WO2004005335A2

WO2004005335A2 - Multimerised growth hormone fusion proteins

Info

Publication number: WO2004005335A2
Application number: PCT/DK2003/000428
Authority: WO
Inventors: Thor Las Holtet
Original assignee: Borean Pharma A/S
Priority date: 2002-07-05
Filing date: 2003-06-23
Publication date: 2004-01-15
Also published as: AU2003239777A1; WO2004005335A3; AU2003239777A8

Abstract

The present invention relates to multimerised growth hormone polypeptides wherein growth hormone is fused with a multimerisation domain, such as a tetranectin trimerising structural element. Thereby there is provided growth hormone multimers, e.g. growth hormone dimers and growth hormone trimers, with increased biological activity.

Description

Multimerised Growth Hormone

Field of Invention

The present invention relates to multimerised growth hormone polypeptides with increased biological activity.

Background of the invention and prior art

A problem encountered in the practice of medicine when using proteins as injectable pharmaceuticals is the frequency at which those injections must be made in order to maintain a therapeutic level of the protein in the circulation. Human growth hormone (hGH, somatotropin or somatropin) has a plasma half-life of about 0.36 hours when it is administered intravenously (Eli Lilly, Humatrope Prescribing Information, PA 1640 AMP). Therefore, the therapeutic plasma level of hGH is rapidly decreased, and frequent intravenous administrations is required.

An alternative route of administration is subcutaneous or intramuscular injection. This route offers slower absorption from the site of administration, thus causing a sustained release effect and thereby an enhanced plasma half-life of 3.8 and 4.9 hours for subcutaneous and intramuscular injection, respectively (Eli Lilly, Humatrope Prescribing Information, PA 1640 AMP). However, significantly lower plasma levels are achieved and, thus, a similar frequency of injection, as is required with intravenous administration, may be necessary to produce a comparable therapeutic effect. Clearly, such a regimen of repeated injections, may be highly inconvenient, or even impracticable, for the patients in need of hGH therapy. Thus, there is a need for alternative growth hormone products which can offer a more suitable administration schedule as compared to the existing short-acting growth hormone therapies.

Human growth hormone, hGH, participates in much of the regulation of normal human growth and development. This monomeric 22,000-dalton pituitary hormone consists of a single chain of 191 amino acid residues and is cross-linked by two disulfide bridges. It exhibits a multitude of biological effects derived from the interaction between hGH and its cell surface receptor (hGHR), which is a single membrane-spanning type I glycoprotein. By virtue of HG-receptor dimerization, hGH causes the activation of the hGHR-associated cytoplasmic tyrosine kinase, JAK2.

hGH preparations have been prepared from human pituitaries, but presently the products on the market are produced by recombinant methods. hGH is primarily used to stimulate linear growth in patients with hypopituitary dwarfism or Turner's syndrome. Growth hormone therapy is also presently used in children to promote growth and in adults to improve muscle strength, reduce fat mass and improve metabolic profiles, which could predispose to cardiovascular disease.

Modification of naturally occurring proteins which have therapeutic value is often attempted in an effort to increase the protein's biological activity, including plasma half- life. Several methods have been employed to increase the biological activity of therapeutic proteins. These methods often focus on increasing the size of the therapeutic agents. For example, the size of a protein can be increased through chemical conjugation with a reagent such as polyethylene glycol (PEG). This procedure, also known as "PEGylation", has been reported with several protein agents, first as a means to reduce antigenicity, but also as a way to increase biological activity.

Another method of increasing a protein's size is through chemical cross-linking with another protein. Chemically cross-linked dimeric human growth hormone (hGH) and bovine growth hormone (bGH) has been described by Mockridge et al. (1998). Using a cross-linking reagent 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), hGH and bGH was randomly derivatized to give predominantly amide-linked dimers but also amide-linked multimers, depending on the concentration of EDC reagent used. The final protein preparation was heterogeneous due to non-specific reaction of the EDC reagent with various amino acids in the protein, including lysine, aspartic acid and glutamic acid residues and the amino- and carboxy-termini. It was found that there was a correlation between the amount of GH dimer present in the heterogeneous protein preparation and biological activity. Clearly, an injection of such a heterogeneous preparation into humans would be undesirable due to the toxic nature of EDC and the potential immunogenic response to the unnatural amide bond formed between the proteins. Generating consistent batches of a purified protein also would be difficult at the manufacturing scale.

WO 01/79480 discloses that the stability, and hence the shelf-life, of hGH may be increased by the fusion of one hGH-molecule to a human serum albumin (HA) molecule which comprises a specific amino acid sequence. It is suggested that the increased stability results in increased biological activity of the HA-hGH fusion protein.

It has now been found by the present inventor, that the fusion of at least one growth hormone polypetide molecule to a multimerisation domain, and thereby the provision of growth hormone multimers, e.g. dimers and trimers, may significantly increase the biological activity, including plasma half life, of growth hormones in general and human growth hormone in particular. Thus, according to the present invention there is now provided alternative growth hormone molecules which may have increased plasma half- life. This increased half life will result in a more suitable administration schedule as compared to the existing short-acting growth hormone therapies.

Summary of the invention

Accordingly, the invention relates in a first aspect to a growth hormone fusion protein comprising at least one growth hormone and a multimerisation domain.

In a further aspect there is provided a polypeptide complex comprising at least two fusion proteins according to the invention.

The present invention also provides a composition comprising the growth hormone fusion protein or the polypeptide complex according to the invention.

In still further aspects the invention pertains to a method of treating a disease or disorder in a mammal comprising the step of administering the growth hormone fusion protein or the polypeptide complex according to the invention to a mammal, and the use of the growth hormone fusion protein or the polypeptide complex for the preparation of a pharmaceutical composition. Finally, there is provided a kit comprising the composition according to the invention, and a method of producing the growth hormone fusion protein and the polypeptide complex according to the invention.

