human, or may be a domestic, companion or zoo animal. While it is particularly contemplated that the constructs of the invention and growth factors expressed using the leader sequences of the invention are suitable for use in medical treatment of humans, they are also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as non-human primates, felids, canids, bovids, and ungulates. It will be appreciated that there is a very high degree of sequence conservation in growth factor sequences between mammalian species. While the invention is described in detail with reference to IGF and EGF, it is to be clearly understood that it is contemplated that the invention described herein is also applicable to other growth factors.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1 shows dose response curves illustrating the ability of (A) LONG 0 R3IGF-I (GroPep Ltd) and (B) metpGH(l- 11) WLSTQdes(1-3) IGF-I to stimulate the growth of L6 myoblasts in culture.
DETAILED DESCRIPTION
Definitions
In the claims of this application and in the description of the invention, except where the context
requires otherwise due to express language or necessary- implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
As used herein, the singular forms "a" , "an" , and "the" include the corresponding plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a polypeptide" includes a plurality of such polypeptides, and a reference to "an amino acid" is a reference to one or more amino acids. Where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all values in between these limits.
For the purposes of this specification, IGF-I, IGF- II and biologically active fragments, functional analogues or derivatives thereof are considered to be equivalent, and are referred to collectively as "IGF" or "IGFs" .
The term λ methionine porcine growth hormone (metpGH) ' means porcine growth hormone, in which methionine has been substituted for the amino acid normally at the N-terminus. The terms "fragment", "analogue", and "derivative" of a growth factor as used herein mean a molecule derived from a growth factor, in which the molecule retains at least one biological function or activity as that of the original growth factor. The phrase "consisting essentially of" means that other amino acids can be added to the amino or carboxyl terminal of the peptide LSTG without affecting the ability of LSTG to increase protein expression.
The term "amino acid" or "amino acid residue", as used herein, refers to naturally occurring L amino acids. The commonly used one-and three-letter abbreviations for amino acids are used herein (Bruce Alberts et al . ,
Molecular Biology of the Cell, Garland Publishing, Inc., New York (3d ed. 1994)) .
An "isolated" construct or desired polypeptide is one which has been identified and separated and/or recovered from a component of the culture in which it is produced. Contaminant components of the culture are materials which would interfere with cell culture, diagnostic or therapeutic uses for the construct or desired polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes . In some embodiments, the construct or desired polypeptide will be purified to
(1) greater than 95% by weight as determined by the Lowry method, and most preferably more than 99% by weight, (2) a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of an automated Edman degradation N-terminal sequenator, or
(3) homogeneity by SDS-PAGE under reducing or non- reducing conditions, as assessed using Coomassie blue or silver stain. Silver stain is more sensitive.
"Percent (%) amino acid sequence identity" with respect to the polypeptide sequences referred to herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
The expression "essentially free" with reference to a component of a cell culture medium means that the component is not deliberately added to or present in the culture medium, and if detectable is present as a result of unavoidable impurities in other components of the culture medium.
The term "antibody" includes all classes and
subclasses of intact immunoglobulins, and also encompasses antibody fragments. The term "antibody" specifically encompasses monoclonal antibodies, including antibody fragment clones .
Abbreviations
DTT dithiothreitol
EDTA ethylene diamine tetraacetic acid
EGF epidermal growth factor FGF fibroblast growth factor
IGF insulin-like growth factor
IPTG isopropylthiogalactoside
MetpGH, MpGH methionine porcine growth hormone
PCR polymerase chain reaction PDGF platelet-derived growth factor
RP-HPLC reversed phase high performance liquid chromatography
TGF transforming growth factor
VEGF vascular endothelial growth factor
The present invention may performed without any undue need for experimentation using, unless otherwise indicated, conventional techniques of molecular biology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following well-known publications, which are incorporated herein by reference:
1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories,
New York, Second Edition (1989) ;
2. DNA Cloning: A Practical Approach, (D. N. Glover, ed., 1985), IRL Press, Oxford,;
3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, particularly
the chapters therein by Gait, ppl-22; Atkinson et 3.1., pp35-81; Sproat et al., pp 83-115; and Wu et al. , pp 135-151;
4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds . , 1985) IRL Press,
Oxford;
5. Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed. , 2000), ISBN 0199637970; 6. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford;
7. Perbal, B., A Practical Guide to Molecular Cloning (1984) ;
8. J. F. Ramalho Ortigao, "The Chemistry of Peptide Synthesis" Jn: Knowledge database of Access to
Virtual Laboratory website (Interactiva, Germany) ;
9. Sakakibara, D., Teichman, J., Lien, E. L. and Fenichel, R. L. (1976) . Biochem. Biophys. Res. Commun. 73 336-342 10. Merrifield, R. B. (1963). J. Am. Chem. Soc. ^5, 2149-
2154. 11. Barany, G. and Merrifield, R. B. (1979) in The
Peptides (Gross, E. and Meienhofer, J. eds.), vol.
2, pp. 1-284, Academic Press, New York. 12. Bodanszky, M. (1984) Principles of Peptide
Synthesis, Springer-Verlag, Heidelberg.
13. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg.
14. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.
15. Handbook of Experimental Immunology, VoIs. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications) .
The construct or fusion protein of the present invention comprises a peptide leader sequence containing or consisting essentially of LSTQ (SEQ ID NO: 19), and an amino sequence encoding a growth factor. It will be appreciated that that the fusion protein of the present invention can be produced by recombinant DNA methodology. For example, a nucleic acid molecule encoding a leader peptide sequence of the present invention can be ligated to a nucleic acid molecule encoding a growth factor. Techniques for ligating such nucleic acids are well known in the art. If required the nucleic acid molecule can be further manipulated to introduce additional restriction enzyme cleavage sites, or to encode sites for enzymatic cleavage of the expressed protein. Once the nucleic acid of the present invention has been constructed it can be introduced into a vector for expression.
