WO2010020766A2

WO2010020766A2 - Interleukin fusion polypeptides

Info

Publication number: WO2010020766A2
Application number: PCT/GB2009/002004
Authority: WO
Inventors: Peter Artymiuk; Richard Ross
Original assignee: Asterion Ltd
Current assignee: Asterion Ltd
Priority date: 2008-08-21
Filing date: 2009-08-17
Publication date: 2010-02-25
Anticipated expiration: 2011-02-21
Also published as: GB0815216D0; WO2010020766A3

Abstract

The disclosure relates to interleukin fusion polypeptides and dimers; nucleic acid molecules encoding said polypeptides and methods of treatment that use said polypeptides/dimers.

Description

lnterleukin Fusion Polypeptides

The invention relates to interleukin fusion polypeptides and dimers; nucleic acid molecules encoding said polypeptides and methods of treatment that use said polypeptides/dimers.

Cytokine receptors can be divided into two separate classes. Class 1 (referred to as the haematopoietic or growth hormone family) receptors are characterised by four conserved cysteine residues in the amino terminal part of their extracellular domain and the presence of a conserved Trp-Ser-Xaa-Trp-Ser motif in the C-terminal part. The receptors consist of two polypeptide chains. Class I receptors can be sub-divided into the GM-CSF sub-family (which includes IL-3, IL-5, GM-CSF, GCSF) and IL-6 sub-family (which includes IL-6, IL-11 and IL-12). In the IL-6 sub-family there is a common transducing subunit (gp130) that associates with one or two different cytokine subunits. The repeated Cys motif is also present in Class 2 (interferon receptor family) the ligands of which are α, β and y interferon but lack the conserved Trp-Ser-Xaa-Trp-Ser motif.

The interleukins represent a large group of cytokines with diverse functions and were first characterised by expression in leukocytes and have since been shown to be expressed in a wide variety of cells, for example macrophages, TH-1 and TH-2 cells, T- lymphocytes, monocytes and bone marrow stroma. Broadly, the function of the immune system depends in a large part on the expression and function of the interleukins. A large number of interleukins have been cloned and functionally characterised; for example interleukins 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 and 33 and within each species of interleukin there exists isotypes that are sequence variants that have subtly different functions.

The interleukin 2 (IL-2) sub-family (includes IL-2, IL-4, IL-7, IL-9, IL-15 and IL21). IL-2 signals through the IL2 receptor α (CD25), IL-2 receptor β (CD122) and a polypeptide chain referred to as yc; yc is a common receptor for all IL-2 subfamily members. Binding of IL-2 activates the Ras/MAPK, JAK/Stat and Pl 3 kinase signal transduction pathways. IL-2 is a 15.5kDa glycosylated polypeptide that stimulates extended T- lymphocyte proliferation and was the first interleukin to be discovered. IL-2 is induced during an immune response to a foreign antigen. The binding of antigen to T cell antigen receptor (TCR) results in the secretion of IL-2 and expression of IL-2 receptors. This results in activation of antigen selected cytotoxic T cells. IL-2 is also involved in T cell maturation in the thymus and for the development of Regulatory T -cells (T_REG_S) which regulate self recognition by T cells. Recombinant IL-2 is used in the treatment of cancer to augment the immune system in particular in the treatment of melanoma and renal cancer. IL-2 also has proposed applications in chronic viral infection and as an adjuvant in vaccines. IL-2 antagonists are used in the treatment of inflammatory diseases, for example rheumatoid arthritis.

This disclosure relates to the identification of interleukin recombinant forms that have improved pharmacokinetics and activity. The new interferon molecules are biologically active, form dimers and have improved stability.

According to an aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence that encodes a polypeptide that has the activity of _s an interleukin wherein said polypeptide comprises an interleukin, or part thereof linked, directly or indirectly, to the interleukin binding domain of an interleukin receptor.

According to an aspect of the invention there is provided a fusion polypeptide comprising: the amino acid sequence of an interleukin, or active binding part thereof, linked, directly or indirectly, to the binding domain of an interleukin receptor.

In a preferred embodiment of the invention said interleukin is IL-2.

In a preferred embodiment of the invention said fusion polypeptide comprises the amino acid sequence represented in Figure 8, optionally including the signal sequence.

In a preferred embodiment of the invention said fusion polypeptide comprises IL2 receptor α.

In an alternative preferred embodiment of the invention said fusion polypeptide comprises IL2 receptor β.

In an alternative preferred embodiment of the invention said fusion polypeptide comprises an IL2 receptor vc. In a preferred embodiment of the invention said fusion polypeptide comprises or consists of IL2 receptor α as represented by the amino acid sequence in Figure 9, optionally including the signal sequence.

In an alternative preferred embodiment of the invention said fusion polypeptide comprises or consists of IL2 receptor β as represented by the amino acid sequence in Figure 10, optionally including the signal sequence.

In an alternative preferred embodiment of the invention said fusion polypeptide comprises or consists of an IL2 receptor yc as represented by the amino acid sequence in Figure 11 , optionally including the signal sequence.

In a preferred embodiment of the invention said interleukin is linked to an interleukin binding domain of an interleukin receptor wherein said interleukin is positioned amino terminal to said binding domain in said fusion polypeptide.

In an alternative preferred embodiment of the invention interleukin is linked to an interleukin binding domain of an interleukin receptor wherein said interleukin is positioned carboxyl-terminal to said binding domain in said fusion polypeptide.

