WO2009081170A2

WO2009081170A2 - Peptide fusion proteins

Info

Publication number: WO2009081170A2
Application number: PCT/GB2008/004279
Authority: WO
Inventors: Peter Artymiuk; Richard Ross; Jon Sayers
Original assignee: Asterion Limited
Priority date: 2007-12-24
Filing date: 2008-12-24
Publication date: 2009-07-02
Also published as: WO2009081170A3; GB0725201D0

Abstract

We disclose fusion proteins comprising a peptide comprising a binding domain for a receptor which is linked to a polypeptide comprising the binding domain to which said peptide binds.

Description

Peptide Fusion Proteins

The invention relates to fusion proteins comprising a peptide comprising a binding domain for a receptor which is linked to a polypeptide comprising the binding domain to which said peptide binds; dimers comprising said fusion protein; and methods to treat diseases that would benefit from administration of said fusion proteins.

Peptides are typically short polymers of amino acids linked to one another via amide bonds; polypeptides are generally consider to be longer chains of amino acids similarly linked to one another by amide bonds. Proteins are polypeptides that are folded into secondary structures (e.g. alpha helix, beta sheet) and form tertiary structures which represent the three dimensional form of the protein. Proteins may interact with other proteins to form quaternary structures. Some proteins function as monomers. The interaction between proteins is fundamental to function and results in biological effects in cells such as regulation of energy metabolism, cell differentiation and cell proliferation. Peptides that interact with receptors to bring about a biochemical response are known as agonists and those that prevent, or hinder, a biochemical response are known as antagonists. Activation of the receptors by peptide-specific binding promotes cell proliferation via activation of intracellular signalling cascades that result in the expression of, amongst other things, cell-cycle specific genes and the activation of quiescent cells to proliferate.

There are many examples of bioactive peptides that function to regulate biological processes.

For example substance P is an 11 amino acid neuro-peptide (Arg Pro Lys Pro GIn GIn Phe Phe GIy Leu Met) that binds the receptor neurokinin 1. Substance P is implicated in many biological processes, for example the transmission of pain, regulation of cell proliferation and vasodilation. Substance P is also implicated wound healing. Further examples of neuro-peptides include neurokinin A and neurokin B. Glucagon is a 29 amino acid peptide involved in regulating sugar metabolism and is secreted by the pancreas when sugar levels are low. Glucagon binds glucagon receptors expressed by the liver which results in mobilization of glucose release by catabolism of glycogen. Glucagon is used in cases of hypoglycemia. Glucagon like peptide 1 binds the GLP-1 receptor, a GPCR, and is important in promoting insulin secretion, sensitivity and can invoke weight loss and used in the treatment of diabetes. GLP-2 promotes bowel growth. Oxyntomodulin which shares homology with glucagon inhibits meal related energy intake. Amylin binds to a CRGP receptor in association with RAMP and is important in promoting insulin sensitivity and also may promote sensitivity to the weight reducing effects of leptin to be used in the treatment of diabetes. Calcitonin is a 32 amino acid peptide produced by the thyroid gland and is involved in the regulation of bone metabolism by promoting bone mineralization, vitamin D biosynthesis and regulating appetite. Calcitonin binds the calcitonin receptor which is a G-protein coupled receptor. Calcitonin is used to treat osteoporosis, hypercalcaemia and Paget's disease. Antidiuretic hormone vasopressin binds to the vasopressin receptors a GPCR and acts to retain water and may be important in memory and is used as an antidiuretic treatment.

The control of reproduction is mediated by the combined effects of hormones which regulate the female ovulation and menstruation. The hypothalamus secretes GnRH or LHRH that regulates the pituitary gland to secrete the hormones follicle stimulating hormone (FSH) and luteinizing hormone (LH). These hormones control the growth of oocytes in the ovaries and the secretion of the female hormones oestradiol and progesterone by the ovaries. FSH is produced and secreted by the anterior pituitary gland. In the ovary FSH stimulates the growth of immature Graafian follicles to maturation and as the follicle matures it begins to release inhibin B which inhibits the secretion of FSH. Active FSH is a glycoprotein comprising two subunits; the ά unit is 92 amino acids in length and the β subunit is 118 amino acids in length. Human FSH has been cloned and characterised, for example see US5, 156, 957. The FSH receptor is part of a large family of transmembrane receptors that regulate the heterotrimetric G proteins. The human FSH receptor contains 678 amino acids. It is organized into three domains: 1) the extracellular NH2 terminal ligand binding domain with six potential N- linked glycosylation sites and a cluster of cysteines at the junction between the extracellular and transmembrane domains; 2) the transmembrane spanning domain composed of seven hydrophobic a helicies that anchor the receptor to the plasma membrane; and 3) the intracellular COOH-terminal domain with a relatively high proportion of serine and threonine residues.