A "vector" is a plasmid or other DNA molecule which is capable of replicating autonomously within a host cell, and as such, is useful for performing two functions in conjunction with compatible host cells (a vector-host system) . One function is to facilitate the cloning of the nucleic acid which encodes the construct or desired polypeptide of the invention, i.e., to produce usable quantities of the nucleic acid. The other function is to direct the expression of the construct or desired polypeptide. One or both of these functions is performed by the vector-host system. The vectors will contain different components, depending upon the function they are to perform as well as the host cell with which they are to be used for cloning or expression. In one embodiment, the vector is a plasmid such as pGHXSC.4. This plasmid has a modified RBS/spacer region and strategic 5 ' -codon alterations downstream from its powerful trc promoter (J. Brosius et al. J. Biol. Chem. 260, 3539, 1985; P. D. Vize & J. R. E. Wells, FEBS Lett. 213, 155, 1987) .
The- vector may be introduced into the host cell by
methods which are also known in the art, for example by transformation, transduction or transfection techniques-.
In some embodiments of the invention, the process may include the preliminary step of introducing coding for a cleavable bond between the first and second DNA sequences in the vector. This may involve subjecting the first DNA sequence to mutagenesis in order to introduce a cleavable sequence at the 3' end thereof. Such mutagenesis methods are known in the art, and include in vitro PCR mutagenesis techniques. A suitable in vitro mutagenesis PCR reaction may utilize an oligonucleotide which introduces a cleavable bond between the first and second DNA sequences. A hydroxylamine-cleavable bond or a subtilisin-cleavable bond may be introduced. Alternatively or additionally an enzymatically-cleavable amino-acid motif may be introduced. Alternatively or additionally an amino acid motif which is susceptible to chemical cleavage may be introduced.
If desired, mutagenesis steps may be included to reduce the size of the sequence encoding the leader sequence and/or to introduce a restriction enzyme recognition site to facilitate additional mutagenesis steps.
The construct of the invention may be modified at the carboxy- or amino-terminal end, without loss of biological activity. Thus it is intended that the present invention includes within its scope constructs which comprise further amino acids in addition to the "core" LSTQ residues of the leader sequence. The construct may also include additional amino acids in the growth factor sequence, such as additions or modifications to this sequence .
The constructs and desired polypeptides described herein may be produced by recombinant DNA techniques known to the person skilled in the art, for example those described in T. Maniatis et al . , in Molecular Cloning, A Laboratory Manual. CSH Lab. N. Y. (1989). Production of the
construct or polypeptide of the invention involves culturing a host cell which has been transformed with a vector according to the invention, and isolating the construct or polypeptide from the culture. Suitable methods for the expression and purification of IGF and analogues thereof are disclosed in US patent No. 5,330, 971. It is to be clearly understood that the present invention extends to biologically active fragments or functional analogues of human IGF, i.e. analogues or derivatives of human IGF in which the wild-type IGF sequence contains additions, deletions or substitutions by other amino acids or amino acid analogues, in which the biological activity of the IGF is retained. IGF analogues suitable for use in the present invention include those described in US patents Nos . 5,077,276, 5,164,370,
5,470,828, and 5,330,971, and in International Patent Application No. PCT/AU99/00292. These analogues include des (1-3) IGF-I, des (1-6) IGF-II, analogues of IGF-I with amino acid substitutions at amino acid position 1 to 3, analogues of IGF-II with amino acid substitutions at amino acid position 1 to 6, porcine growth hormone-IGF fusion proteins and IGF analogues incorporating heparin-binding motifs which enable the IGF analogue to bind to fixed surfaces . Methods for the identification, production and biological characterisation of active fragments, functional analogues or derivatives of growth factors are well known to those of ordinary skill in the art, and can be addressed with no more than routine experimentation. In one form the growth factor fragment, functional analogue or derivative has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, or 99% amino acid sequence homology, usually at least 70% amino acid sequence homology, preferably at least 90%, and even more preferably at least 95% amino acid sequence homology with the native growth factor amino acid sequence in
question. In some embodiments, the biologically active fragment, functional analogue or derivative of the growth factor demonstrates biological activity.
In some embodiments, the growth factor may have 100% amino acid sequence homology to the amino acid sequence of the corresponding human growth factor. For example the IGF may have 100% amino acid sequence homology to the amino acid sequence of human IGF-I. The amino acid sequence for human IGF-I is as follows: GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLE MYCAPLKPAKSA (SEQ ID NO: 1)
Furthermore, the IGF may have up to 100% amino acid sequence homology to the amino acid sequence of human IGF- II. The amino acid sequence for human IGF-II is as follows:
AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLE T YCATPAKSE (SEQ ID NO: 7). Although there are species differences between IGF-II sequences, these are subtle; see the bovine (Francis et al . (1988) Biochem. J. 251, 95- 102), ovine (Francis et al . (1989) Endocrinology. 24,
1173-1183), porcine (Francis et al . (1989) J. Endocrinol. 122, 681-687), and murine (Stempien et al . (1986) DNA. 5, 357-361) sequences.
Methods for assessing amino acid sequence identity are well known in the art. For example, a suitable program for determining percentage sequence identity is BLAST 2.0 Sequence Comparison (NIH) . Preferably, the limiting parameters imposed for such a task are the default settings for the program. Therefore the person skilled in the art would readily, and with a reasonable expectation of success, be able to predict which alterations to the protein sequence would affect the structure of the growth factor, and affect its biological activity. Preferably the alteration does not alter the growth factor domain responsible for affinity to the corresponding growth factor receptor.