Detailed disclosure of the invention

The primary objective of the present invention is to provide alternative growth hormone molecules which are growth hormone fusion proteins comprising at least one growth hormone and a multimerisation domain providing for growth hormone multimers.

It has been found by the present inventor, that the fusion of at least one growth hormone polypeptide to a multimerisation domain, provides for growth hormone multimers, e.g. growth hormone dimers and growth hormone trimers, having significantly increased biological activity as compared to native growth hormone, such as human growth hormone.

In the present context "increased biological activity" includes prolonged plasma half-life, i.e. a longer circulating half-life relative to native monomeric growth hormone. It will be appreciated that by "half-life" or "plasma half-life" is meant the time for a drug concentration in the plasma to be reduced by one-half, typically measured after administration of a selected dose. By "plasma" herein is meant either plasma or serum.

The plasma half-life of the growth hormone multimers according to the invention is preferably increased compared to that of native monomeric growth hormone. Preferably, the plasma half-life is increased by at least 5 %, such as at least 10 %, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 40% such as at least 50%, for example at least 75%, such as at least 100%. In even more preferred embodiments, the plasma half life is increased at least about 3, 4, 5, 6, 7, 8, 9, 10, 15 or 18 times as compared to native monomeric growth hormone. In even more preferred embodiments the, plasma half-life is increased at least about 20, 30, 40 or 50 times, as compared to native monomeric growth hormone.

An increased plasma half-life may have profound implications for the use of the growth hormone multimers according to the invention in the treatment of various indications. It is therefore expected that the clinical effect of the growth hormone multimers according to the invention is superior to the effect of native monomeric growth hormone, such as human growth hormone.

Also within the meaning of increased biological activity is a higher potency, i.e., a smaller quantity of the growth hormone fusion protein or the growth hormone multimer is required relative to the naturally occurring growth hormone to achieve a specified level of biological activity. It is also contemplated that the growth hormone multimers according to the invention may have improved stability, and hence improved shelf-life, as compared to native monomeric growth hormone.

Increased biological activity can also encompass a combination of the above-described activities, for example, a growth hormone fusion protein or growth hormone multimer with higher potency that also exhibits a prolonged circulating half-life and improved stability. Because the growth hormone fusion proteins of the present invention may have increased biological activity, it is contemplated that the frequency with which they must be administered may be reduced, or the amount administered to achieve an effective dose can be reduced.

It is also within the scope of the present invention that the growth hormone fusion proteins according to the invention, may provide for growth hormone multimers having increased affinity for growth hormone receptors. Such an increased affinity can result in an increased stimulation of the signal generated by the activation of the receptors. This may imply that a reduced quantity of growth hormone fusion proteins would then be necessary over the course of treatment, as compared to the quantity necessary if native growth hormone was used. As used herein, the term "growth hormone" or "GH" is intended to refer to either natural or recombinant pituitary growth hormone, regardless of the source. The term is limited only in that the material must demonstrate pituitary growth hormone biological activity in a recipient such as a human. Therefore, the term also applies to physiologically active equivalents, fragments, or portions of the complete growth hormone molecule. Examples include human growth hormone (hGH), which is natural or recombinant GH with the human native sequence (somatotropin or somatropin), and recombinant growth hormone (rGH), which refers to any GH or variant produced by means of recombinant DNA technology. Further examples include bovine growth hormone (bGH) and porcine growth hormone (pGH).

In accordance with the invention, the growth hormone is linked to a multimerisation domain. In the present context, the term "multimerisation domain" is a peptide, a protein or part of a protein which is capable of interacting with other, similar or identical multimerisation domains. The interaction is of the type that produces multimeric proteins or polypeptides. Such an interaction may be caused by covalent bonds between the components of the multimerisation domains as well as by hydrogen bond forces, hydrophobic forces, van derWaals forces and salt bridges. In useful embodiments, the multimerisation domain peptide is a dimerising domain, a trimerising domain, a tetramerising domain, a pentamerising domain or a hexamerising domain.

One example of a multimerisation domain is disclosed in WO 95/31540, which describes polypeptides comprising a collectin neck region. The amino acid sequence constituting the collectin neck region may be attached to any polypeptide of choice. Trimers can then be made under appropriate conditions with three polypeptides comprising the collectin neck region amino acid sequence.

In further embodiments, the multimerisation domain of the fusion protein according to the invention may comprise coiled-coil dimerization domains such as leucine zipper domains which are found in certain DNA-binding polypeptides. Advantageously, the multimerisation domain according to the invention may also comprise a dimerization domain which is an immunoglobulin Fab constant domain, such as an immunoglobulin heavy chain CH1 constant region or an immunoglobulin light chain constant region.

In a presently preferred embodiment, the multimerisation domain is derived from tetranectin, and more specifically comprises the tetranectin trimerising structural element (hereafter termed TTSE) which is described in detail in WO 98/56906. The amino acid sequence of TTSE is shown in SEQ ID NO 17. The trimerising effect of TTSE is caused by a coiled coil structure which interacts with the coiled coil structure of two other TTSEs to form a triple alpha helical coiled coil trimer which is exceptionally stable even at relatively high temperatures. The term TTSE is also intended to embrace variants of a TTSE of a naturally occurring member of the tetranectin family of proteins, variants which have been modified in the amino acid sequence without adversely affecting, to any substantial degree, the capability of the TTSE to form alpha helical coiled coil trimers. Thus, the fusion protein according to the invention may comprise a TTSE as a multimerisation domain, which comprises a sequence having at least 68% amino acid sequence identity with the sequence of SEQ ID NO SEQ ID NO 17, such as at least 75%, including at least 87%, such as at least 92%. In accordance herewith, the cystein residue No. 50 of the TTSE (SEQ ID NO 17) may advantageously be mutagenised to serine, threonine, methionine or to any other amino acid residue in order to avoid formation of an unwanted inter-chain disulphide bridge, which could lead to unwanted multimerisation.