Growth hormone (GH) is an anabolic cytokine hormone important for linear growth in childhood and normal body composition in adults. The regulation of growth hormone activity is complex and involves a number of interacting polypeptide and peptide agonists and antagonists. Growth hormone can mediate its effects either directly by binding growth hormone receptor or indirectly by stimulating production of Insulin-like growth factor -1 (IGF-1) A major role of growth hormone is therefore the stimulation of the liver to produce IGF-1. In addition secretion of growth hormone is controlled by two peptide hormones with opposing activities. Growth hormone releasing hormone (GHRH) is a 44 amino acid peptide produced by the arcuate nucleus of the hypothalamus. It functions to stimulate growth hormone production by the anterior pituitary gland. Somatostatin is a peptide hormone that opposes the effects of GHRH and is processed from a larger pre- propeptide to a 14 and 28 amino acid form. Somatostatin is secreted by neuroendocrine cells of the periventricular nucleus of the hypothalamus into the hypothalamo- hypophysial portal system that connects with the anterior pituitary gland where it inhibits secretion of growth hormone. Somatostatin also inhibits the release of thyroid stimulating hormone and suppresses the secretion of gastrin, secretin, insulin and glucagon. There are other peripheral signals that act at the hypothalamus and pituitary to control appetite and growth hormone release and include Ghrelin that stimulates both appetite and growth hormone release and acts through the growth hormone secretagogue receptor.

Somatostatin binds 5 different receptors which are called SSR1 , 2, 3, 4 and 5. SSR1 has a higher affinity for somatostatin 14 than 28 and is a multiple transmembrane domain G protein linked receptor (as is each of SSR2, 3, 4 and 5). SSR1 is expressed in a number of tissues that include the fetal kidney and liver, the adult pancreas, brain, lung and stomach. SSR2 binds both somatostatin 14 and 28 and exists in two isoforms that result from differential splicing. SSR3 also binds both somatostatin 14 and 28 and has a more restricted expression pattern being expressed by the brain, pituitary gland and pancreas. SSR4 preferentially binds somatostatin 14 and has a similar expression pattern to SSR1. SSR5 preferentially binds somatostatin 28 and is expressed in the broadest range of fetal and adult tissues. The SSR receptors are integral membrane receptors with a domain structure comprising multiple extraxcellular and cytoplasmic domains.

Somatostatin is commercially available as Sandostatin® (also called octreotide, an octapeptide) and exerts pharmacologic actions similar to the natural hormone somatostatin. Sandostatin® suppresses GnRH, decreases splanchnic blood flow, and inhibits release of serotonin, gastrin, vasoactive intestinal peptide, secretin, motilin, and pancreatic polypeptide. Sandostatin® is used to treat the symptoms associated with metastatic carcinoid tumours and Vasoactive Intestinal Peptide (VIP) secreting adenomas .Sandostatin® substantially reduces growth hormone and/or IGF-I levels in patients with acromegaly An alternative to Sandostatin® is lanreotide which is a long acting form of Sandostatin® . The melanocortin 4 receptor, a CGPR, is important in regulating energy intake and agonists decrease appetite. Thyrotrophin releasing hormone (TRH) from the hypothalamus regulates the release of thyroid stimulating hormone (TSH) from the pituitary which stimulates the release of thyroid hormones from the thyroid gland. TSH binds to the TSH receptor a GPCR and TSH has been used in the diagnosis and screening of patients with thyroid cancer.

A problem associated with therapeutic peptides and small therapeutic polypeptides is that the subject administered the peptide agent clears the molecule in some cases in a matter of minutes. This is due in part to proteolytic degradation and renal filtration. In many cases this is addressed by providing a combined formulation of peptide agent with a second agent that modulates the release of the peptide after administration. For example lanreotide is marketed as a long acting acetate salt and is disclosed in US5, 411 , 943. US6, 150, 333 discloses an analogue of lanreotide which is a compound comprising the peptide of the formula H-β-D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂ which includes two cysteine amino acid residues which are bonded and an acetate salt thereof. The cyclised peptide has increased stability. In US4, 041 , 155 is discloses a pegylated form of somatostatin. The addition of polyethylene glycol to peptides and polypeptides is a known process for increasing molecule weight and thereby decrease systemic clearance.