While experimentation may be required to verify the
biological activity of fragments, analogues or derivatives of growth factors such as IGF, the degree of experimentation required is merely routine in the art. For example, skilled persons will readily be able to use the rat L6 myoblast protein synthesis assay (G. L. Francis et al. Biochem. J. 233, 207-213 1986) to identify biologically active fragments or functional analogues of IGFs.
The polypeptide of the invention may be produced using methodology which is known to the person skilled in the art . Representative methods are described in the examples provided herein, but it will be clearly understood that other suitable methods are known in the art. Following isolation of the polypeptide, the leader sequence may be cleaved from the second amino acid sequence to release the relevant protein for subsequent use. Therefore the polypeptide is engineered to contain a chemical or enzymatic cleavage site. In order to isolate the construct or desired polypeptide product from the culture medium after cell disruption, e.g. by cell lysis, conventional procedures are used. These include but are not limited to chromatography involving ion exchange chromatography, affinity chromatography, reversed phase high performance liquid chromatography (RP-HPLC) or size exclusion chromatography techniques and/or by sedimentation, e.g. centrifugation, and other known techniques for the purification of polypeptides. Where the construct or desired polypeptide is expressed as an insoluble aggregate and/or is denatured, solubilization and/or renaturation may be effected using conventional techniques. For example, as would be understood by the person skilled in the art the construct or desired polypeptide may be folded and purified using a method such as :
(a) dissolution of inclusion bodies in 8 M Urea, 40
mM Glycine, 1 mM EDTA, 0.1 M Tris, 20 raM DTT at pH 9.1;
(b) desalting into 8 M urea/50 mM glycine pH 9.2 on Sephadex G-25;
(c) oxidative refolding with 2 M Urea, 10 mM Glycine, 0.1 M Tris, 1.3 mM EDTA, 1 mM 2-hydroxymethyl disulphide and 0.4 mM DTT at pH 9.1, at room temperature for 90 minutes with a final protein concentration of 0.125 mg/ml .
(d) recovering the construct or desired polypeptide from the refolding mixture by C4 RP-HPLC using 0.1% TFA and a propan-1-ol gradient;
(e) ion exchange chromatography on Mono S resin using an ammonium acetate gradient at pH 4.8;
(f) C18 RP-HPLC in 0.13% heptafluorobutyric acid (HFBA) and elution with an acetonitrile gradient; and
(g) desalting into 0.1 M acetic acid.
The isolation of the constructs and desired polypeptides of the invention may be performed by any convenient method. Many suitable methods are known in the art. For example, the isolation of the polypeptide may be performed by ion-exchange chromatography, size exclusion chromatography, affinity chromatography or reverse phase high performance liquid chromatography.
In some embodiments of this aspect of the invention, prior to isolation, the construct or desired polypeptide is subjected to a dissolution and refolding step to release a biologically active product, especially where the host cell is a prokaryotic cell and the construct or desired polypeptide is expressed as an inclusion body. The construct or desired polypeptide produced according to the methods of the invention may be isolated as inclusion bodies within the engineered unicellular organisms. In some circumstances the construct or desired polypeptide may be secreted rather than being expressed as an inclusion body. The construct or desired polypeptide may be subjected to further processing steps as required. In some embodiments of this aspect of the invention, the
isolating step may comprise one or more of the following steps:
(i) homogenisation of the cultured host cell to release inclusion bodies; (ϋ) dissolution of inclusion bodies;
(iii) refolding of the construct or desired polypeptide;
(iv) cation exchange chromatography;
(v) reverse phase high performance liquid chromatography;
(vi) size exclusion chromatography; and
(vii) ultrafiltration.
Following the isolation step, the construct or desired polypeptide may be subjected to a freeze-drying step. The construct or desired polypeptide may also be sterilized, for example by microfiltration using an apparatus such as a Milli-GV filter unit (membrane area 4cm 2 ) .
In some embodiments, the isolated product is cleaved so as to release the growth factor or desired polypeptide from the leader sequence. Preferably the cleavage is performed using chemical or enzymatic cleavage. The construct may be engineered to contain a chemical or enzymatic cleavage site, using methods known in the art, such as those described in US Patent No. 5,330,971.
In some embodiments, the process may include the preliminary step of introducing a nucleic acid sequence which encodes a cleavable bond between the first and second DNA sequences in the vector. The process may also include the preliminary steps of:
(a) providing a vector;
(b) providing a nucleic acid molecule encoding a construct according to the invention; (c) digesting the vector DNA to isolate a fragment of DNA which includes a first DNA sequence encoding an amino acid sequence comprising the amino acid sequence
LSTQ ;
(d) subjecting the first DNA sequence to mutagenesis to introduce a cleavable sequence at the 3 1 end thereof; and (e) ligating the fragment containing the first DNA sequence to a second DNA sequence encoding a growth factor polypeptide .
Preferably the digestion includes a subsequent purification step to provide a purified DNA fragment . The second DNA sequence may be synthetic or recombinant, or may be obtained from a natural source. In a further embodiment of this aspect of the invention, the process may further include the preliminary step of chemically synthesising the second DNA sequence. The chemical synthesis may be undertaken in any conventional manner. Furthermore, the digestion and ligation steps may be undertaken in any conventional manner.
In another embodiment, the process may include the preliminary steps of (a) providing a vector;
(b) providing
(i)a first DNA sequence encoding a construct according to the invention, and
(ii) a second DNA sequence joined at the 3' end of the first DNA sequence, wherein the second DNA sequence encodes a desired polypeptide;
(c) digesting the vector DNA to isolate a fragment of DNA which includes the first DNA sequence;
(d) subjecting the first DNA sequence to mutagenesis to reduce the size of the amino acid sequence if required and/or to introduce a recognition site,- and
(e) ligating the fragment containing the first DNA sequence to the second DNA sequence .