In a preferred embodiment of the invention said interleukin is linked to the binding domain of the of the interleukin receptor 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 or 6 copies of the peptide GIy GIy GIy GIy Ser.

Preferably said peptide linking molecule consists of 5 copies of the peptide GIy GIy GIy GIy Ser.

In a still further alternative embodiment of the invention said polypeptide does not comprise a peptide linking molecule and is a direct fusion of interleukin and the interleukin binding domain of the interleukin receptor. According to an aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence selected from: i) a nucleic acid sequence as represented in Figure 1a; ii) a nucleic acid sequence as represented in Figure 2a; iii) a nucleic acid sequence as represented in Figure 3a; iv) a nucleic acid sequence as represented in Figure 4a; v) a nucleic acid sequence as represented in Figure 5a; vi) a nucleic acid sequence as represented in Figure 6a; vii) a nucleic acid sequence as represented in Figure 7a; or a nucleic acid molecule comprising a nucleic sequence that hybridizes under stringent hybridization conditions to Figures 1a, 2a, 3a, 4a, 5a, 6a or 7a and which encodes a polypeptide that has interleukin 2 modulating activity.

Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T_n, is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize)

Hybridization: 5x SSC at 65°C for 16 hours Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize)

Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1 x SSC at 55°C-70°C for 30 minutes each Low Stringency (allows sequences that share at least 50% identity to hybridize) Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

In a preferred embodiment of the invention said nucleic acid molecule encodes a polypeptide that has agonist activity.

In a preferred embodiment of the invention said nucleic acid molecule encodes a polypeptide that has antagonist activity.

In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 1a.

In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 2a.

In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 3a.

In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 4a.

In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 5a.

In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 6a.

In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 7a.

According to an aspect of the invention there is provided a polypeptide encoded by the nucleic acid according to the invention. According to a further aspect of the invention there is provided a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: Figure 1b, 2b, 3b, 4b, 5b, 6b or 7b, optionally including the signal sequence. .

According to an aspect of the invention there is provided a homodimer consisting of two polypeptides wherein each of said polypeptides comprises: i) a first part comprising interleukin 2, or a receptor binding domain thereof, optionally linked by a peptide linking molecule to ii) a second part comprising at least one interleukin 2 binding domain or part thereof, of an interleukin receptor.

In a preferred embodiment of the invention said homodimer comprises two polypeptides comprising or consisting of the amino acid sequence represented in Figure 1 b, 2b, 3b, 4b, 5b, 6b or 7b, optionally including the signal sequence. .

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 an aspect of the invention there is provided a vaccine composition comprising a nucleic acid molecule or polypeptide according to the invention and an antigenic molecule.

It will be apparent to the skilled artisan that the polypeptides of the invention are potent adjuvants. An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells. Examples of adjuvants include Freunds adjuvant, muramyl dipeptides or liposomes. An adjuvant is therefore an immunomodulator. The fusion polypeptides of the invention may be administered either as a polypeptide adjuvant or as a nucleic acid molecule in the example of DNA vaccination. The composition may include a carrier. Some polypeptide or peptide antigens contain B-cell epitopes but no T cell epitopes. Immune responses can be greatly enhanced by the inclusion of a T cell epitope in the polypeptide/peptide or by the conjugation of the polypeptide/peptide to an immunogenic "carrier" protein such as key hole limpet haemocyanin or tetanus toxoid which contain multiple T cell epitopes.

In a preferred embodiment of the invention said antigenic molecule is a viral polypeptide antigen. Viral pathogens are a major source of disease in humans and animals, for example live stock animals. Viral antigens are derived from a viral pathogens such as Human Immunodeficiency Virus; Human T Cell Leukaemia Virus, Ebola virus or other haemorrhagic fever virus, human papilloma vims (HPV) that cause cervical cancer and other cancers, papovavirus, rhinovirus, poliovirus, herpesvirus, adenovirus, Epstein Barr virus, influenza virus A, B or C, Hepatitis B and C viruses, Variola virus, rotavirus or SARS coronavirus.

In an alternative preferred embodiment of the invention said antigenic molecule is a cancer antigen.

The term "antigenic molecule" refers to a nucleotide sequence, the expression of which in a target cell results in the production of a cell surface antigenic protein capable of recognition by the immune system. The antigenic molecule is derived from a tumour cell specific antigen; ideally a tumour rejection antigen. Tumour rejection antigens are well known in the art and include, for example, the MAGE, BAGE, GAGE and DAGE families of tumour rejection antigens, see Schulz et al Proc Natl Acad Sci USA, 1991, 88, pp991- 993. It has been known for many years that tumour cells produce a number of tumour cell specific antigens, some of which are presented at the tumour cell surface. These are generally referred to as tumour rejection antigens and are derived from larger polypeptides referred to as tumour rejection antigen precursors. Tumour rejection antigens are presented via HLA's to the immune system. The immune system recognises these molecules as foreign and naturally selects and destroys cells expressing these antigens. If a transformed cell escapes detection and becomes established a tumour develops. Vaccines have been developed based on dominant tumour rejection antigen's to provide individuals with a preformed defence to the establishment of a tumour.

According to a further aspect of the invention there is provided a method to treat a human subject suffering from a viral infection comprising administering an effective amount of a polypeptide according to the invention. According to a further aspect of the invention there is provided a method to treat a human subject suffering from cancer comprising administering an effective amount of a polypeptide according to the invention.

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 tumours of mesenchymal derivation.

In a preferred method of the invention said cancer is melanoma.

In an alternative preferred method of the invention said cancer is renal cancer.