The present disclosure addresses the problem of maintaining systemic levels of peptide/polypeptide therapeutics by reducing loss via proteolytic cleavage, renal filtration or any other means by which a pharmaceutically effective amount of peptides are reduced.

This disclosure relates to fusion proteins comprising a domain that comprises a peptide that binds a receptor and a domain that comprises a domain of a receptor to which the peptide binds. Optionally the peptide domain and the receptor domain are linked by a peptide linking domain that may be flexible or a linker comprising an inflexible or partially flexible helical region. Alternatively the peptide domain and the receptor domain are a direct translational fusion via a peptide bond. The fusion proteins of the invention have delayed clearance. According to an aspect of the invention there is provided a fusion protein comprising: i) a peptide comprising a binding domain for a receptor; linked to ii) a polypeptide comprising the binding domain of said receptor to which said peptide binds.

In a preferred embodiment of the invention said peptide is a peptide hormone.

In an alternative preferred embodiment of the invention said peptide is a peptide mimetic.

In a preferred embodiment of the invention said fusion protein is an agonist.

In an alternative preferred embodiment of the invention said fusion protein is an antagonist.

In a preferred embodiment of the invention said peptide is between 9 and 120 amino acids in length.

In a preferred embodiment of the invention said peptide is between 20 and 40 amino acids in length; preferably between 20-30 amino acids or between 30 and 40 amino acids in length.

In a preferred embodiment of the invention said peptide is linked to said receptor binding domain and is positioned amino terminal to said receptor binding domain in said fusion protein.

In an alternative preferred embodiment of the invention said peptide is linked to said receptor binding domain and is positioned carboxyl-terminal to said binding domain in said fusion protein.

In a preferred embodiment of the invention said peptide is linked to the receptor binding domain by a peptide linker; preferably a flexible peptide linker.

In a preferred embodiment of the invention said peptide linking molecule comprises at least one copy of the peptide GIy GIy GIy GIy Ser. In a preferred embodiment of the invention said peptide linking molecule comprises 2, 3, 4, 5 copies of the peptide GIy GIy GIy GIy Ser.

In an alternative preferred embodiment of the invention said peptide linker comprises an inflexible helical region.

In an embodiment of the invention a flexible non-helical region is located at or near the amino-terminal end of the peptide linker molecule thereby allowing the orientation of the peptide binding domain located at the amino-terminal end of the peptide linker molecule in relation to said receptor binding domain.

In an alternative embodiment of the invention a flexible non-helical region is located at or near the carboxyl-terminal end of the peptide linker molecule thereby allowing the orientation of the peptide binding domain located at the carboxyl-terminal end of the peptide linker molecule in relation to said receptor binding domain.

In a still further embodiment of the invention a flexible non-helical region is located at or near the amino and the carboxyl-terminal ends of the peptide linker molecule linking the peptide and receptor binding domains.

In a preferred embodiment of the invention the inflexible helical region comprises at least one copy of the motif A(EAAAK)_xA.

The length of the inflexible non-helical region is extendable by increasing the number of repeats of this A (EAAAK)_xA motif.

In a preferred embodiment of the invention, x in the A (EAAAK)_xA motif is less than 5 copies. Even more preferably still x is selected from 1 , 2, 3, 4 or 5 copies.

In a preferred embodiment of the invention said linker consists of an inflexible alpha helical linker between said peptide binding domain and said receptor binding domain.

In a still further alternative embodiment of the invention said fusion protein does not comprise a peptide linking molecule and is a direct fusion of the peptide and the receptor binding domain. In a preferred embodiment of the invention said peptide hormone is selected from the group consisting of: growth hormone releasing hormone anti-diuretic hormone; oxytocin; gonadotropin releasing hormone, corticotrophin releasing hormone; calcitonin, glucagon, amylin, A-type natriuretic hormone, B-type natriuretic hormone, ghrelin, neuropeptide Y, neuropeptide YY3-36, somatostatin; or homologues or analogues thereof.

In a preferred embodiment of the invention said fusion protein comprises growth hormone releasing hormone.