In some embodiments of the seventh aspect of the invention, the osmolality regulator maintains the medium at 200-350 mOsm. For example the osmolality regulator maintains the medium at 200, 210, 220, 230, 240, 250, 260,
270 , 280 , 290 , 300 , 310 , 320 , 330 , 340 , or 350 mOsm .
The concentration of the energy source can be within the range of 1,000-10,000 mg/liter. For example, the concentration of the energy source is 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000,
6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, or 10,000 mg/liter. In some embodiments, the energy source is a monosaccharide .
In some embodiments, the amino acids are selected from the group consisting of L-alanine, L-arginine, L- asparagine, L-aspartic acid, L-cystine, L-cysteine L- glutamic acid, glycine, L-histidine, L-isoleucine, L- leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-cysteine and L-valine.
In some embodiments, the iron source is an inorganic ferric or ferrous salt which is provided in a concentration of from 0.25-5 mg/liter. For example the iron source may be at a concentration of 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25,
3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5.0 mg/liter. Examples of recombinant iron sources include lactoferrin and transferrin or fragments thereof which are capable of sequestering iron and binding to the transferrin receptor. Examples of organic iron sources include topoplone, α- thujaplacin, β-thujaplacin, γ-thujaplacin, 3-hydroxypyran- 4-one, 3-hydroxy-2-methylpyran-4-one, 2-Ethyl-3- hydroxypyran4 -one , 3 , 5-dihydroxy-2-methylpyran-4-one, 2- benzyl-3 -hydroxypyran-4 -one, 2-ethyl-3 -hydroxy- 6- methylpyran-4-one and 2-hydroxy-1, 4 -naphthoquinone (Akers et al. J Bacteriol. 1980; 141(1): 164-168)
The medium may further comprise L-glutamine. Preferably the concentration of L-glutamine is within the range of 400-600 mg/liter. For example the concentration of L-glutamine is 400, 425, 450, 475, 500, 525, 550, 575, or 600 mg/liter.
In some embodiments the medium further comprises a
lipid such as cholesterol, a steroid or a fatty acid in an amount of 0.05-10 mg/1. For example, the concentration of the lipid factor is 0.05, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mg/liter.
The pH of the medium can be maintained by use of a buffer at about 6.5 to about 7.5. For example the pH may be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. The medium may further comprise a peptide or protein digest, hydrolysate or extract.
In some embodiments, the medium is essentially free of hypoxanthine and thymidine . The medium may further comprise methotrexate . It has been found that the process described above produces biologically active growth factors such as IGF in high yield.
Quantification of the polypeptide on the basis of its specific molar absorbency may be performed by a calibrated RP-HPLC method, such as that described in Francis et al . (1992) J. MoI. Endo. (8) 213-223.
Determination of the potency of a construct in which the growth factor is IGF may comprise an assay which tests the stimulation of protein synthesis in rat L6 myoblasts, as described in G. L. Francis et al . Biochem. J. 233, 207- 213 (1986) , or by radioimmunoassay, using conventional methods. Suitable assays for other growth factors within the scope of the invention are well known in the art.
The construct of the invention may be administered by any suitable route, for example by parenteral injection. Although in critical care situations the preferred method of administration may be via addition to parenteral fluids, other methods may be appropriate. The person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will
depend on the nature and state of the condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered. The carrier or diluent, and other excipients, will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case . Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA (2000) and The British National Formulary 43rd ed. (British Medical Association and Royal Pharmaceutical Society of Great Britain, 2002; http://bnf.rhn.net), the contents of which are hereby incorporated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's "The Pharmacological
Basis of Therapeutics" (7th ed. , 1985).
The dose rates and times for the administration of the construct to human subjects may for example be approximately 0.01 to 10 mg/kilogram/day. Dose rates of approximately 0.1 to 1 mg/kilogram/day are preferred.
The construct may be administered alone or with various diluents, carriers or excipients which have been chosen with respect to the intended method of administration and are well known. Acceptable diluents, carriers or excipients are nontoxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids,- antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween (polysorbate) , Pluronics or polyethylene glycol (PEG) .
The invention will now be further described in detail by way of reference only to the following examples . These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus the invention encompasses any and all variations which become evident as a result of the teaching provided herein.
Example 1 Identification of the LSTQ sequence as a cleavage site
The expression of heterologous proteins using recombinant DNA technology in some cases requires a "fusion partner" to prevent degradation and to increase expression in E. coll. This has been achieved by inclusion of the first 11 amino acids of porcine growth hormone at the N-terminus as the fusion partner (Francis et al . , (1992), Journal of Molecular Endocrinology 8:213-223). Furthermore, the porcine growth hormone-derived fusion partner has been shown to promote the correct folding of the fusion protein into its preferred biologically active form (Milner et al . , (1995), Biochemical Journal 308:865- 871) . For some applications it may be necessary to remove the fusion partner from the protein of interest, for example for pharmaceutical or veterinary use . To achieve this, a linker sequence which may be cleaved specifically can be inserted between the fusion partner and the desired protein, as is the case in the present invention.