In a further embodiment, the TTSE multimerisation domain (SEQ ID NO 17) may e.g. be modified by (i) the incorporation of polyhistidine sequence and/or a cleavage site for the Blood Coagulating Factor X_a, (ii) replacing Cys 50 with Ser, and (iii) by including a C- terminal KGS sequence. An example of such a modified TTSE is given as SEQ ID NO 18, and is designated TripA. Similarly, the TTSE multimerisation domain may be modified by truncating the amino acid sequence and removing the C-terminal amino acid residues C50 and L51. This modified TTSE is in the present context designated TripD. In accordance with the invention, the growth hormone may either be linked to the N- or the C-terminal amino acid residue of the multimerisation domain. However, it is also envisaged that in certain embodiments it may be advantageous to link a growth hormone to both the N-terminal and the C-terminal of the multimerisation domain of the fusion protein, and thereby providing a fusion protein comprising two growth hormones.

It will be appreciated that a flexible molecular linker (or spacer) optionally may be interposed between, and covalently join, the growth hormone and the multimerisation domain. Preferably, the linker is a polypeptide sequence of about 1-20 amino acid residues, such as about 2-10 amino acid residues, including 3-7 amino acid residues. In useful embodiments the linker is essentially non-immunogenic, not prone to proteolytic cleavage and does not comprise amino acid residues which are known to interact with other residues (e.g. cystein residues). Preferred examples of spacer or linker peptides include those, which have been used to link proteins without substantially impairing the function of the linked proteins or at least without substantially impairing the function of one of the linked proteins. More preferably the linkers or spacers have been used to link proteins comprising coiled-coil structures.

The following are examples of linker sequences, which are believed to be suitable for linking growth hormone to the multimerisation domain of the present invention.

Tetranectin based linker: The linker may include the tetranectin amino acid residues 53- 56, which in tetranectin forms a beta-strand, and the residues 57-59 which forms a turn in tetranectin (Nielsen et al., 1997). The sequence of the segment is GTKVHMK. This linker has the advantage that it in native tetranectin is bridging the trimerisation domain with the CRD-domain, and hence it is contemplated to be well suited for connecting the trimerisation domain to another domain in general. Furthermore, the resulting construct is not expected to be more immunogenic than the construct without a linker. The tetranectin based linker is highly preferred when the multimerisation domain is TTSE.

Fibronectin based linker: The linker may be chosen as a sub-sequence from the connecting strand 3 from human fibronectin, this corresponds to amino acid residues 1992-2102 (SWISS-PROT numbering, entry P02751). Preferably the subsequence: PGTSGQQPSVGQQ covering amino acid residues number 2037-2049 is used, and within that subsequence the segment GTSGQ corresponding to amino acid residues 2038-2042 is more preferable. This construct has the advantage that it is know not to be highly prone to proteolytic cleavage and is not expected to be highly immunogenic bearing in mind that fibronectin is present at high concentrations in plasma.

Human lgG3 upper hinge based linker: The 10 amino acid residue sequence derived from the upper hinge region of murine lgG3, PKPSTPPGSS, has been used for the production of antibodies dimerised trough a coiled coil (Pack et al., 1992) and may be useful as a spacer peptide according to the present invention. Even more preferable may be a corresponding sequence from the upper hinge region of human lgG3. Sequences from human lgG3 are not expected to be immunogenic in humans.

Possible examples of flexible amino acid linker sequences include SGGTSGSTSGTGST, AGSSTGSSTGPGSTT or GGSGGAP. These sequences have previously been used for the linking of designed coiled coils to other protein domains (Mutter et al., 2000).

In one useful embodiment, the linker interposed between the growth hormone and the multimerisation domain may be the amino acid sequences GSA or GSQEGSA. Thus, in useful embodiments the growth hormone fusion protein according to the invention is TripA-GSA-hGH (SEQ ID NO 4), MOTripA-GSA-hGH (SEQ ID NO 6), TripA-GSQEGSA- hGH (SEQ ID NO 9), MOTripA-GSQEGSA-hGH (SEQ ID NO 11), TripA-hGH (SEQ ID NO 16) or TripD-GSQEGSA-hGH (SEQ ID NO 23).

The growth hormone fusion proteins and the multimer polypeptide complex according of the present invention may be expressed in any suitable standard protein expression system by providing a host cell capable of expressing the fusion proteins and/or the multimer polypeptide complex in recoverable amounts. Preferably, the expression systems are systems from which the desired protein may readily be isolated and refolded in vitro. As a general matter, prokaryotic expression systems are preferred since high yields of protein can be obtained and efficient purification and refolding strategies are available. Thus, it is well within the abilities and discretion of the skilled artisan, without undue experimentation, to choose an appropriate or favourite expression system. Similarly, once the primary amino acid sequence for the growth hormone fusion proteins of the present invention is chosen, one of ordinary skill in the art can easily design appropriate recombinant DNA constructs which will encode the desired proteins, taking into consideration such factors as codon biases in the chosen host, the need for secretion signal sequences in the host, the introduction of proteinase cleavage sites within the signal sequence, and the like. These recombinant DNA constructs may be inserted in-frame into any of a number of expression vectors appropriate to the chosen host. The choice of an appropriate or favourite expression vector is, again, a matter well within the ability and discretion of the skilled practitioner. Preferably, the expression vector will include a strong promoter to drive expression of the recombinant constructs.

Accordingly, the growth hormone fusion proteins and the polypeptide complex may be produced by a method which comprises the steps of (i) providing a recombinant vector comprising the isolated nucleic acid sequence encoding the fusion protein of the invention which is, optionally, operatively linked to a promotor, (ii) transforming a host cell with this recombinant vector, (iii) culturing the host cell under conditions to express the fusion protein, and (iv) optionally isolating the growth hormone fusion protein and/or polypeptide complex.