In an alternative preferred embodiment of the invention said fusion protein comprises somatostatin or homologue or analogue thereof; preferably somatostatin is somatostatin 14. Alternatively somatostatin is somatostatin 28.

In a preferred embodiment of the invention said fusion protein comprises the amino acid sequence: AGCKNFFW KTFTSC.

In an alternative preferred embodiment of the invention said fusion protein comprises the amino acid sequence: SANSNPAMAPRERKAGCKNFFW KTFTSC.

In a preferred embodiment of the invention said receptor binding domain is an extracellular receptor binding domain.

In a preferred embodiment of the invention said receptor binding domain comprises a somatostatin binding domain of a somatostatin 1 receptor.

In a preferred embodiment of the invention said receptor binding domain comprises a somatostatin binding domain of a somatostatin 2 receptor.

In a preferred embodiment of the invention said receptor binding domain comprises a somatostatin binding domain of a somatostatin 3 receptor.

In a preferred embodiment of the invention said receptor binding domain comprises a somatostatin binding domain of a somatostatin 4 receptor.

In a preferred embodiment of the invention said receptor binding domain comprises a somatostatin binding domain of a somatostatin 5 receptor. In a preferred embodiment of the invention said extracellular receptor binding domain comprises or consists of a somatostatin binding domain.

In a preferred embodiment of the invention said extracellular receptor binding domain comprises or consists of a somatostatin binding domain as illustrated by the amino acid sequence in Figure 1 with reference to table 2.

In an alternative preferred embodiment of the invention said fusion protein comprises of the follicle stimulating hormone (FSH) α subunit.

In a preferred embodiment of the invention said fusion protein comprises or consists (FSH) α subunit as represented by the amino acid sequence in Figure 2.

It is noted that the FSH α subunit is common to the hormones luteinising (LH) hormone and thyroid stimulating hormone (TSH) and therefore a claim to FSH α subunit is equivalent to a claim to LH and FSH. It is the β subunit of the respective hormones that confers receptor specificity.

In a further alternative preferred embodiment of the invention said fusion protein comprises or consists of the follicle stimulating hormone (FSH) β subunit.

In a preferred embodiment of the invention said fusion protein comprises or consists of the follicle stimulating hormone (FSH) β subunit as represented by the amino acid sequence in Figure 3.

In a preferred embodiment of the invention said fusion protein comprises or consists of the extracellular domain of follicle stimulating hormone receptor (FSHR).

In a preferred embodiment of the invention said fusion protein comprises or consists of the extracellular domain of follicle stimulating hormone receptor (FSHR) as represented by the amino acid sequence in Figure 4 with reference to Table 3.

In an alternative preferred embodiment of the invention said fusion protein comprises or consists of the LH β subunit. In a preferred embodiment of the invention said fusion protein comprises or consists of the LH β subunit as represented in Figure 5.

In a preferred embodiment of the invention said fusion protein comprises or consists of the extracellular domain of the LH receptor.

In a preferred embodiment of the invention said fusion protein comprises or consists of the extracellular domain of the LH receptor as represented in Figure 6 with reference to Table 5.

In a preferred embodiment of the invention said fusion protein comprises or consists of a TSH β subunit.

In a preferred embodiment of the invention said fusion protein comprises or consists of the amino acid as represented in Figure 7.

In a further preferred embodiment of the invention said fusion protein comprises of the extracellular domain of the TSH receptor.

In a preferred embodiment of the invention said fusion protein comprises or consists of the extracellular domain of the TSH receptor as represented in Figure 8 with reference to Table 6.

According to an aspect of the invention there is provided a nucleic acid molecule that encodes a fusion protein according to the invention.

According to a further aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the invention.

In a preferred embodiment of the invention said vector is an expression vector adapted to express the nucleic acid molecule according to the invention.

A vector including nucleic acid (s) according to the invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome for stable transfection.

Preferably the nucleic acid in the vector is operably linked to an appropriate promoter or other regulatory elements for transcription in a host cell. The vector may be a bi- functional expression vector which functions in multiple hosts. By "promoter" is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in eukaryotic or prokaryotic cells. "Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.

In a preferred embodiment the promoter is a constitutive, an inducible or regulatable promoter.

According to a further aspect of the invention there is provided a cell transfected or transformed with a nucleic acid molecule or vector according to the invention.

Preferably said cell is a eukaryotic cell. Alternatively said cell is a prokaryotic cell.

