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EP1991679A2 - Varianten des humanen interferon-gamma (ifngamma) - Google Patents

Varianten des humanen interferon-gamma (ifngamma)

Info

Publication number
EP1991679A2
EP1991679A2 EP07731116A EP07731116A EP1991679A2 EP 1991679 A2 EP1991679 A2 EP 1991679A2 EP 07731116 A EP07731116 A EP 07731116A EP 07731116 A EP07731116 A EP 07731116A EP 1991679 A2 EP1991679 A2 EP 1991679A2
Authority
EP
European Patent Office
Prior art keywords
ifnγ
variant
pharmaceutical composition
composition according
agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07731116A
Other languages
English (en)
French (fr)
Inventor
José BERENGUER
Marc Delcourt
Hélène CHAUTARD
Thierry Menguy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biomethodes SA
Original Assignee
Biomethodes SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FR0602064A external-priority patent/FR2898359B1/fr
Priority claimed from FR0603150A external-priority patent/FR2899589A1/fr
Application filed by Biomethodes SA filed Critical Biomethodes SA
Publication of EP1991679A2 publication Critical patent/EP1991679A2/de
Withdrawn legal-status Critical Current

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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
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention falls within the field of protein enhancement. It relates to the improvement of human ⁇ interferon (IFN ⁇ ), as well as compositions comprising an improved IFN ⁇ , a nucleic acid encoding it, and their uses.
  • IFN ⁇ human ⁇ interferon
  • IFN ⁇ is a cytokine of 166 amino acids.
  • the molecule has a signal peptide allowing its membrane translocation and its secretion, a cleavage site and a so-called mature protein part.
  • the signal peptide of IFN ⁇ is made up of the first 23 or 20 first amino acids according to the authors. Indeed, there is doubt in the literature on the presence of the amino acid triplet Cys-Tyr-Cys (CYC) in N-terminal of the mature sequence.
  • CYC Cys-Tyr-Cys
  • IFN ⁇ exists as a homodimer in which the two subunits are not covalently bound. Each subunit has two N-glycosylation sites (positions 48 and 120 of the 166 aa precursor).
  • each of these monomers has six alpha helices with a compact part consisting of the first 4 most N-terminal alpha helices (alpha A, B, C, and D helices) and a C-terminal part consisting of two isolated alpha helices and closely interaction with the second IFN ⁇ monomer.
  • IFN ⁇ is the typical example of a pleiotropic cytokine with a broad spectrum of activities. Indeed, interferons (IFNs) are endowed with activities such as inhibition of viral replication, inhibition of cell multiplication and induction of apoptosis.
  • IFNs interferons
  • stimulation of macrophages by IFN ⁇ induces the following responses:
  • MHC major histocompatibility complex
  • IFN ⁇ increased cytokine production and endogenous IFN production.
  • the action of IFN ⁇ on T lymphocytes is to promote their differentiation thus modulating the specific immune response.
  • IFN ⁇ is a molecule developed as a human therapeutic agent in the treatment of many diseases, of various natures.
  • Commercial IFN ⁇ (Actimmune, and Biogamma) are now used for two main therapeutic indications: chronic granulomatosis and idiopathic pulmonary fibrosis, in combination with oral prednisolone.
  • Many new secondary therapeutic indications are currently being developed at different clinical phases, in particular for their role as immunosuppressors, for example in addition to pegylated IFN ⁇ / ribavirin in the context of the treatment of Hepatitis C.
  • IFNs The main adverse effects of IFNs are dose-dependent and therefore closely related to the rate of administration. These effects are cumulative and worsen over time. In addition to the acute toxicity resulting after injection (2-8 hours after subcutaneous injection), nausea and vomiting, the most frequent adverse effects are flu-like symptoms (chills, headache, asthenia), inflammatory reactions at the site of injection and elevation of liver transaminases. The most serious adverse effects are cases of depression, lymphopenia or rare cases of necrosis at the subcutaneous injection site. In patients treated with high doses of IFN, diabetes may occur after the start of treatment. In addition, the tolerance of IFN injection is sometimes limited in time and results in the development of neutralizing antibodies (in approximately 10-20% of patients).
  • US 4,832,959 contains polypeptides with partial sequences of human IFN ⁇ including residues 1-127, 5-146 and 5-127 of mature IFN ⁇ and having the 3 additional amino acids CYC.
  • US 6,120,762 discloses a peptide fragment comprising residues 118-157 of precursor PIFN ⁇ and its use.
  • WO2004005341 describes the methods for generating and producing a series of active mutants of PIFN ⁇ comprising the 143 amino acids of the mature form of IFN ⁇ without CYC with a variation comprising at least one of the mutations in the S 155 and S 165 group and at least a mutation in the group Rl 60, Rl 62 and Rl 63. These mutants would be useful especially in the treatment of idiopathic pulmonary fibrosis.
  • EP 0 219 781 discloses the use of partial sequences of human IFN ⁇ including amino acids 3-124 of the mature protein. The importance of the last 20 amino acids on the activity and stability of IFN ⁇ has been and still is a source of controversial studies. Human IFN ⁇ s with the truncated C-terminus have been described by Slodowski et al who performed truncations of different size (from 10 to 20 truncated amino acids) (Eur J. Biochem 202: 1133-1140, 1991). .
  • WO 2004/022593 in silico analysis of the sequences of numerous therapeutic proteins, including IFN ⁇ , for the existence of proteolysis sites sensitive to proteases present in human serum (such as trypsin, endoproteinase Asp-N, chymotrypsin and proline endopeptidase).
  • proteolysis sites sensitive to proteases present in human serum such as trypsin, endoproteinase Asp-N, chymotrypsin and proline endopeptidase.
  • the mutations expected to provide protection against the proteases mentioned are: L53V, L53I, K57Q, K57N, K60Q, K60N, E61Q, E61N, E61H, E62Q, E62N, E62H, K78Q, K78N, K81Q, K81N, K84Q, K84N, D85Q, D85N, D86Q.
  • IFN ⁇ in monomeric form in particular
  • IFN ⁇ in monomeric form in particular
  • WO 92/08737 discloses PIFN ⁇ variants comprising an additional N-terminal methionine in position -1, the first 132 amino acids of the mature sequence without CYC, the 133rd amino acid being a leucine instead of a glutamine.
  • US 6,046,034 discloses thermostable variants of human IFN ⁇ for which pairs of cysteines have been incorporated at specific locations in the IFN ⁇ structure so as to create inter-monomer and intra-monomer disulfide bridges and thus provide stabilization of the IFN ⁇ homodimer.