When one or more growth hormones is/are coupled to a multimerisation domain, and thereby forming the fusion protein according to the invention, multimers of the growth hormone are formed under appropriate conditions resulting in a multimeric polypeptide complex. In this way growth hormone dimers, trimers, tretramers, pentamers, hexamers or even higher -mers can be prepared depending on the type of multimerisation domain being linked to the growth hormone. Thus, in one aspect of the invention there is provided a multimeric polypeptide complex comprising at least two fusion proteins, such as at least three, including at least four, such at least five, including at least six fusion proteins. The presence of a growth hormone multimer, such as a growth hormone trimer, may be ascertained by well known techniques such as gelfiltration, SDS-PAGE, or native SDS gel electrophoresis depending on the nature of the multimer.

The growth hormone fusion protein in accordance with the invention, may be used for the preparation of a pharmaceutical composition by any suitable method well known in the art. The composition may together with the growth hormone fusion protein, comprise one or more acceptable carriers therefore, and optionally other therapeutic ingredients. The carriers must be acceptable in the sense of being compatible with the other ingredients and not deleterious to the recipient thereof. In general, methods for the preparation of pharmaceutical compositions include the step of bringing into association the active ingredient and a carrier.

The therapeutic application of the present invention comprises the treatment of a disease or disorder in an animal, by administering a therapeutically effective amount of the growth hormone fusion protein or the polypeptide complex according to the invention to the animal in need thereof. Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular animal or patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.

Typically, the therapeutically effective amount or dosage is in the range from at least about 0.001 to 500 milligrams per kilogram of animal, and preferably from at least about 0.1 to 100 milligrams per kilogram of animal per single or multiple administration, depending upon the specific activity of contained in the composition.

Preferred doses can optionally include 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 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 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500 mg/kg/administration, or any range, value or fraction thereof.

As a non-limiting example, treatment of animals, including humans can be provided as a onetime or periodic dosage of the growth hormone fusion protein or the polypeptide complex of the present invention at an amount of 0.1 to 100 mg/kg per day, such as 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 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 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/kg, per day, on at least one of day 1 , 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, or 40, or alternatively or additionally, at least one of week 1 , 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 , or 52, or alternatively or additionally, at least one of 1 , 2, 3, 4, 5, 6„ 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or any combination thereof, using single, infusion or repeated doses.

In a presently preferred embodiment the disease or disorder is growth hormone deficiency, turner syndrome and metabolic disorder.

The growth hormone fusion protein or the polypeptide complex according to the present invention may be directly administered to the animal by any suitable technique, including parenterally, and can be administered locally or systemically. The specific route of administration depends, e.g., on the medical history of the animal. Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial, and intraperitoneal administration.

For parenteral administration, the growth hormone fusion protein or the polypeptide complex according to the invention can be formulated as a solution, suspension, emulsion or lyophilised powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. The vehicle or lyophilised powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by known or suitable techniques.

The animals potentially treatable by the growth hormone fusion protein or the polypeptide complex herein, include mammals such as bovine, ovine, and porcine animals. The preferred mammal herein is a human. In particular, the growth hormone fusion protein or the polypeptide complex herein may be used for stimulating linear growth in human pediatric patients who lack adequate normal endogenous growth hormone and patients with Turner syndrome.

The invention will now be described by way of illustration in the following non-limiting examples.

Examples

EXAMPLE 1

Cloning, Expression and Purification of trimeric human growth hormone fusion protein

Trimeric growth hormone derivative TripA-GSA-hGH

The Expression vector pT76HFXTripA-GSA-hGH, was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHN: 5- GCT CAC GGG ATC CGC TTT CCC AAC CAT TCC CTT AT-3 [SEQ ID NO:1] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind 111 cut E. coli expression vector, pT76HFXtripa (Lorentsen et al., 2000) using standard procedures. The nucleotide sequence of the resulting TripA-GSA-hGH, is given as SEQ ID NO:3 and the amino acid sequence encoded by the TripA-GSA-hGH insert is given in SEQ ID NO:4. The recombinant human Growth Hormone derivative TripA-GSA-hGH was produced by growing and expressing the plasmid pT76HFXTripA-GSA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier et al. (1986). Exponentially growing cultures at 37°C were at OD600=0.8 infected with bacteriophage lambdaCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before the cells were harvested by centrifugation. The cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base). Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Following gel filtration on Sephadex G-25 (Amersham Biosciences) into 8 M Urea, 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 5mM 2-mercaptoethanol, the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (Ni2+NTA-agarose, Qiagen).

Upon application of the crude protein extract on the Ni2+NTA-agarose column, the trimeric fusion protein, TripA-GSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5mM 2- mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.

The fusion protein was refolded on the Ni2+NTA-agarose column using the cyclic refolding procedure described by Thøgersen et al. (International Patent Application W09418227) with a gradient manager profile as described below in Table 1 and 0.5 M NaCl, 50 mM Tris-HCl pH 8, 1 mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B. TABLE 1