According to a further aspect of the invention there is provided a method to treat a human subject suffering from an inflammatory condition comprising administering an effective amount of a polypeptide antagonist according to the invention.

In a preferred method of the invention said inflammatory condition is an autoimmune disease; preferably said auto-immune disease is rheumatoid arthritis. In a further preferred embodiment of the invention said polypeptide is administered at two day intervals; preferably said polypeptide is administered at weekly, 2 weekly or monthly intervals.

According to an aspect of the invention there is provided the use of a polypeptide according to the invention for the manufacture of a medicament for the treatment of viral infection.

According to an aspect of the invention there is provided the use of a polypeptide according to the invention for the manufacture of a medicament for the treatment of cancer.

In a preferred embodiment of the invention said cancer is melanoma; preferably malignant melanoma.

In an alternative preferred embodiment of the invention said cancer is renal cancer.

According to an aspect of the invention there is provided the use of a polypeptide antagonist according to the invention for the manufacture of a medicament for the treatment of an autoimmune disease.

In a preferred embodiment of the invention said autoimmune disease is rheumatoid arthritis.

According to a further aspect of the invention there is provided a monoclonal antibody that binds the polypeptide or dimer according to the invention.

Preferably said monoclonal antibody is an antibody that binds the polypeptide or dimer but does not specifically bind interleukin 2, or interleukin 2 receptor individually.

The monoclonal antibody binds a conformational antigen presented either by the polypeptide of the invention or a dimer comprising the polypeptide of the invention.

In a further aspect of the invention there is provided a method for preparing a hybhdoma cell-line producing monoclonal antibodies according to the invention comprising the steps of: i) immunising an immunocompetent mammal with an immunogen comprising at least one polypeptide according to the invention; ii) fusing lymphocytes of the immunised immunocompetent mammal with myeloma cells to form hybridoma cells; iii) screening monoclonal antibodies produced by the hybridoma cells of step

(ii) for binding activity to the polypeptide of (i); iv) culturing the hybridoma cells to proliferate and/or to secrete said monoclonal antibody; and v) recovering the monoclonal antibody from the culture supernatant.

Preferably, the said immunocompetent mammal is a mouse. Alternatively, said immunocompetent mammal is a rat.

The production of monoclonal antibodies using hybridoma cells is well-known in the art. The methods used to produce monoclonal antibodies are disclosed by Kohler and Milstein in Nature 256, 495-497 (1975) and also by Donillard and Hoffman, "Basic Facts about Hybridomas" in Compendium of Immunology V.ll ed. by Schwartz, 1981, which are incorporated by reference.

According to a further aspect of the invention there is provided a hybridoma cell-line obtained or obtainable by the method according to the invention.

According to a further aspect of the invention there is provided a diagnostic test to detect a polypeptide according to the invention in a biological sample comprising:

i) providing an isolated sample to be tested; ii) contacting said sample with a ligand that binds the polypeptide or dimer according to the invention; and iii) detecting the binding of said ligand in said sample.

In a preferred embodiment of the invention said ligand is an antibody; preferably a monoclonal antibody.

According to an aspect of the invention there is provided a fusion polypeptide comprising: the amino acid sequence of an interleukin, or active binding part thereof, linked, directly or indirectly, to the binding domain of an interleukin receptor wherein said interleukin forms a complex with a polypeptide comprising or consisting of the amino acid sequence represented in Figure 11 , optionally including the signal sequence.

In a preferred embodiment of the invention said interleukin is selected from the group consisting of: IL-2, IL-4, IL-7, IL-9, IL-15 and IL21.

In a preferred embodiment of the invention said binding domain of an interleukin receptor comprises or consists of the amino acid sequence represented in Figure 11.

According to a further aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence that encodes a polypeptide that has the activity of interleukin 7 wherein said polypeptide comprises interleukin 7, or part thereof linked, directly or indirectly, to the interleukin 7 binding domain of an interleukin 7 receptor.

According to an aspect of the invention there is provided a fusion polypeptide comprising: the amino acid sequence of interleukin 7, or active binding part thereof, linked, directly or indirectly, to the binding domain of an interleukin 7 receptor.

In a preferred embodiment said fusion polypeptide comprises or consists of the amino acid sequence represented in Figure 12, optionally including the signal sequence.

In a preferred embodiment of the invention said fusion polypeptide comprises IL7 receptor α.

In an alternative embodiment of the invention said fusion polypeptide comprises IL2 receptor y.

Preferably said fusion polypeptide comprises or consists of the amino acid sequence represented in Figure 13, optionally including the signal sequence.

Preferably said fusion polypeptide comprises or consists of the amino acid sequence represented in Figure 14, optionally including the signal sequence.

In a preferred embodiment of the invention interleukin 7 is linked to an interleukin 7 binding domain of an interleukin 7 receptor wherein said interleukin 7 is positioned amino terminal to said binding domain in said fusion polypeptide. In an alternative embodiment of the invention interleukin 7 is linked to an interleukin 7 binding domain of an interleukin 7 receptor wherein said interleukin 7 is positioned carboxyl-terminal to said binding domain in said fusion polypeptide.

In a preferred embodiment of the invention interleukin 7 is linked to the binding domain of the of the interleukin 7 receptor by a peptide linker.

Preferably 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 consists of 4 or 5 copies of the peptide GIy GIy GIy GIy Ser.

In a preferred embodiment of the invention said polypeptide does not comprise a peptide linking molecule and is a direct fusion of interleukin 7 and the interleukin binding domain of the interleukin 7 receptor.

