WO2010020766A2 - Polypeptides de fusion d'interleukine - Google Patents

Polypeptides de fusion d'interleukine Download PDF

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Publication number
WO2010020766A2
WO2010020766A2 PCT/GB2009/002004 GB2009002004W WO2010020766A2 WO 2010020766 A2 WO2010020766 A2 WO 2010020766A2 GB 2009002004 W GB2009002004 W GB 2009002004W WO 2010020766 A2 WO2010020766 A2 WO 2010020766A2
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acid sequence
nucleic acid
interleukin
polypeptide
represented
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PCT/GB2009/002004
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English (en)
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WO2010020766A3 (fr
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Peter Artymiuk
Richard Ross
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Asterion Limited
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Publication of WO2010020766A3 publication Critical patent/WO2010020766A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5418—IL-7
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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).
  • 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 ⁇ , ⁇ 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.
  • 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 ⁇ (CD25), IL-2 receptor ⁇ (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.
  • T cell antigen receptor TCR
  • TCR T cell antigen receptor
  • IL-2 is also involved in T cell maturation in the thymus and for the development of Regulatory T -cells (T RE G 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.
  • 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.
  • 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.
  • said interleukin is IL-2.
  • said fusion polypeptide comprises the amino acid sequence represented in Figure 8, optionally including the signal sequence.
  • said fusion polypeptide comprises IL2 receptor ⁇ .
  • said fusion polypeptide comprises IL2 receptor ⁇ .
  • said fusion polypeptide comprises an IL2 receptor vc.
  • said fusion polypeptide comprises or consists of IL2 receptor ⁇ as represented by the amino acid sequence in Figure 9, optionally including the signal sequence.
  • said fusion polypeptide comprises or consists of IL2 receptor ⁇ as represented by the amino acid sequence in Figure 10, optionally including the signal sequence.
  • 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.
  • 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.
  • 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.
  • said interleukin is linked to the binding domain of the of the interleukin receptor by a peptide linker; preferably a flexible peptide linker.
  • said peptide linking molecule comprises at least one copy of the peptide GIy GIy GIy GIy GIy Ser.
  • said peptide linking molecule comprises 2, 3, 4, 5 or 6 copies of the peptide GIy GIy GIy GIy Ser.
  • said peptide linking molecule consists of 5 copies of the peptide GIy GIy GIy GIy GIy Ser.
  • 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
  • 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:
  • nucleic acid molecule encodes a polypeptide that has agonist activity.
  • nucleic acid molecule encodes a polypeptide that has antagonist activity.
  • said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 1a.
  • said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 2a.
  • said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 3a.
  • said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 4a.
  • said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 5a.
  • said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 6a.
  • said nucleic acid molecule comprises or consists of a nucleic acid sequence as represented in Figure 7a.
  • polypeptide encoded by the nucleic acid according to 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. .
  • 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.
  • 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. .
  • a vector comprising a nucleic acid molecule according to 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.
  • 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.
  • 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.
  • the promoter is a constitutive, an inducible or regulatable promoter.
  • a cell transfected or transformed with a nucleic acid molecule or vector according to the invention there is provided a cell transfected or transformed with a nucleic acid molecule or vector according to the invention.
  • said cell is a eukaryotic cell.
  • said cell is a prokaryotic cell.
  • 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.
  • 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 e.g. COS cell, CHO cell
  • composition comprising a polypeptide according to the invention including an excipient or carrier.
  • said pharmaceutical composition is combined with a further therapeutic agent.
  • compositions of the present invention are 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.
  • 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.
  • 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).
  • the pharmaceutical compositions of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
  • 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.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • compositions may be combined, if desired, with a pharmaceutically- acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human.
  • 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.
  • 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.
  • compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • 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.
  • the acceptable solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • 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.
  • a vaccine composition comprising a nucleic acid molecule or polypeptide according to the invention and an antigenic molecule.
  • 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.
  • an immunogenic "carrier” protein such as key hole limpet haemocyanin or tetanus toxoid which contain multiple T cell epitopes.
  • 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.
  • Human Immunodeficiency Virus Human T Cell Leukaemia Virus, Ebola virus or other haemorrhagic fever virus
  • HPV human papilloma vims
  • said antigenic molecule is a cancer antigen.
  • 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.
  • tumour rejection antigens 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.
  • a method to treat a human subject suffering from a viral infection comprising administering an effective amount of a polypeptide according to the invention.
  • a method to treat a human subject suffering from cancer comprising administering an effective amount of a polypeptide according to the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • sarcoma is art recognized and refers to malignant tumours of mesenchymal derivation.
  • said cancer is melanoma.
  • said cancer is renal cancer.
  • 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.
  • said inflammatory condition is an autoimmune disease; preferably said auto-immune disease is rheumatoid arthritis.
  • said polypeptide is administered at two day intervals; preferably said polypeptide is administered at weekly, 2 weekly or monthly intervals.
  • a polypeptide according to the invention for the manufacture of a medicament for the treatment of viral infection.
  • said cancer is melanoma; preferably malignant melanoma.
  • said cancer is renal cancer.
  • a polypeptide antagonist according to the invention for the manufacture of a medicament for the treatment of an autoimmune disease.
  • said autoimmune disease is rheumatoid arthritis.
  • a monoclonal antibody that binds the polypeptide or dimer according to the invention.