Gradient Manager Profile

Step Time 1 Flow %A ' %B Step Tine Fiow %A %B

1 0 2 100 0 49 720 2 100 0

2 45 2 100 0 50 765 2 100 0

3 46 2 0 100 51 766 2 74 26

4 52 2 0 100 52 772 2 74 26

5 60 2 100 0 53 780 2 100 0

6 105 2 100 0 54 825 2 100 0

7 106 2 8 92 55 826 2 76 24

8 113 2 8 92 56 832 2 76 24

9 120 2 100 0 57 840 2 too 0

10 16S 2 100 0 58 885 2 100 0

It 166 2 20 80 59 886 2 78 22

12 172 2 20 80 60 892 2 78 22

13 180 2 100 0 61 900 2 100 0

14 225 2 100 0 62 945 2 100 0

15 226 2 28 72 63 946 2 80 20

16 232 2 28 72 64 952 2 80 20

17 240 2 100 0 65 960 2 100 0

18 285 2 100 0 66100S 2 100 0

19 286 2 34 66 671006 2 82 18

20 292 2 34 66 681012 2 82 18

21 300 2 100 0 691020 2 100 0

22 345 2 100 0 701065 2 100 0

23 346 2 42 58 711066 2 84 16

24 352 2 42 58 721072 2 84 16

25 360 2 100 0 731080 2 100 0

26 405 2 100 0 741125 2 100 0

27 406 2 50 50 751126 2 86 14

28 412 2 50 50 761132 2 86 14

29 420 2 100 0 771140 2 100 0

30 465 2 100 0 781185 2 100 0

31 466 2 54 46 791186 2 88 12

32 472 2 54 46 801192 2 88 12

33 480 2 100 0 811200 2 100 0

34 525 2 100 0 821245 2 100 0

3S S26 2 58 42 831246 2 90 10

36 532 2 58 42 8412S2 2 90 10

3? 540 2 100 0 851260 2 100 0

38 S85 2 100 0 861305 2 100 0

39 586 2 62 38 871306 2 95 5

40 592 2 62 38 881312 2 95 5

41 600 2 100 0 891319 2 100 0

42 645 2 100 0 901364 2 100 0

43 646 2 G6 34

44 652 2 66 34

45 660 2 100 0

46 705 2 TOO 0

47 706 2 70 30

48 713 2 70 30 After completion of the cyclic folding procedure the TripA-GSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 10 mM EDTA pH 8.

After elution from the Ni2+NTA column the TripA-GSA-hGH protein was gelfiltrated in to a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G- 25, and the protein was then applied onto a Q-Sepharose (Amersham Biosciences) ion exchange column. Protein was eluted over 5 column volumes with a linear gradient from 8 M urea, 5 mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 500 mM NaCl and 50 mM Tris-HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.

The monomeric fraction eluted from the ion exchange column was gelfiltrated in to a buffer containing 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa is inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.

After cleavage the recombinant protein was diluted with 10 volumes of H₂0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein were eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8. Fractions containing the cleaved purified recombinant protein TripA-GSA-hGH was gelfiltrated into mannitol buffer (220 mM mannitol, 3.24 mM histidin and 32 mM phenol) on a Sephadex G-25 column.

Trimeric growth hormone derivative HOTripA-GSA-hGH

The Expression vector pT76HFXI10TripA-GSA-hGH, was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHN: 5- GCT CAC GGG ATC CGC TTT CCC AAC CAT TCC CTT AT-3 [SEQ ID NO:1] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind III cut E. coli expression vector, pT76HFXI10Tripa using standard procedures. The resulting nucleotide sequence of the MOTripA-GSA-hGH insert is given as SEQ ID NO:5. The amino acid sequence encoded by the MOTripA-GSA-hGH insert is given in SEQ ID NO:6.

The recombinant human growth hormone derivative MOTripA-GSA-hGH was produced by growing and expressing the plasmid pT76HFXI10TripA-GSA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier and Moffat, J. Mol. Biol., 189: 113-130, 1986. Exponentially growing cultures at 37°C were at OD600 0.8 infected with bacteriophage lambdaCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base). Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Following gel filtration on Sephadex G-25 (Amersham Biosciences) into 8 M Urea, 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 5mM 2-mercaptoethanol, the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (NI2+NTA- agarose, Qiagen).

Upon application of the crude protein extract on the Ni2+NTA-agarose column, the fusion protein, MOTripA-GSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.

The fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in Table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8, and 5 mM reduced gluthatione as buffer B. After completion of the cyclic folding procedure the HOTripA-GSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 10 mM EDTA pH 8.

After elution from the Ni2+NTA column the HOTripA-GSA-hGH protein was gelfiltrated into a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose (Amersham Biosciences) ion exchange column. Protein were eluted over 5 column volumes with a linear gradient from 8 M urea, 5 mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 500 mM NaCl and 50 mM Tris-HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.

The ion exchange protein was gelfiltrated into a buffer containing 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa was inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.

After cleavage the recombinant protein was diluted with 10 volumes of H₂0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein was eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8. Fractions containing the cleaved purified recombinant protein HOTripA-GSA-hGH was gelfiltrated into mannitol buffer (220 mM mannitol, 3.24 mM histidin and 32 mM phenol) on a Sephadex G-25 column.

Trimeric growth hormone derivative TripA-GSQEGSA-hGH

The Expression vector pT76HFXTripA-GSQEGSA-hGH, was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHgsqegsaN: 5- GCT CAC GGG ATC CCA GGA AGG CTC CGC TTT CCC AAC CAT TCC CTT AT -3 [SEQ ID NO: 7] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind III cut vector, pT76HFXtripa (Lorentsen et al. (2000)) using standard procedures. The nucleotide sequence of the TripA-GSQEGSA-hGH insert is given as SEQ ID NO:8 and the amino acid sequence encoded by the TripA-GSQEGSA-hGH is given as SEQ ID NO:9.

The recombinant human Growth Hormone TripA-GSQEGSA-hGH was produced by growing and expressing the plasmid pT76HFXTripA-GSQEGSA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier et al. (1986). Exponentially growing cultures at 37°C were at OD600 0.8 infected with bacteriophage lambdaCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base). Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Following gel filtration on Sephadex G-25 (Pharmacia, LKB, Sweden) into 8 M Urea, 1 M NaCl, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol, the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (Ni2+NTA-agarose, Qiagen).

Upon application of the crude protein extract on the Ni2+NTA-agarose column, the fusion protein, TripA-GSQEGSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.

The fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in Table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B. After completion of the cyclic folding procedure the TripA-GSQEGSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 20 mM EDTA pH 8.