According to a further aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence selected from: i) a nucleic acid sequence as represented in Figure 15b; ii) a nucleic acid sequence as represented in Figure 16b; iii) a nucleic acid sequence as represented inFigure 17b; iv) a nucleic acid sequence as represented inFigure 18b; v) a nucleic acid sequence as represented inFigure 19b; vi) a nucleic acid sequence as represented inFigure 19c; vii) a nucleic acid sequence as represented inFigure 20b; viii) a nucleic acid sequence as represented in Figure 20c ix) a nucleic acid sequence as represented in Figure 21b x) a nucleic acid sequence as represented inFigure 21c; xi) a nucleic acid sequence as represented inFigure 22b; xii) a nucleic acid sequence as represented inFigure 22c; xiii) a nucleic acid sequence as represented inFigure 23b; xiv) a nucleic acid sequence as represented inFigure 23c; xv) a nucleic acid sequence as represented inFigure 24b; xvi) a nucleic acid sequence as represented inFigure 24c; xvii) a nucleic acid sequence as represented inFigure 25b; xviii) a nucleic acid sequence as represented inFigure 25c; xix) a nucleic acid sequence as represented inFigure 26b;

XX) a nucleic acid sequence as represented inFigure 26c or a nucleic acid molecule comprising a nucleic sequence that hybridizes under stringent hybridization conditions to Figure 15b, 16b, 17b, 18b, 19b, 19c, 20b, 20c, 21b, 21c, 22b, 22c, 23b, 23c, 24b, 24c, 25b, 25c, 26b or 26c and which encodes a polypeptide that has interleukin 7 activity.

In an alternative preferred embodiment of the invention said nucleic acid molecule encodes a polypeptide that has antagonist activity.

According to an aspect of the invention there is provided a polypeptide encoded by the nucleic acid according to the invention.

According to an aspect of the invention there is provided a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence represented in Figure 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a or 26a, optionally including the signal sequence.,

According to a further aspect of the invention there is provided a homodimer consisting of two polypeptides wherein each of said polypeptides comprises: i) a first part comprising interleukin 7, or a receptor binding domain thereof, optionally linked by a peptide linking molecule to ii) a second part comprising at least one interleukin 7 binding domain or part thereof, of an interleukin 7 receptor. In a preferred embodiment of the invention said homodimer comprises two polypeptides comprising or consisting of the amino acid sequence represented in Figure 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a or 26a, optionally including the signal sequence.

According to a further aspect of the invention there is provided a method to treat a human subject suffering from autoimmune disease comprising administering an effective amount of a polypeptide according to the invention.

In a preferred method of the invention said autoimmune disease is multiple sclerosis.

In an alternative method of the invention said autoimmune disease is rheumatoid arthritis.

According to a further aspect of the invention there is provided a method to treat a human subject suffering from lymphopenia comprising administering an effective amount of a polypeptide according to the invention.

Preferably, lymphopenia is the result of a disease or condition selected from the group consisting of: AIDS, tuberculosis, chemotherapy, radiotherapy, critical illness, sepsis, bone marrow suppression, vitamin deficiency.

According to a further aspect of the invention there is provided a method to treat a human subject suffering from a viral infection comprising administering an effective amount of a polypeptide according to the invention.

Preferably, said viral infection is caused by a virus selected from the group consisting of: HIV, HCV, HSV, hepatitis A, B, C.

According to a further aspect of the invention there is provided a method to treat a human subject suffering from cancer comprising administering an effective amount of a polypeptide according to the invention.

In a preferred method of the invention said polypeptide is administered at two day intervals. In an alternative preferred method of the invention said polypeptide is administered at weekly intervals.

In a further preferred method of the invention said polypeptide is administered at 2 weekly intervals.

In a further preferred method of the invention said polypeptide is administered at monthly intervals.

According to an aspect of the invention there is provided a pharmaceutical composition comprising a combination of at least two polypeptides wherein the first polypeptide is a fusion polypeptide comprising the amino acid sequence of interleukin 2, or active binding part thereof, linked, directly or indirectly to the binding domain of an interleukin 2 receptor and the second polypeptide is a fusion polypeptide comprising the amino acid sequence of interferon α 2b, or active binding part thereof, linked directly or indirectly to the binding domain of an interferon receptor.

In a preferred embodiment of the invention said first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: Figure 1b, 2b, 3b, 4b, 5b, 6b or 7b, optionally including the signal sequence, and said second polypeptide comprises or consists of the amino acid sequence represented in Figure 27b, optionally including the signal sequence .

According to a further aspect of the invention there is provided the use of a combination of at least two polypeptides wherein the first polypeptide is a fusion polypeptide comprising the amino acid sequence of interleukin 2, or active binding part thereof, linked, directly or indirectly, to the binding domain of an interleukin 2 receptor and the second polypeptide is a fusion polypeptide comprising the amino acid sequence of interferon α 2b, or active binding part thereof, linked directly or indirectly to the binding domain of an interferon receptor in the treatment of cancer.

In a preferred embodiment of the invention said first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: Figure 1b, 2b, 3b, 4b, 5b, 6b or 7b, optionally including the signal sequence and said second polypeptide comprises or consists of the amino acid sequence represented in Figure 27b optionally including the signal sequence.. In a preferred embodiment of the invention said cancer is melanoma.