  • the only pair of cysteines that preserve the biological activity of IFN ⁇ is E30C-S92C, which links helices A and D of the same monomer, while the other pairs of inter-monomeric cysteines destroy the biological activity of IFN ⁇ .
  • these mutants also have a truncated C-terminus corresponding to the Delta 10 mutant.
  • WO 99/03887 discloses protein variants of the structural super-family of growth hormone (of which IFN ⁇ is a part).
  • IFN ⁇ is described as an example of this super-family, but no experimental example of modifications is described in the case of IFN ⁇ .
  • WO 01/36001 discloses novel IFN ⁇ molecules modified by insertions of glycosylation sites and / or derivation by PEG type entities. These molecules have improved properties such as improved half-life and / or improved bioavailability.
  • WO03002152 discloses a pharmaceutical composition containing a sulfoalkyl ether interferon cyclodextrin derivative, the stability of which would be improved. None of these variants are currently available as a drug. It is for this reason that there is still a strong demand for an improved IFN ⁇ , and in particular an IFN ⁇ having a better stability under physiological conditions. This gain in stability under physiological conditions can be evaluated by a gain of stability at high temperature.
  • the present invention relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a thermostable variant of human IFN ⁇ or a functional fragment thereof comprising at least one substitution selected from the group consisting of S63C, E62C, F159C, D99Y, E116C, L158C, S74G, R162C, S122D, L126P, N58R, and T95V, the variant not carrying a non-peptide group attached to the residue (s) introduced (s) by the first substitution (s).
  • the variant differs from a polypeptide having a sequence selected from SEQ ID Nos. 2, 4 and 6 by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. residue (s), preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residue (s).
  • the variant has a single substitution.
  • the variant further comprises at least one other substitution selected from the group consisting of M157C, G41S and M100N.
  • the variant may comprise or a combination of two substitutions selected from the group consisting of S63C, E62C, F159C, D99Y, El 16C 5 L158C, S74G, R162C, S122D, MlOON, L126P, N58R, T95V, M157C and G41S.
  • the variant comprises or has a combination of substitutions selected from the group consisting of S63C + E62C, S63C + F159C, S63C + D99Y, S63C + E116C, S63C + L158C, S63C + S74G, S63C + R162C, S63C + S122D, S63C + MlOON, S63C + L126P, S63C + N58R, S63C + T95V, S63C + M157C, S63C + G41S, E62C + F159C, E62C + D99Y, E62C + El 16C, E62C + L158C, E62C + S74G, E62C + R162C, E62C + S122D, E62C + MlOON, E62C + L126P, E62C + N58R, E62C + T95V, E62C + M157C, E62C + G41S, F159C + D99Y, E62C + El 16C,
  • the variant comprises or has a combination of substitutions selected from the group consisting of S63C + E62C, S63C + F159C, S63C + D99Y, S63C + E116C, S63C + L158C, S63C + S74G, S63C + R162C, S63C + S122D, S63C + M100N, S63C + L126P, S63C + N58R, S63C + T95V, S63C + M157C, S63C + G41S.
  • the variant comprises or has the combination S63C + G41S.
  • the variant does not have a deletion of 1 to 11 residues at the C-terminus.
  • the variant has a deletion of 1 to 11 residues at the C-terminus.
  • the variant does not carry a non-peptide group selected from the group consisting of a polymer molecule, a lipophilic molecule, and an organic derivatization agent.
  • the variant carries a non-peptide group selected from the group consisting of a polymer molecule, a lipophilic molecule, and an organic derivatization agent.
  • the non-peptide group in question is especially a polymer molecule, preferably a polyethylene glycol.
  • the variant is glycosylated.
  • the variant is not glycosylated.
  • the pharmaceutical composition further comprises at least one other active ingredient.
  • the at least one other active ingredient is preferably selected from the group consisting of an antibody, an antitumor or chemotherapy agent, a glucocorticoid, an antihistamine agent, an adrenocortical hormone, an antiallergic agent, a vaccine , a broncodilator, a steroid, a beta-adrenergic agent, an immunomodulatory agent, a cytokine such as interferon alpha or beta, interleukin 1 or 2, TNF (tumor necrosis factor), hydroxyurea, an agent alkylating agent, a folic acid antagonist, an antimetabolite of nucleic acid metabolism, a fusal poison, an antibiotic, a nucleotide analogue, a retinoid, a lipoxygenase and cyclooxygenase inhibitor, a fumaric acid and its salts, an analgesic, a spasm
  • the at least one other active ingredient is a type I interferon, in particular alpha or beta interferon.
  • the pharmaceutical composition may be formulated for oral, parenteral (eg, subcutaneous, intramuscular, intravenous, or intradermal), sublingual, topical, local, intratracheal, intranasal, transdermal, rectal, intraocular, or intraatrial administration.
  • the present invention also relates to a pharmaceutical composition according to the present invention as a medicament.
  • the present invention relates to a product comprising a pharmaceutical composition according to the present invention and another active ingredient for a combined preparation for simultaneous, sequential or separate use as an antiviral, antiproliferative or immunomodulatory drug.
  • the other active ingredient is selected from the group consisting of an antibody, an antitumor or chemotherapy agent, a glucocorticoid, an antihistamine agent, an adrenocortical hormone, an antiallergic agent, a vaccine, a broncodilator, a steroid, a beta-adrenergic agent, an immunomodulatory agent, a cytokine such as interferon alpha or beta, interleukin 1 or 2, TNF (tumor necrosis factor), hydroxyurea, an alkylating agent, a folic acid antagonist, an antimetabolite of nucleic acid metabolism, a fusal poison, an antibiotic, a nucleotide analogue, a retinoid, a lipoxygenase and
  • the other active ingredient is a type I interferon, in particular alpha or beta interferon.
  • the two active ingredients can be administered by the same route of administration or by two separate routes of administration.
  • the medicament is for the treatment of a selected pathology among asthma, chronic familial granulomatosis, idiopathic pulmonary fibrosis, an atypical mycobacterium infection, kidney cancer, osteopetrosis, scleroderma generalized, chronic hepatitis virus B or C, septic shock, allergic dermatitis, and rheumatoid arthritis.
  • the present invention further relates to the use of a pharmaceutical composition according to the present invention for the preparation of an antiviral, antiproliferative or immunomodulatory drug.
  • the medicament is for the treatment of a selected pathology among asthma, chronic familial granulomatosis, idiopathic pulmonary fibrosis, atypical mycobacterium infection, kidney cancer, osteopetrosis, scleroderma. generalized, chronic hepatitis virus B or C, septic shock, allergic dermatitis, and rheumatoid arthritis.