The cleavage reaction may be achieved by either chemical means or by using a protease. For example the
enzyme α-lytic protease from the soil bacterium Lysobacter enzymogenes is a good candidate for this role, following mutagenesis to contain the substitutions
Alal90/His213/Leu218, giving rise to the so-called mutant 9 (Graham et al, (1993) Biochemistry 32 6250-6258; Haggett et al., 1994) Archives of Biochemistry and Biophysics 314:132-141). These authors confirmed that the strongly preferred site for cleavage with this mutant was following a Met or His residue, although molecular modelling suggested that Asn and Gin could in principle emulate the former residues in providing a suitable cleavage point. However, the rates of hydrolysis of ester and amide substrates involving GIn by mutant 9 were negligible compared with those for the preferred Met or His residues . While these studies focused on the linkage motif composition at the cleavage point, which is designated Pl, other studies attempted to construct a preferred cleavage motif containing the upstream residues P2 , P3 and P4 in the linker (Lien, S.: PhD thesis, University of Adelaide 2001; Lien et al, . (2003) Journal of Protein Chemistry 22:155-166). These authors employed a combinatorial approach using phage display in order to select optimal linkers for fusion proteins. A randomized library of fusion motif-containing phage was screened, and optimal linker motifs for cleavage by mutant 9 selected for further evaluation. The linkers selected in each case were, in order of preference, Asp-Ser-Thr-Met (DSTM) and Ile-Asn-Ala-His (INAH), for positions P4 , P3 , P2 and Pl, and, in keeping with the previously- established substrate preferences for mutant 9 at Pl. Although increased cleavage was observed when the amino acid at the Pl position was Gin, this was not identified as being of any importance, and there was no suggestion of using GIn at Pl with Leu, Ser and or Thr. The present inventors have independently reviewed these observations, and have formulated a further hypothesis regarding the role played by each part of the
motif, which led to the present invention. Firstly, the choice of Leu as the P4 residue departs from the unexpected preference for Asp identified by Lien et al . It is now proposed by the inventors that its prominence in the results of the phage display selection for the P4 position reported by Lien et al arose because of undefined factors in the screening assay, and that the more conservative hydrophobic amino acid Leu would perform better. The polar residues, Ser and Thr at P3 and P2 respectively, were retained from the Asp-Ser-Thr-Met motif, in the belief that these polar residues might contribute to exposure of the motif to the cleaving protease by reducing the hydrophobic character. The latter changes are not as extreme as introducing a charged amino acid like Asp into the linker.
It has been accepted in the art that mutant 9 has a preference of for cleavage at His or Met compared to GIn, as reported by Haggett et al . , (1994) (Archives of Biochemistry and Biophysics 314:132-141). Consequently our finding that our proposed linker motif, Leu-Ser-Thr-Gln
(LSTQ) , provided a readily cleavable substrate for mutant 9 α-lytic protease was unexpected.
Example 2 Production of DNA encoding metpGH(l- lpVNLSTQdes (1-3) IGF-I by mutagenesis
A chemically-synthesised nucleic acid encoding des (1-3) IGF-I was assembled in the plasmid pCR-Script. In addition to 39 changes to the 201 native des (1-3) IGF-I nucleotides for the purpose of optimised E. coli codon usage, the sequence was modified to introduce a nucleic acid sequence encoding the amino acid sequence LSTQ at a site 5' to the des (1-3) IGF-I gene. Mutagenesis was performed using the PCR technique. The nucleotide sequence encoding the VNdes (1-3) IGF-I polypeptide with the encoded protein product is shown below:
Hpal
GTT AAC ACC CTG TGC GGC GCG GAA CTG GTG GAT GCG CTG CAG TTT GTG TGC GGC GAT CGT GGC TTT TAT TTT AAC AAA CCG ACC GGC TAT GGC AGC AGC AGC CGT CGT GCG CCG CAG ACC GGC ATT GTG GAT GAA TGC TGC TTT CGT AGC TGC GAT CTG CGT CGT CTG GAA ATG TAT TGC GCG CCG CTG AAA CCG GCG AAA AGC GCG TGA TAA GCT T (SEQ ID NO: 40) HindiII
An oligonucleotide 29 base pairs long was used to introduce a HindiII (AAGCTT) recognition site 3' to the stop codons of the nucleotide sequence encoding the des(l- 3) IGF-I polypeptide. The oligonucleotide sequence is shown below:
5' AGGTCGAAGCTTATCACGCGCTTTTCGCC 3' (SEQ ID NO: 41)
A second oligonucleotide 38 base pairs long was used to introduce the LSTQ sequence by mutagenesis. The oligonucleotide sequence is shown below:
5' TTAGACGTTAACCTGAGCACCCAGACCCTGTGCGGCGC 3' (SEQ ID NO: 42)
The protein sequence encoded by the 5' end of the PCR fragment is shown below:
V N L . S T Q T L C G
GTT AAC CTG AGC ACC CAG ACC CTG TGC GGC- ( SEQ ID NO : 43 ) Hpal enzymatic cleavage des (1-3 ) IGF-I recognition site
A mutant clone of pCR-Script comprising DNA encoding VNLSTQdes (1-3 ) IGF- I was selected, double stranded DNA was prepared, the DNA was digested with Hpal and Hindi II , and was subsequently isolated . This fragment was then cloned into the expression plasmid for MetpGH (1 -11) VNLSTQEGF from
which the Hpal-Hindlll fragment (LSTQEGF) had been removed (pGHXSC.4) to give the expression plasmid for MetpGH(l- lUVNLSTQdes (1-3) IGF-I (see Example 4).
Example 3 Introduction of PAPM 5' to the nucleic acid sequence encoding des (1-3) IGF-I
Mutation to introduce a nucleic acid sequence encoding the amino acid sequence PAPM was carried out essentially as described in Example 2. An identical 29 base pair oligonucleotide (SEQ ID NO: 29) was used to introduce a HindIII (AAGCTT) restriction site 3' to the stop codons of the nucleotide sequence encoding the des(l- 3 ) IGF-I polypeptide . However, in this Example a second oligonucleotide 33 base pairs long was used to introduce a DNA sequence encoding PAPM to create VNPAPMdes (1-3) IGF-I . The oligonucleotide sequence is shown below:
5' TTAGACGTTAACCCGGCGCCGATGACCCTGTGC 3' (SEQ ID NO: 44)
The protein sequence encoded by the 5' end of the PCR fragment is shown below:
V N P A P M T L C
GTT AAC CCG GCG CCG ATG ACC CTG TGC- (SEQ I NO:45) Hpal enzymatic cleavage des (1-3) IGF-I recognition site
A mutant clone of pCR-Script comprising DNA encoding VNPAPMdes (1-3) IGF-I was selected and cloned into the Hpal- HindIIl treated MetpGH (1-11) VNLSTQEGF expression vector (pGHXSC.4) to provide the expression vector for MetpGH(l- 11) VNPAPMdes (1-3) IGF-I, using the method described in Example 2.