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 figures:

Table 1 is a summary of LR nomenclature;

Figure 1a is the nucleic acid sequence of LR 6A1 ; Figure 1b is the amino acid sequence of LR 6A1;

Figure 2a is the nucleic acid sequence of LR 6B1; Figure 2b is the amino acid sequence of LR 6B1;

Figure 3a is the nucleic acid sequence of LR 6C1 ; Figure 3b is the amino acid sequence of LR 6C1;

Figure 4a is the nucleic acid sequence of LR 6D1 ; Figure 4b is the amino acid sequence of LR 6D1; Figure 5a is the nucleic acid sequence of LR 6E1; Figure 5b is the amino acid sequence of LR 6E1; Figure 6a is the nucleic acid sequence of LR 6E2; Figure 6b is the amino acid sequence of LR 6E2;

Figure 7a is the nucleic acid sequence of LR 6F1 ; Figure 7b is the amino acid sequence of LR 6F1;

Figure 8 is the amino acid sequence of human IL2 (bold capitals indicate the signal sequence; non bold capitals indicate the mature protein);

Figure 9 is the amino acid sequence of human IL2 alpha receptor (bold capitals indicate the signal sequence; non-bold capitals indicate the mature protein; italics lower case indicates the transmembrane domain; and bold lower case indicates the cytoplasmic domain;

Figure 10 is the amino acid sequence of human IL2 beta receptor;

Figure 11 is the amino acid sequence of human IL2 gamma receptor;

Figure 12 is the amino acid sequence of human IL7 [1-25 signal 26-177 mature protein]

Figure 13 is the amino acid sequence of human IL7R alpha;

Figure 14 is the amino acid sequence of human IL2R gamma;

Figure 15a is the amino acid sequence of LR fusion IL7ss-IL7-(G₄S)₄-IL7Ralpha; Figure 15b is the nucleic acid sequence;

Figure 16a is the amino acid sequence of LR fusion IL7ss-IL7-(G₄S)₅-IL2Rgamma; Figure 16b is the nucleic acid sequence;

Figure 17a is the amino acid sequence of LR fusion IL7Rass-IL7Ralpha-(G₄S)₄-IL7; Figure 17b is the nucleic acid sequence;

Figure 18a is the amino acid sequence of LR fusion IL2gss-IL2gamma-(G₄S)₅-IL7; Figure 18b is the nucleic acid sequence; Figure 19a is the amino acid sequence of LR fusion IL7-(G₄S)₄-IL7Ralpha; Figure 19b is the nucleic acid sequence; Figure 19c is a modified nucleic acid sequence optimised for bacterial expression;

Figure 20a is the amino sequence of LR fusion IL7-(G₄S)₅-IL2Rgamma; Figure 20b is the nucleic acid sequence; Figure 20c is a modified nucleic acid sequence optimised for bacterial expression;

Figure 21a is the amino sequence of LR fusion IL7Ralpha-(G₄S)₄-IL7; Figure 21b is the nucleic acid sequence; Figure 21c is a modified nucleic acid sequence optimised for bacterial expression;

Figure 22a is the amino acid sequence of LR fusion IL2gamma-(G₄S)₅-IL7; Figure 22b is the nucleic acid sequence; Figure 22c is a modified nucleic acid sequence optimised for bacterial expression;

Figure 23a is the amino acid sequence of LR fusion IL7-(G₄S)₄-IL7Ralpha-Histag; Figure 23b is the nucleic acid sequence; Figure 23c is a modified nucleic acid sequence optimised for bacterial expression;

Figure 24a is the amino acid sequence of LR fusion IL7-(G₄S)₅-IL2Rgamma-Histag; Figure 24b is the nucleic acid sequence; Figure 24c is a modified nucleic acid sequence optimised for bacterial expression;

Figure 25a is the amino acid sequence of LR fusion IL7Ralpha-(G₄S)₄-IL7-Histag; Figure 25b is the nucleic acid sequence; Figure 25c is a modified nucleic acid sequence optimised for bacterial expression;

Figure 26a is the amino acid sequence of LR fusion IL2gamma-(G₄S)₅-IL7-Histag; Figure 26b is the nucleic acid sequence; Figure 26c is a modified nucleic acid sequence optimised for bacterial expression;

Figure 27a is the nucleic acid sequence of LR a7B1 ; Figure 27b is the amino acid sequence of LR a7B1 ; Figure 28 illustrates a western blot of CHO cell expressed a7B1. Samples were prepared as described in the presence of DTT. Lane 1: Ladder, Lane 2: a7B1 (1Ox concentrated media from stable cell line), Lane 3: GAP, Lane 4: Positive control, 250ng rh- IFNalρha2B. A7B1 separates as a distinct band of approximate MW 75-10OkDa: Non glycosylated MW = 45.5kDa. IFN control has a MW of 19.2kDa;

Figure 29 illustrates the biological activity of interferon α 2b; and

Figure 30a & b illustrates the biological activity of interferon α 2b chimera A7B1.

Figure: 31 PCR was used to generate DNA consisting of the gene of interest flanked by suitable restriction sites (contained within primers R1-4). b) The PCR products were ligated into a suitable vector either side of the linker region, c) The construct was then modified to introduce the correct linker, which did not contain any unwanted sequence (i.e. the non-native restriction sites);

Figure: 32 Oligonucleotides were designed to form partially double-stranded regions with unique overlaps and, when annealed and processed would encode the linker with flanking regions which would anneal to the ligand and receptor, b) PCRs were performed using the "megaprimer" and terminal primers (R1 and R2) to produce the LR-fusion gene. The R1 and R2 primers were designed so as to introduce useful flanking restriction sites for ligation into the target vector; and

Figure 33 illustrates expression of IL7 fusion protein 11A recombinant expression.