  • the present invention relates to a nucleic acid encoding a thermostable variant of IFN ⁇ as described in the compositions above. It also relates to a nucleic acid expression cassette according to the present invention, a vector comprising a nucleic acid or an expression cassette according to the present invention, and a host cell comprising a nucleic acid, an expression cassette or a vector according to the present invention. Finally, it relates to the use of such a nucleic acid, of such an expression cassette, of such a vector or of such a host cell to produce a thermostable variant of IFN ⁇ as described in the compositions herein. - above.
  • FIG. 1 Diagram of the pNCK vector used for the generation of mutant libraries and their selection in Thermus thermophiles.
  • FIG. 2 Results of the functional analysis of the simple mutants of the IFN ⁇ selected by Thermus thermophilus - thermostability analysis (% of residual activity panel A), total relative activity with respect to the wild-type protein (panel B) and index of product-defined improvement (residual activity by relative total activity against wild-type protein / 100) (panel C).
  • FIG. 3 Results of functional analysis of simple mutants of IFN ⁇ resulting from the double and multiple positions selected by Thermus thermophilus - Thermostability analysis (% of residual activity panel A), total relative activity with respect to the wild-type protein (panel B) and product-defined improvement index (residual activity by relative total activity against wild-type protein / 100) (panel C).
  • Figure 4 The era of the functional analysis results of single point mutants of IFN-g generated systematically and have been upgraded to their stability and / or activity - heat stability analysis (% residual activity of panel A) activity total relative to wild-type protein (panel B) and product-defined improvement index (residual activity by relative total activity against wild-type protein / 100) (panel C).
  • Figure 5 2nd part of the functional analysis results of single point mutants of IFN-g generated systematically and have been upgraded to their stability and / or activity - heat stability analysis (% residual activity of panel A) activity total relative to wild-type protein (panel B) and product-defined improvement index (residual activity by relative total activity against wild-type protein / 100) (panel C).
  • Figure 6 3rd part of the functional analysis results of single point mutants of IFN-g generated systematically and have been upgraded to their stability and / or activity - heat stability analysis (% residual activity of panel A) activity total relative to wild-type protein (panel B) and product-defined improvement index (residual activity by relative total activity against wild-type protein / 100) (panel C).
  • FIG 7 Evaluation of the thermostability of protein variants of human PIFN ⁇ by measuring their half-life in vitro during kinetics of thermal denaturation at 59 ° C in the presence of a concentration of adjusted FBS. These measures consist in monitoring the retention of PIFN ⁇ activity as a function of denaturation time at 59 ° C. These data allow us to calculate the "in vitro half-life" of these molecules under these conditions. These half life calculations are listed in Table 3.
  • Figure 8 Pharmacokinetics of thermostable variants of human IFN ⁇ after intravenous administration. The amount of IFN ⁇ is monitored by an ELISA test on C57BL / 6 mouse serum samples after intravenous injection of 100 ⁇ l at 10 ⁇ g / ml.
  • Figure 9 Example of pharmacokinetics of thermostable variants of human PIFN ⁇ after subcutaneous administration. Plasma IFN ⁇ gamma concentration is monitored by ELISA quantification on C57BL / 6 mouse serum samples after subcutaneous injection of 100 ⁇ l at 6.7 ⁇ g / ml. Table 1: Mutants from the primary selection of the PIFN ⁇ library in Thermus thermophilus. The numbers correspond to the position of the mutation in the form of the 166 residue precursor.
  • Table 2 Mutants validated in secondary selection test in Thermus thermophilus. The numbers correspond to the position of the mutation in the form of the 166 residue precursor.
  • Table 3 Summary of "in vitro" half-life calculations of thermostable variants of IFN ⁇ in thermal inactivation experiments at 59 ° C and calculation of half-life improvement ratio of variants compared to half - life of wild IFN ⁇ produced in CHO.
  • Table 4 Summary of terminal half-life calculations for the elimination of thermostable variants of IFN ⁇ during intravenous injection experiments and calculation of the half-life improvement ratio of the variants with respect to the half-life of IFN ⁇ wild product in CHO. Terminal elimination half-lives (Tl / 2i.v) were calculated using Kinetica Vs 4.4 software by modeling IV bolus administration in a non-compartmentalized system.
  • Table 5 Recapitulation of total area under the curve (AUC early sc) and elimination half-lives (T1 / 2 sc) in subcutaneous injection experiments. Calculation of the respective improvement ratios of the total area under the curve and the half-lives of the variants relative to the total area under the curve and the half-life of wild-type IFN ⁇ produced in CHO. These parameters were calculated using the Kinetica Vs 4.4 software. Detailed description of the invention
  • the present invention relates to variants of human gamma interferon (IFN ⁇ ) whose stability, in particular the thermal stability, is increased compared to wild-type IFN ⁇ .
  • IFN ⁇ human gamma interferon
  • the PIFN ⁇ protein variants of this invention were obtained by coupling the generation of a wide variety of mutations by directed evolution to a direct selection method of the improved variants for their thermostability. The stability against the thermal denaturation of the improved candidates as well as the conservation of their activity was validated by biological tests.
  • the variants of this invention are alternatives to recombinant IFN ⁇ currently used in the therapeutic field, particularly in the treatment of chronic granulomatosis and idiopathic pulmonary fibrosis.
  • the numbering adopted in the present application will be that which takes into account all the residues of IFN ⁇ with the signal peptide sequence included (total amino acid numbering of 1 for the N-terminal methionine signal peptide included at 166 for glutamine of the C-terminus interferon - the sequence SEQ ID No. 2).
  • the position of the substitution in the other two forms can easily be determined by those skilled in the art.
  • substitution indicates the substitution of the cysteine residue at position 21 of SEQ ID No. 2 with a glycine.
  • substitution and “mutation” are interchangeable.
  • sign indicates a combination of two substitutions.
  • the present invention relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof comprising at least one substitution described in Table 1, Table 2 and Figures 2 to 9.
  • the present invention preferably relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof comprising at least one substitution selected from one of the groups consisting of:
  • variant or the fragment thereof has one or more substitutions as indicated with respect to the SEQ ID Nos. 2, 4 and 6 polypeptide sequences, but that it may have other modifications, in particular substitutions, deletions or additions.
  • thermostable variant of human IFN ⁇ or a functional fragment thereof comprising a combination of substitutions selected from the groups mentioned above.
  • the combination may consist of 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions selected from this group.
  • thermostable variant of human PIFN ⁇ according to the present invention may comprise other mutations not described in this group, preferably substitutions, in particular some known in the art.