After elution from the N.2+NTA column the TripA-GSQEGSA-hGH protein was gelfiltrated in to a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose ion exchange column. Protein were eluted over 5 column volumes with a linear gradient from 8 M urea, 5mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 400 mM NaCl and 50 mM Tris- HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.

The monomeric fraction eluted from the ion exchange column was gelfiltrated into a buffer containing 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa was inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.

After cleavage the recombinant protein was diluted with 10 volumes of H₂0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein was eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8. Fractions containing the cleaved purified recombinant protein TripA-GSQEGSA-hGH was gelfiltrated into mannitol buffer (220 mM mannitol, 3.24 mM histidin and 32 mM phenol) on a Sephadex G-25 column.

Trimeric growth hormone derivative MOTripA-GSQEGSA-hGH

The Expression vector pT76HFXI10TripA-GSQEGSA-hGH, was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHgsqegsaN: 5- GCT CAC GGG ATC CCA GGA AGG CTC CGC TTT CCC AAC CAT TCC CTT AT -3 [SEQ ID NO: 7] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind III cut vector, pT76HFXI10tripa using standard procedures. The nucleotide sequence of MOTripA- GSQEGSA-hGH insert is given as SEQ ID NO: 10. The amino acid sequence encoded by the MOTripA-GSQEGSA-hGH insert is given as SEQ ID NO:11.

The recombinant human Growth Hormone HOTripA-GSQEGSA-hGH was produced by growing and expressing the plasmid pT76HFXI10TripA-GSQEGSA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier et al. (1986). Exponentially growing cultures at 37°C were at OD600 0.8 infected with bacteriophage lambdaCEβ at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base). Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Following gel filtration on Sephadex G-25 (Amersham Biosciences) into 8 M Urea, 0,5 M NaCl, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol, the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (Ni2+NTA-agarose, Qiagen).

Upon application of the crude protein extract on the Ni2+NTA-agarose column, the fusion protein, MOTripA-GSQEGSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2- mercaptoethanol, until the optical density (OD) at 280 nm of the eluate was stable.

The fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in Table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1 mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B. After completion of the cyclic folding procedure the MOTripA-GSQEGSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 10 mM EDTA pH 8.

After elution from the Ni2+NTA column the MOTripA-GSQEGSA-hGH protein was gelfiltrated into a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose ion exchange column. Protein was eluted over 5 column volumes with a linear gradient from 8 M urea, 5 mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 500 mM NaCl and 50 mM Tris-HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers ( due to inter-hGHdomain disulfide bridge formation) eluted later.

The ion exchange protein was gelfiltrated in to a buffer contaning 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa was inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.

After cleavage the recombinant protein was diluted with 10 volumes of H₂O and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein were eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8. Fractions containing the cleaved purified recombinant protein was gelfiltrated into PBS mannitol buffer (220mM mannitol, 3.24mM histidin and 32mM phenol) buffer on a Sephadex G-25 column.

Trimeric growth hormone derivative TripA-hGH

The DNA fragment TripA-hGH (SEQ ID NO: 12) was amplified from a so-called assembly reactions combining the two DNA fragments BamHI-TripA and hGH-Hind. The BamHI-TripA fragment was amplified from pT76HFXtripa vector (with the oligonucleotide primers AssFXTripAN: 5-GCT CGA CGG ATC CAT CCA GGG AAG AGG TGA GCC ACC AAC CCA GAA GC-3 [SEQ ID NO: 13] and AssTripAC 5-GGT TGG GAA CTT CAG GGA GAC CGT GTG C-3 [SEQ ID NO:14 ]), and the hGH-Hind fragment was amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers AsshGHN: 5-CTC CCT GAA GTT CCC AAC CAT TCC CTT ATC C-3 [SEQ ID NO: 15] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]).

The Expression vector pT76HFXTripA-hGH, was constructed by ligation of the BamH I and Hind III restricted DNA fragment Ass-TripA-hGH (SEQ ID NO:12), into a BamH I and Hind III cut E. coli expression vector, pT76H (Christensen et al., 1991) using standard procedures. The resulting nucleotide sequence of the TripA-hGH insert is given as SEQ ID NO:12, and the amino acid sequence encoded by the TripA-hGH insert is given as SEQ ID NO:16.

The recombinant human Growth Hormone derivative TripA-hGH was produced by growing and expressing the plasmid pT76HFXTripA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier et al. (1986). Exponentially growing cultures at 37°C were at OD600 0.8 infected with bacteriophage lambdaCEδ at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base). Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Following gel filtration on Sephadex G-25 (Amersham Biosciences) into 8 M Urea, 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 5mM 2-mercaptoethanol, the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (Ni2+NTA-agarose).

Upon application of the crude protein extract on the Ni2+NTA-agarose column, the fusion protein, TripA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable. The fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1 mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B.

After completion of the cyclic folding procedure the TripA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50mM Tris-HCl pH 8 and 10 mM EDTA pH 8.

After elution from the NΪ2+NTA column the TripA-hGH protein was gelfiltrated into a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose ion exchange column. Protein was eluted over 5 column volumes with a linear gradient from 8 M urea, 5 mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 500 mM NaCl and 50 mM Tris-HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.

After cleavage the recombinant protein was diluted with 10 volumes of H₂0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein were eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8. Fractions containing the cleaved purified recombinant protein TripA-hGH was gelfiltrated into mannitol buffer (220mM mannitol, 3.24mM histidin and 32mM phenol) buffer on a Sephadex G-25 column. Trimeric growth hormone derivative TripD-GSQEGSA-hGH

The Expression vector pT76HFXTtipd was constructed by ligation of the Bgl II and Hind III restricted DNA fragment FXtripd (SEQ ID NO:19) amplified from the expression vector pT76HFXtripa (Lorentsen et al., 2000) with the oligonucleotide primers N- pT7H6FXGTripA (SEQ ID NO:20) and C-pT7H6FXGTrip49 (SEQ ID NO:21 ) in to a BamH I and Hind III cut vector pT76H (Christensen et al., 1991).