In a preferred embodiment of the invention said cell is selected from the group consisting of; a fungal cell (e.g. Pichia spp, Saccharomyces spp, Neurospora spp); insect cell (e.g. Spodoptera spp); a mammalian cell (e.g. COS cell, CHO cell); a plant cell.

According to a further aspect of the invention there is provided a pharmaceutical composition comprising a polypeptide according to the invention including an excipient or carrier.

In a preferred embodiment of the invention said pharmaceutical composition is combined with a further therapeutic agent.

When administered the pharmaceutical composition of the present invention is administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. The pharmaceutical compositions of the invention can be administered by any conventional route, including injection. The administration and application may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, intra-articuar, subcutaneous, topical (eyes), dermal (e.g a cream lipid soluble insert into skin or mucus membrane), transdermal, or intranasal.

Pharmaceutical compositions of the invention are administered in effective amounts. An "effective amount" is that amount of pharmaceuticals/compositions that alone, or together with further doses or synergistic drugs, produces the desired response. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods or can be monitored according to diagnostic methods.

The doses of the pharmaceuticals compositions administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject (i.e. age, sex). When administered, the pharmaceutical compositions of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. When used in medicine salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

The pharmaceutical compositions may be combined, if desired, with a pharmaceutically- acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction that would substantially impair the desired pharmaceutical efficacy. The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir or an emulsion.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation that is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 , 3-butane diol. Among the acceptable solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

According to a further aspect of the invention there is provided a method to treat a disease or condition in a subject comprising administering an effective amount of a fusion protein according to the invention. Peptide hormones are used to treat a number of diseases or conditions. For example antdiuretic hormone is used in the treatment of diabetes insipidus. Oxytoxin is used to induce parturition in pregnancy. Gonadotropin releasing hormone (GRH) is has been used to treat congenital deficiencies in GRH and also in the treatment of prostate cancer. Calcitonin is used in the treatment of osteoporosis, hypercalcaemia and Paget's disease. The peptides A-type natriuretic hormone, B-type natriuretic hormone are used in the treatment of high blood pressure.

Somatostatin is involved in a broad range of disease indications; for example in inhibiting cancer cell growth.

As used herein, the term "cancer" refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "cancer" includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "carcinoma" also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation.

According to an aspect of the invention there is provided a method to treat a subject suffering from a cancer comprising administering an effective amount of a somatostatin fusion protein according to the invention. In a preferred method of the invention said cancer is a carcinoid cancer

Carcinoid syndrome results from secondary symptoms of cancers that affect the gastrointestinal tract. These cancers are typically endocrine and result in the production of hormones that affect undesirable effects such as flushing, diarrhoea, abdominal pain, fibrosis, bronchoconstriction.

Somatostatin and somatostatin analogues are effective treatments for thyrotrophic adenomas, neuroendocrine tumours, hepatomas, lung cancer and melanoma.

According to an aspect of the invention there is provided a method to treat a subject suffering from acromegaly comprising administering an effective amount of a somatostatin fusion protein according to the invention.

According to an aspect of the invention there is provided a method to treat a subject suffering side effects of cancer chemotherapy comprising administering an effective amount of a somatostatin fusion protein according to the invention.

According to an aspect of the invention there is provided a method to treat a subject in need of treatment for hypogonadism comprising providing an effective amount of a FSH fusion protein according to the invention.

According to an aspect of the invention there is provided a method to treat a subject in need of treatment for hypogonadism comprising providing an effective amount of a LH fusion protein according to the invention.

In a preferred method of the invention said subject is male.

In an alternative preferred method of the invention said subject is female.

In a preferred method of the invention hypogonadism is the result of a disease or condition selected from the group consisting of: Kallman syndrome, hypothalamic suppression, hypopituitarism, hyperprolactinemia, gonadotropin deficiency. In a preferred method of the invention of the invention said method includes the administration of at least one further agent effective in the treatment of hypogonadism.

In a preferred method of the invention said further agent is the LH fusion protein according to the invention.