  • the present invention relates to a thermostable variant of human PIFN ⁇ or a functional fragment thereof comprising at least one substitution selected from the group consisting of C21W, Q24A, D25V, P26D, V28C, G41I, G41S, H42D, G49K, T50Y, L51H, K57S, N58R, N58C, N58H, N58Y, W59F, K60H, K60R, E61K, E62C, S63R, S63C, Y76D, E98K, K109C, K109L, K109Q, KIOH, E135V, M140P, A146K, A146M, A147R , A147G, A147L, A147M, A147P, A147S, A147E, M157W, M157Q, M157L, L158C, L158I, L158W, F159C, F159V, R160A, R162D, R162Q and R162E or
  • the present invention relates to a thermostable variant of human PIFN ⁇ or a functional fragment thereof comprising at least one of mutations selected from the group consisting of Q24A, P26D, V28C, G41I, G41S, H42D, G49K, T50Y.
  • the present invention relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof comprising at least a first substitution selected from the group consisting of S63C, E62C, F159C, D99Y, E116C , L158C, S74G, R162C, S122D, L126P, N58R, and T95V.
  • the variant does not carry a non-peptide group attached to the residue (s) introduced by said one or more first substitutions.
  • This variant may further comprise at least one other substitution selected from the group consisting of M157C, G41S and M100N.
  • the present invention also relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof having either a single C23S or M157C substitution, or a C23S or M157C substitution in combination with one or more substitutions. selected from the group consisting of C21G, C21W, Y22D, Y22T, Y22S, Q24A, D25V, P26D, V28C, G41I, G41S, H42D, S43C, S43G, S43T, G49K, T50Y, L51H, L51I, K57S, N58R, N58C, N58H, N58Y, W59F, K60H, K60R, E61K, E62C, S63R, S63C, Y76D, E98K, MlOON, K109C, K109Q, K109L, KI HOl, T119Y, Tl 19P 5 Y121T, S122H, S122P, K131I, E
  • the present invention relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof having either a single C23S or M157C substitution, or a C23S or M157C substitution in combination with one or more selected substitutions. from the group consisting of S63C, E62C, F159C, D99Y, E116C, L158C, S74G, R162C, S122D, L126P, N58R, M100N, T95V and G41S.
  • the present invention relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof having either a single M157C substitution or an M157C substitution in combination with one or more substitutions selected from group consisting of S63C, E62C, F159C, D99Y, E116C, L158C, S74G, R162C, S122D, L126P, N58R, MlOON, T95V and G41S.
  • the present invention relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof having a single substitution selected from a group consisting of:
  • the substitution is selected from the group consisting of C21W, C23S, Q24A, D25V, P26D, V28C, G41I, G41S, H42D, G49K, T50Y, L51H, K57S, N58R, N58C, N58H, N58Y, W59F, K60H, K60R, E61K, E62C, K109L, K109Q, K110H, E135V, M140P, A146K, A146M, A147R, M157L, L158C, L158I, L158W, F159C, F159V, R160A, R162D, R162Q and R162E.
  • substitution can be selected from the group consisting of C23S, Q24A, P26D, V28C, G41I, G41S, H42D, G49K, T50Y, L51H, K57S, N58R, N58C, N58H, N58Y, K60H, K60R, E61K, E62C, S63C, K109C, A146K, A146M, A147R, A147G, A147L, A147M, A147P, A147S, A147E, M157Q, M157C, M157L, L1 581, F159C, F159V, R16OA, R162E, R162Q and R162D. .
  • the present invention relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof. having a single substitution selected from the group consisting of S63C, E62C, F159C, D99Y, El16C, L158C, S74G, R162C, S122D, L126P, N58R, and T95V.
  • the present invention relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof having a combination of substitutions selected from the group consisting of C21G + F159C, A147E + R162D, M100N + T119Y, Y76D. + K131I, T50Y + Y121T + M140P, P26D + S122P,
  • the present invention relates to a thermostable variant of human IFN ⁇ or a functional fragment thereof comprising or having a combination of two substitutions selected from the group consisting of S63C, E62C, F159C, D99Y, E116C , L158C, S74G, R162C, S122D, MlOON, L126P, N58R, T95V, M157C and G41S, preferably a combination selected from the group consisting of S63C + E62C, S63C + F159C, S63C + D99Y, S63C + E116C, S63C + L158C , S63C + S74G, S63C + R162C, S63C + S122D, S63C + M100N, S63C + L126P, S63C + N58R, S63C + T95V, S63C + M157C, S63C + G41S, E62C + F159C, E62C + D99Y, E62C + E
  • the present invention relates to a variant comprising or having a combination of substitutions selected from the group consisting of S63C + E62C, S63C + F159C, S63C + D99Y, S63C + E116C, S63C + L158C, S63C + S74G, S63C + R162C, S63C + S122D, S63C + M100N, S63C + L126P, S63C + N58R, S63C + T95V, S63C + M157C, S63C + G41S, preferably the combination S63C + G41S.
  • variants SEQ ID Nos. 1-6 describe the protein sequences of the precursor and mature human IFN ⁇ as well as the nucleic sequences coding therefor.
  • the variant according to the present invention corresponds to the precursor protein of 166 amino acids (SEQ ID No. 2), or to the mature protein with or without the tripeptide CYC (SEQ ID Nos. 6 and 4, respectively) comprising at least one substitution or a combination of substitutions according to the present invention.
  • variant is meant in particular a polypeptide differing from a polypeptide having a sequence selected from the sequences SEQ ID Nos. 2, 4 and 6 by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or Residue (s), preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residue (s).
  • “functional fragment” is meant a fragment of human PIFN ⁇ exhibiting the activity of human IFN ⁇ .
  • this fragment may correspond to the precursor or mature human IFN ⁇ , with or without the CYC tripeptide, with a C-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, preferably from 1 to 15 residues, even more preferably from 1 to 11 residues.
  • the fragment may comprise 100, 110, 120, 130 or 140 consecutive amino acids of human IFN ⁇ .
  • human IFN ⁇ activity is meant the ability to bind to the human IFN ⁇ receptor and to induce signal transduction induced by binding human IFN ⁇ to its receptor as determined in vitro or in vivo.
  • the activity of PIFN ⁇ can be measured by the methods described hereinafter in the description and in the examples.
  • thermostability is meant the ability of the protein to retain its activity after being subjected to the action of heat. For example, the protein can be incubated for 10 minutes at 59 ° C. The thermostability of the variant is then estimated by the percentage of residual activity after this pretreatment. This measurement of the thermostability of a variant is then compared to the same value obtained using the wild-type IFN ⁇ subjected to the same conditions.