The Expression vector pT76HFXTripD-GSQEGSA-hGH, was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHgsqegsaN: 5- GCT CAC GGG ATC CCA GGA AGG CTC CGC TTT CCC AAC CAT TCC CTT AT -3 [SEQ ID NO: 7] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind III cut vector, pT76HFXtripd using standard procedures. The nucleotide sequence of the TripD- GSQEGSA-hGH insert is given as SEQ ID NO:22 and the amino acid sequence encoded by the TripD-GSQEGSA-hGH is given as SEQ ID NO:23.

The recombinant human growth hormone TripD-GSQEGSA-hGH was produced by growing and expressing the plasmid pT76HFXTripA-GSQEGSA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier et al. (1986). Exponentially growing cultures at 37°C were at OD600=0.8 infected with bacteriophage lambdaCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base). Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol. Following gel filtration on Sephadex G-25 (Pharmacia, LKB, Sweden) into 8 M Urea, 1 M NaCl, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol, the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (Ni2+NTA-agarose, Qiagen). Upon application of the crude protein extract on the Ni2+NTA-agarose column, the fusion protein, TripD-GSQEGSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.

The fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in Table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B.

After completion of the cyclic folding procedure the TripD-GSQEGSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 20 mM EDTA pH 8.

After elution from the N.2+NTA column the TripD-GSQEGSA-hGH protein was gelfiltrated in to a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose ion exchange column. Protein were eluted over 5 column volumes with a linear gradient from 8 M urea, 5mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 400 mM NaCl and 50 mM Tris- HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.

After cleavage the recombinant protein was diluted with 10 volumes of H₂0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein was eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8. Fractions containing the cleaved purified recombinant protein TripD-GSQEGSA-hGH, were gelfiltrated into mannitol buffer (220mM mannitol, 3.24mM histidin and 32mM phenol) on a Sephadex G-25 column.

EXAMPLE 2

Comparative functional in vitro analysis of the trimeric human growth hormone derivatives TripA-hGH, TripA-GSA-hGH, TripA-GSQEGSA-hGH. TripD-GSQEGSA- hGH and somatropin.

The trimeric human growth hormone derivatives TripA-hGH, TripA-GSA-hGH, TripA- GSQEGSA-hGH, TripD-GSQEGSA-hGH were expressed, refolded, and purified as described in Example 1.

The biological activity of the trimeric hGH derivatives and a commercially available somatropin (monomeric human growth hormone, "Norditropin Simplexx, 5 mg/1.5 ml, Novo Nordisk A S, Denmark) was assayed in vitro using the eluted stain assay (ESTA) measuring Nb2 cell proliferation after incubation with the human growth hormone derivatives essentially as described by Ealy et al. (1995).

The in vitro Nb2 cell proliferation assay was conducted as follows: Nb2-11 rat lymphoma cells (obtained from the European Collection of Cell Cultures, ecacc) were grown in RPMI 1640 medium supplemented with antibiotics at 1% (penicillin 100 U/mL and streptomycin 100 μg/mL), 2 mM L-glutamine, 10% horse serum and 10% fetal calf serum at standard conditions. The cells were transferred to "quiescent" medium consisting of RPMI 1640 medium supplemented with antibiotics at 1%, 2 mM L- glutamine, 10% horse serum and 1 % fetal calf serum 24 hours prior to assay start. At the start of the assay Nb2-11 cells were transferred to "assay" medium (quiescent medium without fetal calf serum) and 2x10⁴ cells were seeded into each well of a 96- well microtiter plates. Fifty microliters samples of each of the trimeric human growth hormone derivatives or controls diluted in assay medium in order to yield a dose response result, were added to each well. Plates were incubated for 48 or 72 hours at standard conditions. To measure cell proliferation MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5- di-phenyl-tetrazolium bromide) was used as part of the CellTiter 96 Non-Radioactive Cell Proliferation Assay Kit (Promega) according to the manufacturers protocol. Experiments were done in triplicate. The results were read on a microtiter plate reader, where the amount of formazan produced, measured as OD492, reflected the level of cellular proliferation. All dilutions were made in triplicate.

The results of the experiment are depicted as mean OD 492 values in Figure 1 (showing the trimeric derivatives TripA-hGH, TripA-GSQEGSA-hGH, TripD-GSQEGSA- hGH) and Figure 2 (showing the trimeric derivative TripA-GSA-hGH). The activity of the growth hormone compounds are given in milli International Unit /litre (mlU/L) and International Unit /litre (IU/L). The differences between the triplicate determinations were found to be less than 10%.

From Figure 1 it can be seen that the trimeric human growth hormone derivatives TripA- hGH, TripA-GSQEGSA-hGH and TripD-GSQEGSA-hGH are capable of stimulating Nb- 2 cell proliferation significantly better or at least as good as monomeric somatropin. From Figure 2 it can be seen that the trimeric derivative TripA-GSA-hGH stimulates Nb- 2 cell proliferation at a level which is about 50 times lower than for somatropin.

EXAMPLE 3

Comparative in vivo analysis of the trimeric human growth hormone derivative TripA- GSA-hGH and somatropin using rat weight gain assay

Samples of trimeric human growth hormone derivatives TripA-GSA-hGH prepared as described in Example 1 and samples of commercially available somatropin ("Norditropin Simplexx, 5 mg/1.5 ml, Novo Nordisk A S, Denmark) in a buffer (220 mM mannitol, 3.24 mM histidin, 32 mM phenol and 1% HSA), were administered to hypophysectomised rats by subcutaneous injection. The rats were operated at four weeks of age, and in accordance with standard procedures, only rats which post-operation did not exhibit significant change in body weight were used. Two doses of each sample were used in the analysis, and the animals were injected twice daily for nine days. The dose levels are shown in Table 2 below.