According to an aspect of the invention there is provided a method to treat a subject suffering from a cancer comprising administering an effective amount of a TSH fusion protein according to any of claims 46-49.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following Tables and figures:

Table 1 is illustrates the domains in full length somatostatin and the full length amino acid sequence;

Table 2 illustrates the domains in full length somatostatin 1 receptor and the full length amino acid sequence;

Table 3 illustrates the domains in full length FSH receptor and the full length amino acid sequence; Table 4 illustrates the domains in full length TSH α and the full length amino acid sequence;

Table 5 illustrates the domains in full length LH receptor and the full length amino acid sequence;

Table 6 illustrates the domains in full length TSH receptor and the full length amino acid sequence;

Table 7 illustrates the domains in full length TSH β and the full length amino acid sequence;

Table 8 illustrates the domains in LH and the full length amino acid sequence;

Figure 1 is the amino acid sequence of the full length somatostatin receptor;

Figure 2 is the amino acid sequence of processed human (FSH) α subunit;

Figure 3 is the amino acid sequence of processed human (FSH) β subunit.

Figure 4 is the amino acid sequence of the extracellular domain of human FSH receptor;

Figure 5 is the amino acid sequence of processed human LH β subunit;

Figure 6 is the amino acid sequence of the extracellular domain of human LH receptor;

Figure 7 is the amino acid sequence of human TSH β;

Figure 8 is the amino acid sequence of human TSH receptor

Materials and Methods

Immunological testing

Immunoassays that measure the binding of peptide fusion protein or receptor to polyclonal and monoclonal antibodies are known in the art. Commercially available antibodies are available to detect the peptide or receptor in samples and also for use in competitive inhibition studies.

Recombinant Production of fusion proteins

The components of the fusion proteins were generated by PCR using primers designed to anneal to the ligand or receptor and to introduce suitable restriction sites for cloning into the target vector. The template for the PCR comprised the target gene and was obtained from IMAGE clones, cDNA libraries or from custom synthesised genes. Once the ligand and receptor genes with the appropriate flanking restriction sites had been synthesised, these were then ligated either side of the linker region in the target vector. The construct was then modified to contain the correct linker without flanking restriction sites by the insertion of a custom synthesised length of DNA between two unique restriction sites either side of the linker region, by mutation of the linker region by ssDNA modification techniques, by insertion of a primer duplex/multiplex between suitable restriction sites or by PCR modification.

Alternatively, the linker with flanking sequence, designed to anneal to the ligand or receptor domains of choice, was initially synthesised by creating an oligonucleotide duplex and this processed to generate double-stranded DNA. PCRs were then performed using the linker sequence as a "megaprimer", primers designed against the opposite ends of the ligand and receptor to which the "megaprimer" anneals to and with the ligand and receptor as the templates. The terminal primers were designed with suitable restriction sites for ligation into the expression vector of choice.

Expression and Purification of Fusion Proteins

Expression was carried out in a suitable system (e.g. mammalian CHO cells, E. coli) and this was dependant on the vector into which the fusion gene was generated. Expression was then analysed using a variety of methods which could include one or more of SDS- PAGE, Native PAGE, western blotting, ELISA.

Once a suitable level of expression was achieved the fusions were expressed at a larger scale to produce enough protein for purification and subsequent analysis. Purification was carried out using a suitable combination of one or more chromatographic procedures such as ion exchange chromatography, hydrophobic interaction chromatography, ammonium sulphate precipitation, gel filtration, size exclusion and/or affinity chromatography (using nickel/cobalt-resin, antibody-immobilised resin and/or ligand/receptor-immobilised resin).

Purified protein was analysed using a variety of methods which could include one or more of Bradford's assay, SDS-PAGE, Native PAGE, western blotting, ELISA.

Characterisation of fusions

Denaturing PAGE, native PAGE gels and western blotting were used to analyse the fusion polypeptides and western blotting performed with antibodies non-conformationally sensitive to the fusion. Native solution state molecular weight information can be obtained from techniques such as size exclusion chromatography using a Superose G200 analytical column and analytical ultracentrifugation.

Claims

1. A fusion protein comprising: i) a peptide comprising a binding domain for a receptor; linked to ii) a polypeptide comprising the binding domain of said receptor to which said peptide binds.

2. A fusion protein according to claim 1 wherein said peptide is a peptide hormone.

3. A fusion protein according to claim 1 wherein said peptide is a peptide mimetic.

4. A fusion protein according to any of claims 1-3 wherein said fusion protein is an agonist.

5. A fusion protein according to any of claims 1-3 wherein said fusion protein is an antagonist.

6. A fusion protein according to any of claims 1-5 wherein said peptide is between 9 and 120 amino acids in length.

7. A fusion protein according to claim 6 wherein said peptide is between 20 and 40 amino acids in length; preferably between 20-30 amino acids or between 30 and 40 amino acids in length.