  • thermostable variants of the present invention retain an activity (condition without pretreatment) which corresponds to at least 10% of the activity of wild-type human IFN ⁇ , preferably at least 20, 30, 40, 50, 60 , 70, 80 or 90% of the activity of wild-type human IFN ⁇ .
  • the thermostable variants of the present invention retain an activity equivalent to that of wild-type human IFN ⁇ , or even increased.
  • non-peptide group is meant a non-peptide molecule that can be attached to the side chain of an amino acid of human IFN ⁇ .
  • This molecule may be a polymer molecule, a lipophilic molecule, a carbohydrate or an organic derivatization agent.
  • the carbohydrate can be attached to IFN ⁇ by glycosylation in vitro or in vivo, for example by N- or O-glycosylation.
  • a lipophilic molecule may be for example a saturated or unsaturated fatty acid, a terpene, a vitamin, a steroid or carotenoid.
  • a polymer molecule may be a polyol, a polyamine, an acid polycarbocyl or a polyalkylene oxide, particularly a polyethylene glycol (PEG). This type of molecule is well known to those skilled in the art.
  • a variant bearing a PEG group will be referred to as pegylated.
  • the variant of human IFN ⁇ according to the present invention may be glycosylated, preferably at positions 48 and 120. In another embodiment, the variant may not be glycosylated. When the variant comprises the G41S substitution, it can be glycosylated at the N39 position by N-glycosylation. In an alternative mode, such a variant may not be glycosylated at this position.
  • the variant may be modified by adding a polymer molecule, in particular by adding polymers (Kita et al., Drug Des., Deliv., 6: 157-167, 1990, EP 236987 and US 5,109,120). or by pegylation (WO99 / 03887, see WO2004005341 "Conjugation of a polymer molecule").
  • the polymer molecule is attached to a position other than the positions 41, 58, 62, 63, 74, 95, 99, 100, 116, 122, 126, 157, 158, 159 and 162.
  • the molecule is not attached to a residue introduced by a substitution made in a variant according to the present invention, in particular a substitution selected from S63C, E62C, F159C, D99Y, M100N , E116C, L158C, S74G, R162C, S122D, L126P, N58R, T95V, G41S and M157C.
  • the variant of the present invention does not carry a polymer molecule. In particular, it is not peggylated.
  • IFN ⁇ in vitro activity
  • the anti-viral response to doses of IFN ⁇ can be measured on different pairs of systems (virus / adherent cell line responding to IFN ⁇ and sensitive to the virus used).
  • the viruses used will be vaccinia virus or lymphocytic choriomeningitis virus (LCMV).
  • Herpes simplex virus (HSV) and cytomegalovirus can also be used.
  • the activity of IFN ⁇ can also be tested using a reporter gene, for example luciferase, under the control of an IFN ⁇ sensitive promoter containing GAS elements (gamma-interferon activation sites) or ISRE (interferon stimulated response element).
  • a reporter gene for example luciferase
  • GAS elements gamma-interferon activation sites
  • ISRE interferon stimulated response element
  • the reporter gene is assayed following stimulation with IFN ⁇ .
  • the pGAS / Luciferase and pISRE / Luciferase vectors are commercially available (# 219091, Stratagene).
  • the pGAS / luciferase vector method is used to measure the activity of IFN ⁇ .
  • thermostability of IFN ⁇ while maintaining its biological activity makes it possible to envisage the development of more effective treatments allowing, with equal biological activity, a reduction of the therapeutic doses used, and thus a reduction of the side effects. associated with the treatment. It also allows higher dose IFN ⁇ treatments to reduce viral infections such as herpes, as these treatments have so far been unenforceable with this type of molecule. Furthermore, the variants according to the present invention may have the advantage of having a longer half-life during the storage of these variants, and therefore a better conservation, compared to wild-type IFN ⁇ , especially at room temperature. .
  • the present invention therefore relates to a pharmaceutical composition comprising a variant of thermostable IFN ⁇ according to the present invention.
  • the present invention preferably relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a thermostable IFN ⁇ variant or a functional fragment thereof comprising at least one selected from the group consisting of S63C, E62C, F159C, D99Y, E116C, L158C, S74G, R162C, S122D, L126P, N58R, and T95V, the variant not carrying a non-peptide moiety attached to the residue (s) introduced by the first substitution (s).
  • the variant differs from a polypeptide having a sequence selected from SEQ ID Nos. 2, 4 and 6 by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. residue (s), preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residue (s).
  • the variant has a single substitution.
  • the variant further comprises at least one other substitution selected from the group consisting of M157C, G41S and M100N.
  • the variant may comprise or have a combination of two substitutions selected from the group consisting of S63C, E62C, F159C, D99Y, E116C, L158C, S74G, R162C, S122D, M100N, L126P, N58R, T95V, M157C and G41S.
  • the variant comprises or has a combination of substitutions selected from the group consisting of S63C + E62C, S63C + F159C, S63C + D99Y, S63C + E116C, S63C + L158C, S63C + S74G, S63C + R162C, S63C + S122D, S63C + M100N, S63C + L126P, S63C + N58R, S63C + T95V, S63C + M157C, S63C + G41S, E62C + F159C, E62C + D99Y, E62C + El 16C, E62C + L158C, E62C + S74G, E62C + R162C, E62C + S122D, E62C + M100N, E62C + L126P, E62C + N58R, E62C + T95V, E62C + M157C, E62C + G41S, F159C + D99Y, F159C, E62C + L158
  • the variant comprises or has a combination of substitutions selected from the group consisting of S63C + E62C, S63C + F159C, S63C + D99Y, S63C + El 16C 5 S63C + L158C, S63C + S74G, S63C + R162C, S63C + S122D, S63C + MlOON, S63C + L126P, S63C + N58R, S63C + T95V, S63C + M157C, S63C + G41S.
  • the variant comprises or has the combination S63C + G41S.
  • the variant does not have a deletion of 1 to 11 residues at the C-terminus.
  • the variant has a deletion of 1 to 11 residues at the C-terminus.
  • the variant does not carry a non-peptide group selected from the group consisting of a polymer molecule, a lipophilic molecule, and an organic derivatization agent.
  • the variant carries a non-peptide group selected from the group consisting of a polymer molecule, a lipophilic molecule, and an organic derivatization agent.
  • the non-peptide group in question is especially a polymer molecule, preferably a polyethylene glycol.
  • the variant is glycosylated.
  • the variant may in particular be glycosylated in the N39 position by N-glycosylation when it comprises the G41S substitution.