TABLE 2

SC: subcutaneous

The rats were weighed daily from the first administration of test sample and in the following nine days. The development of body weight was analyzed using standard statistical procedures calculating the mean weight gain for each group of rats relative to the mean weight at day 0.

The results of the weight gain assay are illustrated in Figure 3. The group of rats injected with 15 μg somatropin per rat showed the highest weight gain, with a maximum increase in weight of 28% on day 9.

The weight gain for rats injected with 19 μg TripA-GSA-hGH per rat approached the weight gain for rats injected with 3.5 μg somatropin per rat, with a maximum increase in weight gain at 18% at day 11 and 19% at day 9, respectively. This result is remarkable as TripA-GSA-hGH in Example 2 was found to have about 50 times lower ability to stimulate Nb-2 cell proliferation as compared to monomeric somatropin. The concentration difference between high- and low dose injections, (i.e. both somatropin and growth hormone derivative) is 4 times.

References

Christensen et al. (1991 ), Febs Letters, 281(1 ,2):181-184.

Ealy P.A., Yateman ME, Sandhus MT, Dattani MK, Hassan SJ, Holt SJ, and Marshall NJ (1995), Growth Regulation 5:36-44.

Lorentsen, RH. et al, (2000), Biochemical Journal 347:83-87.

Mockridge et al. (1998), European Journal of Endocrinology, 138:449-459.

Mutter, K. M., Arndt, K. M. and Alber, T. (2000), Methods in Enzymology, 328:261-281.

Nielsen BB, Kastrup JS, Rasmussen H, Holtet TL, Graversen JH, Etzerodt M, Thøgersen HC, Larsen IK (1997), FEBS-Letter, 412:388-396.

Pack P. and Pluckthun (1992), Biochemistry 31 :1579-1584.

Studier and Moffat (1986), Journal of Molecular Biology 189: 113-130.

Claims

1. A growth hormone fusion protein comprising at least one growth hormone and a multimerisation domain.

2. A fusion protein according to claim 1.wherein the growth hormone is selected from the group consisting of human growth hormone (hGH), bovine growth hormone (bGH) and porcine growth hormone (pGH).

3. A fusion protein according to claim 1 , wherein the multimerisation domain is a peptide selected from the group consisting of a dimerising domain, a trimerising domain, a tetramerising domain, a pentamerising domain and a hexamerising domain.

4. A fusion protein according to claim 1 , comprising at least two growth hormones.

5. A fusion protein according to claim 1 , wherein the at least one growth hormone is linked to the N-terminal amino acid residue of the multimerisation domain.

6. A fusion protein according to claim 1 , wherein the at least one growth hormone is linked to the C- terminal amino acid residue of the multimerisation domain.

7. A fusion protein according to claim 1 , wherein the multimerisation domain is a trimerising domain derived from tetranectin.

8. A fusion protein according to claim 7, wherein the trimerising domain derived from tetranectin comprises a sequence having at least 68% amino acid sequence identity with the sequence of SEQ ID NO SEQ ID NO 17.

9. A fusion protein according to claim 8, wherein the amino acid sequence identity is at least 75%, such as at least 87% including at least 92%.

10. A fusion protein according to claim 7, wherein the trimerising domain derived from tetranectin comprises the amino acid sequence SEQ ID NO 17.

11. A fusion protein according to claim 1 , further comprising a linker between the growth hormone and the multimerisation domain.

12. A fusion protein according to claim 7, wherein the trimerising domain derived from tetranectin comprises the amino acid sequence SEQ ID NO 18.

13. A fusion protein according to claim 12, wherein said protein is selected from the group consisting of TripA-GSA-hGH (SEQ ID NO 4), HOTripA-GSA-hGH (SEQ ID NO 6), TripA-GSQEGSA-hGH (SEQ ID NO 9), MOTripA-GSQEGSA-hGH (SEQ ID NO 11 ), TripA-hGH (SEQ ID NO 16), TripD-GSQEGSA-hGH (SEQ ID NO 23).

14. A polypeptide complex comprising at least two fusion proteins according to any of claims 1-13.

15. A polypeptide complex according to claim 14 comprising at least three fusion proteins, such as at least four, including at least five, such as at least six fusion proteins.

16. A polypeptide complex according to claim 14 or 15, having a plasma half life that is increased at least about 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 times, as compared to native monomeric growth hormone.

17. A composition comprising the growth hormone fusion protein according to any of claims 1-13.

18. A composition comprising the polypeptide complex according to any of claims 14- 16.

19. A method of treating a disease or disorder in an animal comprising the step of administering an effective amount of the growth hormone fusion protein according to any of claims 1 -13 or the polypeptide complex according to any of claims 14-16 to said animal.

20. A method according to claim 19, wherein said effective amount is 0.001-50 mg/kilogram of said animal.

21. A method according to claim 19, wherein the disease or disorder is selected from the group consisting of growth hormone deficiency, turner syndrome and metabolic disorder.

22. Use of a growth hormone fusion protein according to any one of claims 1-13, for the preparation of a pharmaceutical composition.

23. A kit comprising the composition of claim 17.

24. A kit comprising the composition of claim 18.

25. A method of producing a growth hormone fusion protein according to any of claims 1-13 or a polypeptide complex according to any of claims 14-16, comprising providing a host cell capable of expressing in recoverable amounts said growth hormone fusion protein and/or polypeptide complex.

26. A growth hormone fusion protein or a polypeptide complex produced by a method according to claim 25.

27. An isolated nucleic acid sequence encoding the fusion protein according to any of claims 1-13.

28. A recombinant vector comprising the isolated nucleic acid sequence according to claim 27.

29. A host cell transformed with a vector according to claim 28.