Example 4 Cloning PAPMpEGF 3' to the MetpQH (1-11) sequence in pGHXSC.4
A chemically-synthesised nucleic acid sequence encoding porcine EGF with codons optimised for E: coli expression contained 35 changes of 159 nucleotides of the native porcine EGF nucleotide sequence. The nucleotide sequence encoding the PAPMpEGF polypeptide is shown below:
Hpal
GTT AAC CCG GCG CCG ATG AAC AGC TAT AGC GAA TGC CCG CCG
AGC CAT GAT GGC TAT TGC CTG CAT GGC GGC GTG TGC ATG TAT
ATT GAA GCG GTG GAT AGC TAT GCG TGC AAC TGC GTG TTT GGC
TAT GTG GGC GAA CGT TGC CAG CAT CGT GAT CTG AAA TGG TGG GAA CTG CGT TAG TAA GCT T (SEQ ID NO: 46)
HindiII
This optimised sequence in the plasmid pSTBlue-1 was digested with Hpal and HindiII and was subsequently isolated. The purified fragment was cloned into
Hpal/HindiII cut MetpGH (1-11) VNPAPHIGF-II expression vector (pGHXSC.4), from which the PAPHIGF-II gene had been removed, to create MetpGH(l-ll) VNPAPMpEGF.
Example 5 Mutagenesis to create MetpGH (1-1) VNLSTQpEGF
Mutagenesis was performed on the optimised pEGF sequence from Example 4, using PCR, in order to replace the nucleic acid encoding the amino acid sequence PAPM with nucleic acid encoding the amino acid sequence LSTQ 5' to the pEGF sequence. An oligonucleotide 30 base pairs long was used to introduce a Hindi11 restriction site.3' to the stop codons of the nucleotide sequence. The oligonucleotide sequence is shown below:
5 ' AGGTCGAAGCTTACTAACGCAGTTCCCACC 3 ' (SEQ ID NO : 47 )
A second oligonucleotide 45 base pairs in length was used to replace the PAPM sequence with LSTQ to create VNLSTQpEGF. The oligonucleotide sequence is shown below:
5' TTAGACGTTAACCTGAGCACCCAGAACAGCTATAGCGAATGCCCG 3' (SEQ ID NO: 48)
The protein sequence encoded by the 5' end of the PCR fragment is shown below:
V N L S T Q N S Y S
GTT AAC CTG AGC ACC CAG AAC AGC TAT AGC- (SEQ ID NO:49)
Hpal enzymatic cleavage porcineEGF recognition site
A mutant clone of pSTBlue-1 containing VNLSTQpEGF was selected and cloned into the expression vector
(pGHXSC.4) in order to provide an expression vector for
MetpGH (1-11) VNLSTQpEGF, using the methods described in Example 4.
Example 5 Production of proteins as inclusion bodies in E. coli
The pGHXSC.4 expression vectors respectively- comprising MetpGH (1-11)VNLSTQdes (1-3) IGF-I (Example 2), MetpGH(l-ll)VNPAPMdes(1-3) IGF-I (Example 3), MetpGH (1- lDVNPAPMpEGF (Example 4), or metpGH(1-11) VNLSTQpEGF (Example 4) , were transformed into the E. coli laclq strain, JMlOl.
Cultures were grown in 3 litres of MinA medium at 37°C, pH 7.0, 60% pO2 and fed with glucose until an A600 of 8.0 was reached. Cultures were then induced with 200 mgs of isopropylthiogalactoside (IPTG) for 6 hrs and fed with an additional 50 g of glucose. Cells were homogenised at 9000 psi, and inclusion bodies were collected by centrifugation at 10,000 rpm at 4oC for 25 minutes. The
inclusion bodies were washed by suspension in 30 mM NaCl, 10 mM KH 2 PO 4 , 0.5 mM ZnCl 2/ collected by centrifugation, and the wet paste stored at -80 0 C.
Example 7 Levels of Expression of MetpGH(l-ll) WLSTQdes (1-3) IGF-I and MetpGH(l-ll) WPAPMdes (1-3) IGF-I in inclusion bodies
Inclusion bodies were dissolved in 8 M urea, 40 mM glycine, 1 mM EDTA, 0.1 M Tris, 20 mM DTT at pH 9.1 at a final concentration of 10 mg/ml, and incubated for 1 hour at room temperature. A sample taken from the dissolved inclusion bodies was diluted 1:10 in 0.1% trifluoroacetic acid (TFA) and analysed by RP-HPLC. The results are shown in Table 1. This demonstrates that there was a 208% increase in yield when the leader sequence comprising LSTQ was used.
Table 1
Example 8 Levels of expression of MetpGH (1-11) VNLSTQpEGF and MetpGH (1-11) VNPAPMpEGF in inclusion bodies
Inclusion bodies were dissolved and analysed by RP- HPLC, as described in Example 7. The results are shown in Table 2.
Table 2
Example 9 Biological activity of MetpGH (1-11) VNLSTQdes (1-3) IGF-I
The protein encoded by the nucleic sequence produced in Example 2 and produced as inclusion bodies as described in Example 6 did not require cleavage to achieve full biological activity equivalent to LONG 0 R 3 IGF-I (GroPep Ltd, Australia) after processing. This is demonstrated as follows.