  • a pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient are well known to those skilled in the art (Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, Ed., Mack Publishing Company [1990], Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard , Eds., Taylor & Francis [2000] and Handbook of Pharmaceutical Excipients, 3rd Edition, A. Kibbe, Ed., Pharmaceutical Press [2000]).
  • a pharmaceutical composition according to the present invention can be formulated in various forms, in particular liquid, gel, lyophilized, powder, compressed solid, and others.
  • the present invention also relates to a variant of the thermostable IFN ⁇ according to the present invention or a composition according to the present invention as a medicament.
  • the pharmaceutical compositions of the invention are suitable or formulated for oral, parenteral (intradermal, intramuscular, intravenous, subcutaneous), sublingual, topical, local, intratracheal, intranasal, transdermal, rectal, intraocular, intraauricular, said active ingredient being administrable in unit dosage form.
  • compositions are solutions adapted for parenteral administration.
  • parenteral formulations also include frozen or freeze-dried forms.
  • the composition will be thawed or dissolved before use.
  • lyophilization the composition will be prepared by adding a pharmaceutically acceptable diluent such as sterile water or a physiological buffer.
  • the unit dosage forms may be, for example, tablets, capsules, granule gels, powders, oral or injectable solutions or suspensions, transdermal patches (patches), sublingual, oral, intratracheal, Intraocular, intranasal, intra-auricular, inhalation, topical, transdermal, subcutaneous, intramuscular or intravenous administration forms, rectal administration forms or implants.
  • the pharmaceutical composition is liquid.
  • Said unit forms are dosed to allow a daily administration of 0.001 to 100 ⁇ g of active ingredient per kg of body weight, according to the dosage form.
  • the dosage appropriate to each patient is determined by the physician according to the mode of administration, the weight and the response of the patient.
  • the IFN ⁇ is administered parenterally, and preferentially by subcutaneous injection.
  • a usual dose of IFN ⁇ by subcutaneous injection is between 1 and 100 ⁇ g / m 2 if the body surface area is greater than 0.5 m 2 and between 0.01 and 10 ⁇ g / kg body weight if the body surface area is less than or equal to 0.5 m 2 .
  • IFN ⁇ is the typical example of a pleiotropic cytokine with a broad spectrum of activities. Indeed, interferons (IFNs) are endowed with activities such as inhibition of viral replication, inhibition of cell multiplication and induction of apoptosis.
  • IFNs interferons
  • stimulation of macrophages by IFN ⁇ induces the following responses:
  • MHC major histocompatibility complex
  • B lymphocytes differentiate of B lymphocytes into antibody secreting plasma cells, which results in the production of type G immunoglobulin and complement activation;
  • PIFN ⁇ The action of PIFN ⁇ on T lymphocytes is to promote their differentiation thus modulating the specific immune response.
  • the main effect sought and developed in clinical phases is mainly the immunomodulatory aspect, the aspect of anti-viral therapeutic molecule being less developed to date.
  • IFN ⁇ is a molecule developed as a therapeutic agent human in the treatment of many quite varied diseases.
  • Commercial IFN ⁇ (Actimmune, and Biogamma) are used for two main therapeutic indications: chronic granulomatosis and idiopathic pulmonary fibrosis, in combination with oral prednisolone.
  • numerous new secondary therapeutic indications are currently being developed at different clinical phases (II and III), in particular for their role as immunosuppressors, for example in addition to PEGylated IFN ⁇ / ribavirin in the part of the treatment of Hepatitis C.
  • kidney cancer There may also be mentioned atypical mycobacterium infections; kidney cancer; osteopetrosis; generalized scleroderma; chronic hepatitis B virus; chronic hepatitis C virus; septic shock ; allergic dermatitis; rheumatoid arthritis; ovarian cancer; fibrosis of the liver; asthma; and lymphoma.
  • IFN ⁇ is also useful in the treatment of various viral infections, has activity against infection by human papillomaviruses, and hepatic infections with virus B and virus C.
  • the present invention relates to the use of a variant of the thermostable IFN ⁇ according to the present invention or of a pharmaceutical composition according to the present invention for the preparation of an antiviral, antiproliferative or immunomodulatory drug.
  • this medicament is intended to treat inflammatory diseases, cancers, infections, bone disorders, autoimmune diseases, etc.
  • the medicament is intended for the treatment of a pathology selected from asthma, chronic familial granulomatosis, idiopathic pulmonary fibrosis, atypical mycobacterial infection, kidney cancer, osteopetrosis, generalized scleroderma, chronic hepatitis B or C virus, septic shock, allergic dermatitis, and rheumatoid arthritis.
  • the medicament is for the treatment of a pathology selected from prurigo, neurodermatitis, type I diabetes, vascular stenosis, basal cell carcinoma, cancer or lymphoma such as ovarian cancer, kidney cancer, leukemia such as hyperproliferative disorder B or T cells, chronic myeloid leukemia and related syndromes, breast cancer, lung cancer, melanoma, colon cancer, brain cancer, pleural cancer, stomach cancer , pancreatic cancer, viral infection, e.g., hepatitis C or B virus, Crohn's disease, psoriasis, multiple sclerosis, and amyotrophic lateral sclerosis.
  • a pathology selected from prurigo, neurodermatitis, type I diabetes, vascular stenosis, basal cell carcinoma, cancer or lymphoma
  • ovarian cancer kidney cancer
  • leukemia such as hyperproliferative disorder B or T cells, chronic myeloid leukemia and related syndromes
  • breast cancer lung cancer, melanoma
  • thermostable variant of IFN ⁇ may be used in combination with another active ingredient, for example an active ingredient selected from an antibody, an antitumor agent or chemotherapy agent, a glucocorticoid, an antihistamine agent, an adrenocortical hormone, an antiallergic agent, a vaccine, a broncodilator, a steroid, a beta-adrenergic agent, an immunomodulatory agent, a cytokine such as interferon alpha or beta, interleukin 1 or 2, TNF ( tumor necrosis factor), hydroxyurea, alkylating agent, folic acid antagonist, nucleic acid metabolism antimetabolite, fusal poison, antibiotic, nucleotide analogue, retinoid, lipoxygenase inhibitor, and cyclooxygenase, fumaric acid and its salts, an analgesic, a spasmolytic, a calcium antagonist and a combination thereof.
  • an active ingredient selected from an antibody, an antitum
  • the additional active principle may be administered before, simultaneously with or after the administration of IFN ⁇ according to the present invention.
  • it can be administered by the same route of administration or by two separate routes of administration.