MATERIALS AND METHODS

lnterleukin Bioassav

Commercially available bioassays can be used to test interleukin, in particular IL-2, see http://www.sbhsciences.com/index.asp. SBH Sciences Inc. Additional bioassays to measure IL-2 activity can be found in Molecules and Cells Springer-Verlag Hong Kong Limited, 2000 volume 10 (5): p575-578; and Journal of Immunological Methods 2002, volume 260 (1-2): p279-283. In addition to in vitro testing of interleukins, systemic testing in animal models is known. For example Shimizu et al (PNAS (1999) 96: 2268- 2273 describes the systemic testing for IL-2 bioactivity as an adjuvant in tumour immunotherapy. Immunological testing

Immunoassays that measure the binding of ligand or receptor to polyclonal and monoclonal antibodies are known in the art. Commercially available antibodies are available to detect the ligand or receptor in samples and also for use in competitive inhibition studies. For example see http://www.exactantigen.com/review/lnterleukin.html, which is incorporated by reference in its entirety.

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 (Fig 30a). 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 (Fig 30b). 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 (Fig 30c).

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 (Fig 31a). 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 (Fig 31b).

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 LR-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 RL-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-PAGE₁ Native PAGE, western blotting, ELISA.

Characterisation of LR-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 LR-fusion. Native solution state molecular weight information can be obtained from techniques such as size exclusion chrmoatography using a Superose G200 analytical column and analytical ultracentrifugation.

Statistics

Two groups were compared with a Student's test if their variance was normally distributed or by a Student-Satterthwaite's test if not normally distributed. Distribution was tested with an F test. One-way ANOVA was used to compare the means of 3 or more groups and if the level of significance was p<0.05 individual comparisons were performed with Dunnett's tests. All statistical tests were two-sided at the 5% level of significance and no imputation was made for missing values.

Construction of Chimeric clones

All clones were ligated using the restriction enzymes Nhe1/ Hindlll, into the mammalian expression plasmid pSecTag-link. Clones were attached to the secretion signal for human interferon for efficient secretion into cell media. The whole gene for a7B1 [Figure 27a] was cloned using gene synthesis and cloned into the mammalian expression vector pSecTag-link to form plFNsecTag-a7B1

Mammalian expression of IFN Chimeric clones

A mammalian expression system has been established using a modification of the invitrogen vector pSecTag-V5/FRT-Hist

Invitroqen's FIp-In system

This system allows for the rapid generation of stable clones into specific sites within the host genome for high expression. This can be used with either secreted or cytoplasmic expressed proteins. FIp-In host cell lines (flp-ln CHO) have a single FIp recombinase target (FRT) site located at a transcriptionally active genomic locus

Stable cell lines are generated by co-transfection of vector (Containing FRT target site) and pOG44 (a [plasmid that transiently expresses flp recombinase) into FIp-In cell line. Selection is with Hygromycin B. There is no need for clonal selection since integration of DNA is directed. Culturing FIp-In Cell lines: followed manufactures instruction using basic cell culture techniques.

Stable transfection of CHO FIp-In cells using Fυqene-6

The day before transfection CHO FIp-In cells were seeded at 6 x 10E5 per 100mm petri dish in a total volume of 10ml of Hams F12 media containing 10% (v/v) Fetal Calf Serum, 1% Penicillin/streptomycin and 4mM L-glutamine. The next day added 570 μl of serum free media (containing no antibiotics) to a 1.5ml polypropylene tube. 30μl of fugene-6 was then added and mixed by gentle rolling. A separate mix of plasmids was set up for each transfection which combined 2μg plasmid of interest with 18μg pOG44 (plasmid contains recombinase enzyme necessary for correct integration of plasmid into host genome). Control plate received no plasmid. This was mixed with fugene-6 by gentle rolling, left @ RmT for 15 minutes, then applied drop-wise to the surface of the each petri dish containing CHO FIp-In cells in F12 media + 10% FCS. The plates were gently rolled to ensure good mixing and left for 24 hrs @ 37°C/5% CO2. The next day media was exchanged for selective media containing hygromycin B @ 600ug/ml. Cells were routinely kept at 60% confluency or less. Cells were left to grow in the presence of 600ug/ml hygromycin B until control plate cells (non transfected cells) had died (i.e. no hygromycin resistance).

SDS-PAGE Analysis

Testing expression from Stable CHO cell lines

Confluent CHO FIp-In cell lines expressing the protein of interest were grown in 75cm2 flasks for approximately 3-4 days in serum free media, at which point samples were taken and concentrated using acetone precipitation. Samples were mixed with an equal volume of laemmli loading buffer in the presence or absence of 25mM DTT and boiled for 5 minutes. Samples were analysed by SDS-PAGE and transferred to a PVDF membrane. After blocking in 5% (w/v) Milk protein in PBS-0.05% (v/v) Tween 20, sample detection was carried out using a specific anti-IGF-1 antibody together with a

Horse Radish Peroxidase (HRP) conjugated secondary antibody. Visualisation was by chemiluminesence on photographic film using an HRP detection kit, Figure 28.