  • 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.
  • a method for preparing a hybhdoma cell-line producing monoclonal antibodies 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
  • the said immunocompetent mammal is a mouse.
  • said immunocompetent mammal is a rat.
  • hybridoma cell-line obtained or obtainable by the method according to the invention.
  • a diagnostic test to detect a polypeptide according to the invention in a biological sample comprising:
  • said ligand is an antibody; preferably a monoclonal antibody.
  • 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.
  • said interleukin is selected from the group consisting of: IL-2, IL-4, IL-7, IL-9, IL-15 and IL21.
  • said binding domain of an interleukin receptor comprises or consists of the amino acid sequence represented in Figure 11.
  • 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.
  • 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.
  • said fusion polypeptide comprises or consists of the amino acid sequence represented in Figure 12, optionally including the signal sequence.
  • said fusion polypeptide comprises IL7 receptor ⁇ .
  • said fusion polypeptide comprises IL2 receptor y.
  • said fusion polypeptide comprises or consists of the amino acid sequence represented in Figure 13, optionally including the signal sequence.
  • said fusion polypeptide comprises or consists of the amino acid sequence represented in Figure 14, optionally including the signal sequence.
  • 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.
  • 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.
  • interleukin 7 is linked to the binding domain of the of the interleukin 7 receptor by a peptide linker.
  • said peptide linking molecule comprises at least one copy of the peptide GIy GIy GIy GIy GIy Ser.
  • said peptide linking molecule comprises 2, 3, 4, 5 or 6 copies of the peptide GIy GIy GIy GIy Ser.
  • said peptide linking molecule consists of 4 or 5 copies of the peptide GIy GIy GIy GIy GIy Ser.
  • 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.
  • 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; 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 in Figure 21c; xi) a nucleic acid sequence as represented in Figure 22b; xii) a nucleic acid sequence as represented in Figure 22c; xiii) a nucleic acid sequence as represented in Figure 22c; xiii)
  • XX a nucleic acid sequence as represented in Figure 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.
  • nucleic acid molecule encodes a polypeptide that has agonist activity.
  • nucleic acid molecule encodes a polypeptide that has antagonist activity.
  • 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.
  • 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.
  • 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.
  • a method to treat a human subject suffering from autoimmune disease comprising administering an effective amount of a polypeptide according to the invention.
  • said autoimmune disease is multiple sclerosis.
  • said autoimmune disease is rheumatoid arthritis.
  • a method to treat a human subject suffering from lymphopenia comprising administering an effective amount of a polypeptide according to the invention.
  • 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.
  • a method to treat a human subject suffering from a viral infection comprising administering an effective amount of a polypeptide according to the invention.
  • said viral infection is caused by a virus selected from the group consisting of: HIV, HCV, HSV, hepatitis A, B, C.
  • a method to treat a human subject suffering from cancer comprising administering an effective amount of a polypeptide according to the invention.
  • polypeptide is administered at two day intervals. In an alternative preferred method of the invention said polypeptide is administered at weekly intervals.
  • polypeptide is administered at 2 weekly intervals.
  • polypeptide is administered at monthly intervals.
  • 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 ⁇ 2b, or active binding part thereof, linked directly or indirectly to the binding domain of an interferon receptor.
  • 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
  • said second polypeptide comprises or consists of the amino acid sequence represented in Figure 27b, optionally including the signal sequence.
  • 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 ⁇ 2b, or active binding part thereof, linked directly or indirectly to the binding domain of an interferon receptor in the treatment of cancer.
  • 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.
  • said cancer is melanoma.
  • 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-(G 4 S) 4 -IL7Ralpha;
  • Figure 15b is the nucleic acid sequence;
  • Figure 16a is the amino acid sequence of LR fusion IL7ss-IL7-(G 4 S) 5 -IL2Rgamma;
  • Figure 16b is the nucleic acid sequence;
  • Figure 17a is the amino acid sequence of LR fusion IL7Rass-IL7Ralpha-(G 4 S) 4 -IL7;
  • Figure 17b is the nucleic acid sequence;
  • Figure 18a is the amino acid sequence of LR fusion IL2gss-IL2gamma-(G 4 S) 5 -IL7;
  • Figure 18b is the nucleic acid sequence;
  • Figure 19a is the amino acid sequence of LR fusion IL7-(G 4 S) 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-(G 4 S) 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-(G 4 S) 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-(G 4 S) 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-(G 4 S) 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-(G 4 S) 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-(G 4 S) 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-(G 4 S) 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- IFNal ⁇ ha2B.
  • IFN control has a MW of 19.2kDa;
  • Figure 29 illustrates the biological activity of interferon ⁇ 2b.
  • Figure 30a & b illustrates the biological activity of interferon ⁇ 2b chimera A7B1.
  • Figure 33 illustrates expression of IL7 fusion protein 11A recombinant expression.
  • 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.
  • 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.
  • 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).
  • 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 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.
  • a suitable system e.g. mammalian CHO cells, E. coli
  • Expression was then analysed using a variety of methods which could include one or more of SDS-PAGE, Native PAGE, western blotting, ELISA.
  • 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).
  • 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 1 Native PAGE, western blotting, ELISA.
  • 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.
  • a mammalian expression system has been established using a modification of the invitrogen vector pSecTag-V5/FRT-Hist
  • 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.
  • 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 ⁇ l of serum free media (containing no antibiotics) to a 1.5ml polypropylene tube. 30 ⁇ l of fugene-6 was then added and mixed by gentle rolling. A separate mix of plasmids was set up for each transfection which combined 2 ⁇ g plasmid of interest with 18 ⁇ g 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).
  • 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
  • HRP Horse Radish Peroxidase conjugated secondary antibody. Visualisation was by chemiluminesence on photographic film using an HRP detection kit, Figure 28.
  • 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.
  • DMEM TranslT-CHO Reagent
  • 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
  • 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 , @ 37 0 C /5% CO2 for 2-3days
  • 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.
  • IL-7 LR-Fusion Expression 11A1 Western blot of 11A1 from stable expressions in CHO FIpIn cells.