  • the present invention relates to a product comprising the thermostable variant of PIFN ⁇ or a pharmaceutical composition according to the present invention and another active ingredient, preferably selected from the above list, for a combined preparation intended for simultaneous, sequential or separated for the treatment of one of the pathologies mentioned above.
  • the combination with an antibody is useful for the treatment of cancer.
  • IFN ⁇ is able to increase the effect of antibodies by ADCC (antibody-dependent cellular cytotoxicity).
  • the antibody is preferably directed against an antigen exposed by the cancer cells.
  • the antibody may be a polyclonal, monoclonal, humanized, or chimeric antibody.
  • the antibody is monoclonal and humanized.
  • the antigen may be CD20.
  • This antibody may be Rituximab.
  • glucocorticoid is useful for the treatment of alveolar lung diseases such as idiopathic pulmonary fibrosis.
  • suitable glucocorticoids are hydrocortisone, cortisone, dexamethasone, betamethasone, prednisolone, methyl prednisolone and their pharmaceutically acceptable salts.
  • a preferred embodiment of the present invention relates to the use of a combination of IFN ⁇ according to the present invention and prednisolone.
  • an antihistamine agent an adrenocortical hormone, an antiallergic agent is useful in particular for the treatment of skin diseases such as a prurigo or a neurodermatitis.
  • the combination with an antiallergic agent, a broncodilator, a steroid, a beta-adrenergic agent, an immunomodulatory agent, or a cytokine is useful in particular for the treatment of asthma.
  • the present invention further relates to a method of antiviral, antiproliferative or immunomodulatory treatment in a patient in need thereof, comprising administering a therapeutically effective amount of a thermostable IFN ⁇ variant or a pharmaceutical composition according to the present invention. to the patient.
  • the method of treatment is intended for the treatment of a pathology mentioned above.
  • the method may further comprise the administration of another active ingredient, preferably selected from those mentioned above.
  • An effective therapeutic amount is the amount necessary to decrease or suppress the symptoms of the disease or to cure or slow the progression of the disease.
  • the patient is preferably a human.
  • the present invention relates to a nucleic acid encoding a thermostable variant of human PIFN ⁇ according to the present invention.
  • the present invention also relates to a cassette for expressing a nucleic acid according to the present invention. It also relates to a vector comprising a nucleic acid or an expression cassette according to the present invention.
  • the vector may be selected from a plasmid and a viral vector.
  • the nucleic acid can be DNA (cDNA or gDNA), RNA, a mixture of both. It can be in simple chain or duplex form or a mixture of both. It can comprise modified nucleotides, comprising, for example, a modified linkage, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to those skilled in the art, including chemical synthesis, recombination, mutagenesis, etc.
  • the expression cassette comprises all the elements necessary for the expression of the thermostable variant of human IFN ⁇ according to the present invention, in particular the elements necessary for transcription and translation in the host cell.
  • the host cell may be prokaryotic or eukaryotic.
  • the expression cassette comprises a promoter and a terminator, optionally an amplifier.
  • the promoter may be prokaryotic or eukaryotic. Examples of preferred prokaryotic promoters are: LacI, LacZ, pLacT, ptac, pARA, pBAD, T3 or T7 bacteriophage RNA polymerase promoters, polyhedrin promoter, phage lambda PR or PL promoter.
  • eukaryotic promoters examples include: early CMV promoter, HSV thymidine kinase promoter, SV40 early or late promoter, mouse L-metallothionein promoter, and LTR regions of some retroviruses.
  • a suitable promoter the skilled person can advantageously refer to the work of Sambrook et al. (1989) or the techniques described by Fuller et al. (1996) Immunology in Current Protocols in Molecular Biology.
  • the present invention relates to a vector carrying a nucleic acid or an expression cassette encoding a thermostable variant of human IFN ⁇ according to the present invention.
  • the vector is preferably an expression vector, i.e. it comprises the elements necessary for the expression of the variant in the host cell.
  • the host cell may be a prokaryote, for example E. coli, or a eukaryotic.
  • the eukaryote may be a lower eukaryote such as a yeast (for example, S cerevisiae) or a fungus (for example of the genus Aspergillus) or a higher eukaryote such as an insect cell (Sf9 or Sf21 for example), mammalian or plant.
  • the cell may be a mammalian cell, for example COS (green monkey cell line) (e.g., COS 1 (ATCC CRL-1650), COS7 (ATCC CRL-1651), CHO (US 4,889,803; US 5,047,335, CHO- Kl (ATCC CCL-61)), mouse cells and human cells.
  • COS green monkey cell line
  • the vector may be a plasmid, a phage, a phagemid, a cosmid, a virus, a YAC, a BAC, an Agrohacterium pTi plasmid, etc.
  • the vector may preferably comprise one or more elements selected from an origin of replication, a multiple cloning site and a selection gene.
  • the vector is a plasmid
  • prokaryotic vectors are the following: pQE70, pQE60, pQE-9 (Qiagen), pbs, pCO4, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), pTrc99A, pKK223-3, pKK233-3 5 pDR540, pBR322, and pRIT5 (Pharmaci a), pET (Novagen).
  • Non-exhaustive examples of eukaryotic vectors are: pWLNEO, pSV2CAT, pPICZ, pcDNA3.1 (+) Hyg (Invitrogen), pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pCI-neo (Stratagene), pMSG, pSVL (Pharmacia); and pQE- (QLAexpress).
  • the viral vectors may be non-exhaustively adeno viruses, AAVs, HSVs, lentiviruses, etc.
  • the expression vector is a plasmid or a viral vector.
  • the IFN ⁇ coding sequence according to the present invention may comprise or not comprise the signal peptide.
  • a methionine may be optionally added at the N-terminus.
  • a heterologous signal peptide can be introduced.
  • This heterologous signal peptide can be derived from a prokaryote such as E. coli or from a eukaryotic, especially a mammalian, insect or yeast cell.
  • the present invention relates to the use of a polynucleotide, an expression cassette or a vector according to the present invention to transform or transfect a cell.
  • the present invention relates to a host cell comprising a nucleic acid, an expression cassette or a vector encoding a thermostable variant of human IFN ⁇ and its use for producing a thermostable variant of recombinant human IFN ⁇ according to the present invention.
  • the term "host cell” encompasses the daughter cells resulting from the culture or growth of this cell. In a particular embodiment, the cell is non-human and non-embryonic.
  • thermostable variant of recombinant human IFN ⁇ comprising the transformation or transfection of a cell with a polynucleotide, an expression cassette or a vector according to the present invention; culturing the transfected / transformed cell; and harvesting the thermostable variant of human IFN ⁇ produced by the cell.