Testing expression from transient tansfections of CHO flpln

CHO FIp-In cells were seeded at 0.25x10E6 cells per well of a 6 well plate in a total volume of 2ml media (DMEM, F12, 10% FCS + P/S + L-glutamine + Zeocin). Cells were left to grow o/n. Cells were then transfected using either TranslT-CHO Reagent (Mirus) or fugene-6 at the specified reagent ratios stated in table 1. Control transfections were set up using 1 B7stop (GH containing chimeric molecule). Briefly, if using TranslT reagent, 20OuI of Serum free media (OPTI MEM) was added to a 1.5ml eppendorff per transfection followed by 2ug DNA. The tubes were left for 15 minutes at RmT. 1 ul of CHO Mojo Reagent was then added, mixed and left for a further 15 minutes. Media was changed to serum free and the transfection mix pippetted dropwise onto the surface of the appropriate well. Briefly, if using Fugene-6 reagent, 94ul of Serum free media (OPTI MEM) was added to a 1.5ml eppendorff per transfection followed by 2ug DNA. The tubes were left for 15 minutes at RmT. Trasfection mix was then pippetted drop wise onto the surface of the appropriate well containing serum free media. All plate were left , @ 37⁰C /5% CO2 for 2-3days

Interferon Bioassav

A stable CHO FIpIn cell line expressing the soluble Interferon Alpha Chimeric protein: A7b1 (AS-80) was grown by ARCBioserv. Control media consisting of non transfected CHO cells were also grown at the same time and treated in the same way. The Bioactivity of each sample was detected using a Human Type I Interferon Activity Detection kit (Neutekbio iLite AlphaBeta Kit: Galway, Ireland, catalogue # 46-88R, Lot# 0810601). Both media were concentrated and filter sterilised prior to undertaking serial dilution of sample. Manufacturer's instructions for kit usage were followed throughout.

The results clearly show that A7b1 has bioactivity and that controls show no bioactivity. The dilutions from 1:1 to 1:128 have reached the maximum RLU for the assay see Figures 30. Further dilutions show a good dose response curve of Bioactivity: This relates to a IU/ml range of ~ 16-20,000.

IL-7 LR-Fusion Expression 11A1 : Western blot of 11A1 from stable expressions in CHO FIpIn cells.

10μl of sample was run on and SDS-PAGE gel (Lane 1). Markers are at 250, 150, 100, 75, 50, 37, 25, 20 and 15kDa. Expected Mw of 10A1 is 44kDa.

lmmunoblot carried out with rabbit anti-IL7 antibody (Abeam.; Cat#: ab9628; dilution = 1 :2500) and anti-rabbit-HRP antibody (Abeam; dilution = 1 :5000) and is illustrated in Figure 33

Claims

1. A nucleic acid molecule comprising a nucleic acid sequence that encodes a polypeptide that has the activity of interleukin 7 wherein said polypeptide comprises interleukin 7, or part thereof linked, directly or indirectly, to the interleukin 7 binding domain of an interleukin 7 receptor.

2. A fusion polypeptide comprising: the amino acid sequence of interleukin 7, or active binding part thereof, linked, directly or indirectly, to the binding domain of an interleukin 7 receptor.

3. A fusion polypeptide according to claim 2 wherein said fusion polypeptide comprises or consists of the amino acid sequence represented in Figure 12, optionally including the signal sequence. .

4. A fusion polypeptide according to claim 2 or 3 wherein said fusion polypeptide comprises IL7 receptor α.

5. A fusion polypeptide according to claim 2 or 3 wherein said fusion polypeptide comprises IL2 receptor y.

6. A fusion polypeptide according to claim 4 wherein said fusion polypeptide comprises or consists of the amino acid sequence represented in Figure 13, optionally including the signal sequence.

7. A fusion polypeptide according to claim 5 wherein said fusion polypeptide comprises or consists of the amino acid sequence represented in Figure 14, optionally including the signal sequence.

8. A fusion polypeptide according to any of claims 2-7 wherein interleukin 7 is linked to an interleukin 7 binding domain of an interleukin 7 receptor wherein said interleukin 7 is positioned amino terminal to said binding domain in said fusion polypeptide.

9. A fusion polypeptide according to any of claims 2-7 wherein interleukin 7 is linked to an interleukin 7 binding domain of an interleukin 7 receptor wherein said interleukin 7 is positioned carboxyl-terminal to said binding domain in said fusion polypeptide.

10. A fusion polypeptide according to any of claims 2-9 wherein interleukin 7 is linked to the binding domain of the of the interleukin 7 receptor by a peptide linker.

11. A fusion polypeptide 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 polypeptide according to claim 11 wherein lsaid peptide linking molecule comprises 2, 3, 4, 5 or 6 copies of the peptide GIy GIy GIy GIy Ser.

13. A fusion polypeptide according to claim 12 wherein said peptide linking molecule consists of 4 or 5 copies of the peptide GIy GIy GIy GIy Ser.

14. A fusion polypeptide according to any of claims 2-9 wherein said polypeptide does not comprise a peptide linking molecule and is a direct fusion of interleukin 7 and the interleukin binding domain of the interleukin 7 receptor.