8. A fusion protein according to any of claims 1-7 wherein said peptide is linked to said receptor binding domain and is positioned amino terminal to said receptor binding domain in said fusion protein.

9. A fusion protein according to any of claims 1-7 wherein said peptide is linked to said receptor binding domain and is positioned carboxyl-terminal to said binding domain in said fusion protein.

10. A fusion protein according to any of claims 1-9 wherein said peptide is linked to the receptor binding domain by a peptide linker, preferably a flexible peptide linker.

11. A fusion protein according to claim 10 wherein said peptide linking molecule comprises at least one copy of the peptide GIy GIy GIy GIy Ser.

12. A fusion protein according to claim 11 wherein said peptide linking molecule comprises 2, 3, 4, 5 copies of the peptide GIy GIy GIy GIy Ser.

13. A fusion protein according to any of claims 1-9 wherein said peptide linker comprises an inflexible helical region.

14. A fusion protein according to claim 13 wherein a flexible non-helical region is located at or near the amino-terminal end of the peptide linker molecule thereby allowing the orientation of the peptide binding domain located at the amino-terminal end of the peptide linker molecule in relation to said receptor binding domain.

15. A fusion protein according to claim 13 wherein a flexible non-helical region is located at or near the carboxyl-termina! end of the peptide linker molecule thereby allowing the orientation of the peptide binding domain located at the carboxyl-terminal end of the peptide linker molecule in relation to said receptor binding domain.

16. A fusion protein according to claim 13 wherein a flexible non-helical region is located at or near the amino and the carboxyl-terminal ends of the peptide linker molecule linking the peptide and receptor binding domains.

17. A fusion protein according to any of claims 13-16 wherein the inflexible helical region comprises at least one copy of the motif A(EAAAK)_xA.

18. A fusion protein according to claim 17 wherein x in the A (EAAAK)_XA motif is less than 5 copies.

19. A fusion protein according to any of claims 1-9 wherein said linker consists of an inflexible alpha helical linker between said peptide binding domain and said receptor binding domain.

20. A fusion protein according to any of claims 1-9 wherein said fusion protein does not comprise a peptide linking molecule and is a direct fusion of the peptide and the receptor binding domain by a peptidic bond.

21. A fusion protein according to any of claims 1-20 wherein said fusion protein comprises or consists of a TSH β subunit.

22. A fusion protein according to claim 21 wherein said fusion protein comprises or consists of the amino acid as represented in Figure 7.

23. A fusion protein according to claim 21 or 22 wherein said fusion protein comprises of the extracellular domain of the TSH receptor.

24. A fusion protein according to claim 23 wherein said fusion protein comprises or consists of the extracellular domain of the TSH receptor as represented in Figure 8 with reference to Table 6.

25. A fusion protein according to any of claims 1-20 wherein said fusion protein comprises of the follicle stimulating hormone (FSH) α subunit.

26. A fusion protein according to claim 25 wherein said fusion protein comprises (FSH) α subunit as represented by the amino acid sequence in Figure 2.

27. A fusion protein according to any of claims 1-20 wherein said fusion protein comprises or consists of the follicle stimulating hormone (FSH) β subunit.

28. A fusion protein according to claim 27 wherein said fusion protein comprises or consists of the follicle stimulating hormone (FSH) β subunit as represented by the amino acid sequence in Figure 3.

29. A fusion protein according to any of claims 25-28 wherein said fusion protein comprises or consists of the extracellular domain of follicle stimulating hormone receptor (FSHR).

30. A fusion protein according to claim 29 wherein said fusion protein comprises or consists of the extracellular domain of follicle stimulating hormone receptor (FSHR) as represented by the amino acid sequence in Figure 4 with reference to Table 3.

31. A fusion protein according to any of claims 1-20 wherein said fusion protein comprises or consists of the LH β subunit.

32. A fusion protein according to claim 31 wherein said fusion protein comprises or consists of the LH β subunit as represented in Figure 5.

33. A fusion protein according to claim 31 or 32 wherein said fusion protein comprises or consists of the extracellular domain of the LH receptor.

34. A fusion protein according to claim 33 wherein said fusion protein comprises or consists of the extracellular domain of the LH receptor as represented in Figure 6 with reference to Table 5.