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Abstract

L'invention concerne des polypeptides et des dimères de fusion d'interleukine; des molécules d'acides nucléiques codant pour lesdits polypeptides et des méthodes de traitement utilisant lesdits polypeptides/dimères.
PCT/GB2009/002004 2008-08-21 2009-08-17 Polypeptides de fusion d'interleukine WO2010020766A2 (fr)

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JP2015525217A (ja) * 2012-06-08 2015-09-03 アルカーメス，インコーポレイテッド アゴニストおよびアンタゴニストとしての循環置換により修飾されるリガンド
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JP2017523789A (ja) * 2014-08-06 2017-08-24 ユニバーシティー オブ マイアミＵｎｉｖｅｒｓｉｔｙ Ｏｆ Ｍｉａｍｉ インターロイキン−２／インターロイキン−２受容体アルファ融合タンパク質および使用方法
CN109689694A (zh) * 2016-05-19 2019-04-26 通用医疗公司 与其受体IL-2Rβ结合的白细胞介素-2作为用来增强自然杀伤细胞和调节性T细胞活性的平台
WO2019183389A1 (fr) * 2018-03-23 2019-09-26 Kite Pharma, Inc. Protéines transmembranaires chimériques et leurs utilisations
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WO2020102728A1 (fr) 2018-11-16 2020-05-22 Neoimmunetech, Inc. Méthode de traitement d'une tumeur au moyen de l'association de la protéine il-7 et d'un inhibiteur de point de contrôle immunitaire
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