15. A nucleic acid molecule comprising a nucleic acid sequence selected from: i) a nucleic acid sequence as represented in Figure 15b; ii) a nucleic acid sequence as represented in Figure 16b; iii) a nucleic acid sequence as represented in Figure 17b; iv) a nucleic acid sequence as represented in Figure 18b; v) a nucleic acid sequence as represented in Figure 19b; vi) a nucleic acid sequence as represented in Figure 19c; vii) a nucleic acid sequence as represented in Figure 20b; xxi) a nucleic acid sequence as represented in Figure 20c xxii) a nucleic acid sequence as represented in Figure 21b xxiii) a nucleic acid sequence as represented inFigure 21c; xxiv) a nucleic acid sequence as represented in Figure 22b; xxv) a nucleic acid sequence as represented in Figure 22c; xxvi) a nucleic acid sequence as represented in Figure 23b; xxvii) a nucleic acid sequence as represented in Figure 23c; xxviii) a nucleic acid sequence as represented in Figure 24b; xxix) a nucleic acid sequence as represented in Figure 24c; xxx) a nucleic acid sequence as represented in Figure 25b; xxxi) a nucleic acid sequence as represented in Figure 25c; xxxii) a nucleic acid sequence as represented in Figure 26b; xxxiii) a nucleic acid sequence as represented inFigure 26c or a nucleic acid molecule comprising a nucleic sequence that hybridizes under stringent hybridization conditions to Figure 15b, 16b, 17b, 18b, 19b, 19c, 20b, 20c, 21b, 21c, 22b, 22c, 23b, 23c, 24b, 24c, 25b, 25c, 26b or 26c and which encodes a polypeptide that has interleukin 7 activity.

16. A nucleic acid molecule according to claim 15 wherein said nucleic acid molecule encodes a polypeptide that has agonist activity.

17. A nucleic acid molecule according to claim 15 wherein said nucleic acid molecule encodes a polypeptide that has antagonist activity.

18. A polypeptide encoded by the nucleic acid according to any of claims 15-17.

19. A polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence represented in Figure 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a or 26a, optionally including the signal sequence.

20. A homodimer consisting of two polypeptides wherein each of said polypeptides comprises: i) a first part comprising interleukin 7, or a receptor binding domain thereof, optionally linked by a peptide linking molecule to ii) a second part comprising at least one interleukin 7 binding domain or part thereof, of an interleukin 7 receptor.

21. A homodimer according to claim 30 wherein said homodimer comprises two polypeptides comprising or consisting ofthe amino acid sequence represented in Figure 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a or 26a, optionally including the signal sequence..

22. A vector comprising a nucleic acid molecule according to claim 1 or any of claims 15-17.

23. A cell transfected or transformed with a nucleic acid molecule according to any of claims 1 or 15-17 or vector according to claim 22.

24. A cell according to claim 23 wherein said cell is a eukaryotic cell.

25. A cell according to claim 23 wherein said cell is a prokaryotic cell.

26. A pharmaceutical composition comprising a polypeptide according to any of claims 2-14 or 18 or 19 including an excipient or carrier.

27. A pharmaceutical composition according to claim 26 wherein said composition is combined with a further therapeutic agent.

28. A method to treat a human subject suffering from an autoimmune disease comprising administering an effective amount of a polypeptide according to any of claims 2-17 or 18 or 19.

29. A method according to claim 28 wherein said autoimmune disease is multiple sclerosis.

30. A method according to claim 28 wherein said autoimmune disease is rheumatoid arthritis.

31. A method to treat a human subject suffering from lymphopenia comprising administering an effective amount of a polypeptide according to any of claims 2-17 or 18 or 19.

32. A method according to claim 31 wherein lymphopenia is the result of a disease or condition selected from the group consisting of: AIDS, tuberculosis, chemotherapy, radiotherapy, critical illness, sepsis, bone marrow suppression, vitamin deficiency.

33. A method to treat a human subject suffering from a viral infection comprising administering an effective amount of a polypeptide according to any of claims 2-17 or 18 or 19 .

34. A method according to claim 33 wherein said viral infection is caused by a virus selected from the group consisting of: HIV, HCV, HSV₁ hepatitis A, B, C.

35. A method to treat a human subject suffering from cancer comprising administering an effective amount of a polypeptide according to any of claims 2-17 or 18 or 19.

36. A method according to any of claims 28-35 wherein said polypeptide is administered at two day intervals.

37. A method according to any of claims 28-35 wherein said polypeptide is administered at weekly intervals.

38. A method according any of claims 28-35 wherein said polypeptide is administered at 2 weekly intervals.

39. A method according any of claims 28-35 wherein said polypeptide is administered at monthly intervals.

40. A pharmaceutical composition comprising a combination of at least two polypeptides wherein the first polypeptide is a fusion polypeptide comprising the amino acid sequence of interleukin 2, or active binding part thereof, linked, directly or indirectly to the binding domain of an interleukin 2 receptor and the second polypeptide is a fusion polypeptide comprising the amino acid sequence of interferon α 2b, or active binding part thereof, linked directly or indirectly to the binding domain of an interferon receptor.

41. A composition according to claim 40 wherein said first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: the amino acid sequence represented in Figure 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a or 26a, optionally including the signal sequence and said second polypeptide comprises or consists of the amino acid sequence represented in Figure 27b, optionally including the signal sequence.

42. The use of a combination of at least two polypeptides wherein the first polypeptide is a fusion polypeptide comprising the amino acid sequence of interleukin 2, or active binding part thereof, linked, directly or indirectly, to the binding domain of an interleukin 2 receptor and the second polypeptide is a fusion polypeptide comprising the amino acid sequence of interferon α 2b, or active binding part thereof, linked directly or indirectly to the binding domain of an interferon receptor in the treatment of cancer.

43. Use according to claim 42 wherein said first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: Figure 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a or 26a, optionally including the signal sequence and said second polypeptide comprises or consists of the amino acid sequence represented in Figure 27b, optionally including the signal sequence.

44. Use according to claim 42 or 43 wherein said cancer is melanoma.