35. A fusion protein according to any of claims 1-20 wherein said peptide hormone is selected from the group consisting of: somatostatin, growth hormone releasing hormone, anti-diuretic hormone; oxytocin; gonadotropin releasing hormone, corticotrophin releasing hormone; calcitonin, glucagon, amylin, A-type natriuretic hormone, B-type natriuretic hormone, ghrelin, neuropeptide Y, neuropeptide YY3-36,; or homologues or analogues thereof.

36. A fusion protein according to claim 35 wherein said fusion protein comprises growth hormone releasing hormone.

37. A fusion protein according to claim 35 wherein said fusion protein comprises somatostatin or homologue or analogue thereof.

38. A fusion protein according to claim 37 wherein somatostatin is somatostatin 14.

39. A fusion protein according to claim 37 wherein somatostatin is somatostatin 28.

40. A fusion protein according to claim 38 wherein said fusion protein comprises the amino acid sequence: AGCKNFFW KTFTSC.

41. A fusion protein according to claim 39 wherein said fusion protein comprises the amino acid sequence: SANSNPAMAPRERKAGCKNFFW KTFTSC.

42. A fusion protein according to any of claims 37-41 wherein said receptor binding domain comprises an extracellular receptor binding domain.

43. A fusion protein according to claim 42 wherein said receptor binding domain comprises a somatostatin binding domain of a somatostatin 1 receptor.

44. A fusion protein according to claim 42 wherein said receptor binding domain comprises a somatostatin binding domain of a somatostatin 2 receptor.

45. A fusion protein according to claim 42 wherein said receptor binding domain comprises a somatostatin binding domain of a somatostatin 3 receptor.

46. A fusion protein according to claim 42 wherein said receptor binding domain comprises a somatostatin binding domain of a somatostatin 4 receptor.

47. A fusion protein according to claim 42 wherein said receptor binding domain comprises a somatostatin binding domain of a somatostatin 5 receptor.

48. A fusion protein according to any of claims 42-47 wherein said extracellular receptor binding domain consists of a somatostatin binding domain.

49. A fusion protein according to any of claims 42-48 wherein said extracellular receptor binding domain comprises or consists of a somatostatin binding domain as illustrated by the amino acid sequence in Figure 1 with reference to table 2.

50. A nucleic acid molecule that encodes a fusion protein according to any of claims 1-49.

51. A vector comprising a nucleic acid molecule according to claim 50.

52. A cell transfected or transformed with a nucleic acid molecule or vector according to claim 50 or 51.

53. A cell according to claim 52 wherein said cell is stably transfected or transformed.

54. A cell according to claim 52 wherein said cell is transiently transfected or transformed.

55. A pharmaceutical composition comprising a fusion protein according to any of claims 1-49 including an excipient or carrier.

56. A composition according to claim 55 wherein said pharmaceutical composition is combined with a further therapeutic agent.

57. A method to treat a subject suffering from a cancer comprising administering an effective amount of a somatostatin fusion protein according to any of claims 35-49.

58. A method according to claim 57 wherein said cancer is a carcinoid cancer

59. A method to treat a subject suffering from acromegaly comprising administering an effective amount of a somatostatin fusion protein according to any of claims 35-49.

60. A method to treat a subject suffering side effects of cancer chemotherapy comprising administering an effective amount of a somatostatin fusion protein according to any of claims 35-49.

61. A method to treat a subject in need of treatment for hypogonadism comprising providing an effective amount of a FSH fusion protein according to any of claims 25-30.

62. A method to treat a subject in need of treatment for hypogonadism comprising providing an effective amount of a LH fusion protein according to any of claims 31-34.

63. A method according to claim 61 or 62 wherein said subject is male.

64. A method according to claim 61 or 62 wherein said subject is female.

65. A method according to any of claims 61-64 wherein hypogonadism is the result of a disease or condition selected from the group consisting of: Kallman syndrome, hypothalamic suppression, hypopituitarism, hyperprolactinemia, gonadotropin deficiency.

66. A method according to any of claims 61-65 wherein said method includes the administration of at least one further agent effective in the treatment of hypogonadism.

67. A method according to claim 66 wherein said agent is a LH fusion protein according to any of claims 31-34.

68. A method according to any of claims 61-65 wherein said method includes the administration of at least one further agent effective in the treatment of hypogonadism.

69. A method according to claim 68 wherein said agent is a FSH fusion protein according to any of clams 25-30.

70. A method to treat a subject suffering from a cancer comprising administering an effective amount of a fusion protein according to any of claims 21 to 26.