Inclusion bodies were dissolved as described in Example 6, and refolded in 2 M urea, 10 mM glycine, 0.1 M Tris, 1.3 mM EDTA, 1 mM 2-hydroxymethyl disulphide and 0.4 mM DTT at pH 9.1 at room temperature for 90 minutes. The protein concentration was 0.125 mg/ml . Refolding was stopped by acidification with hydrochloric acid to pH 1.5, filtered, pumped on to a cation exchange column and eluted using an ammonium acetate gradient at pH 4.8. Further purification involved RP-HPLC on Source 30-RPC using 0.1% TFA in an isopropanol gradient, ion exchange chromatography on SP Fast Flow using an ammonium acetate gradient at pH 4.8, and a final desalting into 0.1 M acetic acid.
The purification procedure yielded a single protein peak, which was confirmed by N-terminal sequence analysis to be MetpGH (1-11) VNLSTQdes (1-3) IGF-I. Biological activity was measured as the percentage stimulation of protein synthesis in rat L6 myoblasts (G.L.Francis et al Biochem. J.233, 207-213, 1986). As shown in Figure 1, this
demonstrated that in growth factor-free medium, the protein had a potency equivalent to that of Long ® R3IGF-I .
Example 10 Effect of changing the leader sequence from QFAHY to LSTQ
The objective of this experiment was to summarise the evidence that modification of the des (1-3) IGF-I fusion protein leader sequence from GFAHY to LSTQ improved des CL3) IGF-I fusion protein expression levels in fermentations.
des (1-3) IGF-I fusion protein cDNA inserts
(a) MetpGH(l-ll)VNGFAHYdes(l-3)IGF-I
The nucleotide sequence (SEQ ID NO: 50) coding for the region of the MetpGH (1-11) VNGFAHYdes (1-3) IGF-I polypeptide (SEQ ID NO: 51.) is shown below.
251 TGTGAGCGGA TAACAATTTC ACACAGGAGG TAATATATGT TCCCAGCCAT
* M F P A M Frame2
301 GcccTTGTCc AGCCTATTTG TTAACGGTTT CGCCCATTAT ACCCTGTGCG
P L S S L F V N G F A H Y T L C G Frame2
351 GTGCTGAACT GGTTGACGCT CTGCAGTTCG TTTGCGGTGA CCGTGGCTTC
A E L V D A L Q F V C G D R G F Frame2
401 TACTTCAACA AACCGACCGG TTACGGTTCT TCTTCTCGTC GTGCTCCGCA Y F N K P T G Y G S S S R R A P Q Frame2
451 GACCGGTATC GTTGACGAAT GCTGCTTCCG TTCTTGCGAC CTGCGTCGTC
T G I V D E C C F R S C D L R R L Frame2
501 TGGAAATGTA CTGCGCTCCG CTGAAACCGG CTAAATCTGC TTGATGATGC
E M Y C A P L K P A K S A * * Frame2
* = stop codon.
The numbering system is based on the expression vector.
(b) MetpGH(l-ll) WLSTQdes(l-3) IGF-I
The MetpGH(l-ll)VNLSTQdes (1-3) IGF-I construct was prepared in three steps. Firstly, the nucleotide sequence encoding VNPAPMdes (1-3) IGF-I from residue VaI12
(incorporating a Hpal restriction site) to the stop codon (incorporating a Hindlll restriction site) was synthesised (Interactivata, Germany) . This produced a nucleotide sequence optimised for expression of the protein in E. coll. This sequence was modified at the linker site by PCR to replace the nucleic acid encoding the amino acid sequence PAPM with that encoding the amino acid sequence LSTQ. Finally, the PCR insert was cloned back into the "MetpGH(l-ll)VN" expression plasmid (pGHXSC.4) via the Hpal and Hindlll restriction sites. The nucleotide sequence (SEQ ID NO: 52) coding for the MetpGH till) VNLSTQdes (1-3) IGF-I polypeptide (SEQ ID NO:53) is shown below.
ATG TTC CCA GCC AT M F P A M Frame2
301 GCCCTTGTCC AGCCTATTTG TTAACCTGAG CACCCAGACC CTGTGCGGCG
P L S S L F V N L S T Q T L C G A Frame2
351 CGGAACTGGT GGATGCGCTG CAGTTTGTGT GCGGCGATCG TGGCTTTTAT
E L V D A L Q F V C G D R G F Y Frame2
401 TTTAACAAAC CGACCGGCTA TGGCAGCAGC AGCCGTCGTG CGCCGCAGAC F N K P T G Y G S S S R R A P Q T Frame2
451 CGGCATTGTG GATGAATGCT GCTTTCGTAG CTGCGATCTG CGTCGTCTGG
G I V D E C C F R S C D L R R L E Frame2
501 AAATGTATTG CGCGCCGCTG AAACCGGCGA AAAGCGCGTG ATAAGCTTGG
M Y C A P L K P A K S A * * Frame2
* = stop codon.
The numbering system is based on the expression vector.
Fermentation of the des (1-3) IGF-I fusion protein expression clones
The MetpGH (1-11) VNGFAHYdes (1-3) IGF-I expression clone was fermented five times at the 14 L scale in a Chemap bioreactor (Bresatec Pty Ltd, Adelaide) . The fermentations were performed in minimal defined medium with a glucose feed. The temperature was maintained at 37°C, the pH at 7.0, and dissolved oxygen at >50%. The cultures typically reached an OD600 of 55 prior to induction with isopropylthiogalactoside (IPTG) and a termination OD600 of 75 at 5 hours after induction. Details of the parameters are described in King et al . 1992 J. MoI. Endocrinol. 8(1):29-41. The MetpGH (1-11) VNLSTQdes (1-3) IGF-I expression clone was fermented at 3 L and 50 L-scales using either 3 L Applicon (Schiedam, the Netherlands) or 50 L Biostat (Sartorius BBI Systems GmbH, Melsungen, Germany) bioreactors . The fermentations were performed in minimal defined medium with a glucose feed. The temperature was maintained at 37 0 C and dissolved oxygen >50%. The culture pH was 6.9 for the 3 L fermentation and 6.6 for the 50 L fermentation. The cultures were induced with IPTG at an OD600 of 7-9 at the 3 L-scale and 30-35 at the 50L-scale. The induction proceeded for 5 hours, yielding cultures with terminal OD600 values of 32-35 at the 3L-scale and 75-80 at the 50L-scale.
Homogenisation
Homogenisation of the culture broth from the fermentation of MetpGH (1-11) VNGFAHYdes (1-3) IGF-I was achieved by 3 passages at 15,000 psig (Bresatec Pty Ltd, Adelaide) .
Homogenisation of the culture from the 3L-scale fermentation of MetpGH (1-11) VNLSTQdes (1-3) IGF-I was achieved by recirculating the culture through a high pressure homogeniser (Rannie, Invensys APV, Australia) set to 950 bar for 5 hours.
Homogenisation of the 50 L-scale fermentation of MetpGH (1-11) VNLSTQdes (1-3) IGF-I was achieved by circulating the broth through a Gaulin Homogeniser
(Invensys APV, Australia) set to 400 bar for 3.5 hours at 1.6 L/min.
Inclusion body recovery
Inclusion bodies were recovered from the homogenised MetpGH (1-11) VNGFAHYdes (1-3) IGF-I culture broth using a continuous centrifuge. In the first pass, the centrifuge feed rate was approximately 290 mL/min. The recovered inclusion body paste was then washed by resuspension in a washing solution (final volume 2.5 L). The washed inclusion bodies were then recovered by a second centrifugation run with a feed rate of 650 mL/min.
Inclusion bodies were recovered from the homogenised 3L MetpGH (1-11) VNLSTQdes (1-3) IGF-I culture using a batch centrifuge (BeckmanCoulter, Australia) set to 9300 RPM. The centrifugation was performed at 4°C for 25 minutes.
Inclusion bodies were recovered from the homogenised 50 L MetpGH (1-11) VNLSTQdes (1-3) IGF-I culture broth using a continuous centrifuge (CEPA, Lahr, Germany) at 20,000 rpm. The homogenate was fed through the centrifuge at lL/min.
Analysis of inclusion body purity and fermentation yield
The expression levels achieved during the fermentation of des (1-3) IGF-I fusion protein production clones were measured by dissolving known amounts of the respective inclusion body pastes (typically 0.3 g) in known volumes of reducing, denaturing buffer (typically 30 mL) in a 50 mL plastic tube. The reducing denaturing buffer contained 8 M urea, 100 mM Tris, 40 mM glycine, 20 mM DTT with a pH of 9.0. The fusion protein extractions proceeded for 60 minutes at room temperature while the reactions were mechanically mixed. After the extractions were complete, aliquots of the solubilized inclusion body extracts were removed and analysed by RP-HPLC. The analyses involved calibrating the HPLC equipment with a known quantity of LONG 0 R 3 IGF-I reference standard to generate an extinction coefficient for the integrated peak area. The extinction coefficient was then used to derive a quantity for the fusion protein peak areas measured in the inclusion body extracts. Table 3 summarises the inclusion body data from the *GFAHY' and λ LSTQ' fermentations.
Table 3
Taking both historical small scale production data and more recent inclusion body RP-HPLC testing data into account, it can be seen that the average expression level for "GFAHY" fermentations was 0.51g/L. In contrast, the average expression level for "LSTQ" was 1.06g/L, which corresponds to an 108% increase in expression for the "LSTQ" construct compared to the "GFAHY" construct.
As shown in Table 3, there was an 108% improvement in expression levels when the linker sequence for the des (1-3) IGF-I fusion protein was changed from "GFAHY" to "LSTQ" . This is consistent with the improvements in expression which were observed when the linker sequence was changed from "PAPM" or "DSTM" to "LSTQ" for proteins such as Met-porcine-epidermal growth factor or des(l-
3) IGF-I (Table 1 and Table 2).
It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.
Example 11 Enzymatic cleavage of the LSTQ linker sequence of the fusion protein to liberate des (1-3) IGF-I
The utility of the fusion protein linker sequence for specific enzymatic cleavage was investigated with partially purified MetpGH (1-11) VNLSTQdes (1-3) IGF-I fusion protein. In this experiment, the MetpGH (1-11) VNLSTQdes ( 1- 3) IGF-I fusion protein was extracted from inclusion bodies, refolded and purified by cation exchange chromatography as described in Example 8. The eluate from the cation exchange chromatography column which contained the MetpGH (1-11) VNLSTQdes (1-3) IGF-I fusion protein was subsequently adjusted to 1.0mg/ml in a solution that contained 2.0 M urea and 0.1 M tris that had been adjusted to a pH of 8.0. To this solution was added the α-lytic protease mutant 9 (Haggett et al . (1994) Arch. Biochem. Biophys. 314: 132-141.) at an enzyme to substrate ratio of 1:500. The enzymatic cleavage reaction proceeded for 8.5 hours at 37 0 C and was then quenched by the addition of concentrated HCl to a pH < 4.0. The progress of the cleavage reaction was monitored using RP-HPLC essentially as described in Francis et al . (1992) J. MoI. Endo. (8) 213-223. Of the 3.5g of MetpGH (1-11) VNLSTQdes (1-3) IGF-I fusion protein that was subjected to enzymatic treatment, 1.4 grams of des (1-3) IGF-I was liberated and characterised by analysis.
It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.
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