  • the method for producing a thermostable variant of recombinant human IFN ⁇ according to the present invention comprising providing a cell comprising a polynucleotide, an expression cassette or a vector according to the present invention; culturing the transfected / transformed cell; and harvesting the thermostable variant of human PIFN ⁇ produced by the cell.
  • the cell may be transformed / transfected transiently or stably by the nucleic acid encoding the variant.
  • This nucleic acid may be contained in the cell as an episome or in a chromosomal form.
  • Methods of producing recombinant proteins are well known to those skilled in the art. For example, the specific modes described in US 5,004,689, EP 446,582, Wang et al. (Sci Sin B 24: 1076-1084, 1994 and Nature 295, page 503) for production in E. coli, and JAMES et al. (Protein Science (1996), 5: 331-340) for mammalian cell production.
  • the present invention may also relate to a pharmaceutical composition
  • a pharmaceutical composition comprising a nucleic acid encoding a thermostable IFN ⁇ variant according to the present invention, an expression cassette, a vector or a host cell according to the present invention, its use for the preparation of a medicine, in particular for treating the diseases mentioned above. It also relates to a method of treating a patient in need thereof comprising administering such a composition in a therapeutically effective amount.
  • thermostable protein variants called THR and described in the French patent application number 0505935.
  • This method is based on the preparation of fusion protein between human PIFN ⁇ variants and a variant of a kanamycin resistance protein with increased thermostability (This double mutant of kanamycin nucleotidyl transferase is described in Liao, Enzyme Microb Technol., 1993, 15, 286-92).
  • the human IFN ⁇ variant library was prepared by the Massive Mutagenesis® method described in FR2813314, and was transformed at high temperature into Thermus thermophilus strain HB27. The transforming clones of this library were selected at increasing concentrations of kanamycin for which the production of the IFN ⁇ (wild) -KNTase fusion no longer allows the cells to grow.
  • Variants of human IFN ⁇ selected by our selection method or systematically generated on all PIFN ⁇ positions were transiently expressed in COS7 animal cells. These proteins are secreted in the culture supernatant. In order to evaluate the stability and the conservation of the activity of these variants, the COS7 cell culture supernatants were subjected to thermal denaturation (10 minutes at 59 ° C.). These proteins (denatured or otherwise) were then activated on transfected HeLa cells containing luciferase as the reporter gene. After 16 hours of stimulation, for each mutant and for each condition, we measured the signal of flrefly luciferase corresponding to the activity of PIFN ⁇ tested.
  • the basal activity of the undenatured variant was also compared to that of the non-mutated IFN ⁇ .
  • THR system The vector that was used included the origins of replication of E. coli and T. Thermophilus, an ampicillin resistance gene that allows for the selection of transformants in E. coli, a gene encoding the thermostable KNTase under control. a promoter active in both E. coli and T. thermophilus (pslpA promoter). See Figure 1. The nucleotide sequence of IFN ⁇ (encoding the mature 146 amino acid SEQ ID NO: 5) was cloned between the NcoI and NotI sites of the N-terminal vector of KNTase.
  • linker a linker peptide which presented the peptide sequence AAAGSSGSI (SEQ ID No. 8) and was encoded by the GCG-GCC-nucleic sequence
  • Eukaryotic expression system For the expression of IFN ⁇ in mammalian cells, we used pORP / IFN ⁇ (Invivogen) in which PIFN ⁇ is cloned into an expression cassette containing the hybrid promoter (EF-l ⁇ -HLTV ) and the strong polyadenylation signal of SV40.
  • thermostable mutants described in the bibliography were constructed and used as positive controls. They encode for:
  • the delta IFN ⁇ protein (C-terminal end of the deleted protein of these last 10 amino acids activated and improved stability, TM + 7.5 ° C and antiviral activity multiplied by 4, Slodowski et al., 1991).
  • mutants are generated at the level of the pNCK-IFN ⁇ and PORF-IFN ⁇ matrices by the Massive Mutagenesis® method described in FR2813314.
  • thermostable mutants by the THR method
  • a library of IFN ⁇ variants cloned into the pNCK vector was generated using Massive Mutagenesis®. Total diversity has been introduced in all positions (from 21 to 166). The library was then transformed at high temperature (70 ° C.) in Thermus thermophilus strain HB27 and selected at 20 or 40 ⁇ g / ml of kanamycin (conditions where the IFN ⁇ (wild) -KNTase fusion no longer allows the cell to push). Mutations identified after sequencing on clones growing on selective medium are listed in Table 1. The different mutants from this primary selection were then uniaxtransformed and their level of resistance was compared to that of the wild-type construct. 21 mutants, giving the strain resistance more or less important but still superior to the wild were thus confirmed (Table 2).
  • HeLa cells (human cervix epitheloid carcinoma cells), COS-7 cells (African green monkey SV40 transformed kidney cells) and CHO cells (Chinese Hamster Ovary) were cultured under standard culture conditions (37 ° C. in the atmosphere).
  • D-MEM Dulbecco's Modified Eagle's Medium Medium
  • IMDM Iscove's Modified Dulbecco's Medium
  • COS7 cells In order to perform transfections of COS7 cells by native or mutated pORF / IFN ⁇ constructs, these cells were trypsinized when they reached 90% confluency. COS7 cells were re-seeded at a ratio of 1 A (that is to say so that they represent, once adhered on the surface, a confluence of about 25%). Transfection of COS7 cells was performed in a 24-well plate with seeding of 30,000 to 60,000 cells per well when the cells reached 70-80% confluency. Transfection was performed with approximately 50ng of DNA and Jet PEI (Polyplus transfection) using a PEI / DNA Jet ratio of 5 leaving 30 minutes at room temperature.
  • Jet PEI Polyplus transfection
  • the reference ELISA kit for determining the total amount of IFN ⁇ is from Clinisciences (# 88-7316-86). We verified that the antibodies used in this
  • IFN ⁇ specifically activates the IFN ⁇ receptors present on HeLa cells.
  • the stimulation of the Jak / Statl pathway of HeLa cells by IFN ⁇ is carried out with, in particular, the consequence of which is the activation of the transcription of genes under the control of promoters possessing GAS sequences. for "Gamma Activated Site”. It is then possible to measure and compare the activities of IFN ⁇ variants by transfecting in HeLa cells a reporter gene system in which luciferase (firefly luciferase) is downstream of a promoter possessing several GAS sites (plasmid pGAS Stratagene Luciferase).
  • TM Promega
  • the lysis was carried out for 10 min with stirring at room temperature so as to release the luciferase produced in response to the specific stimulation of IFN ⁇ .
  • Measurement of activity was initiated by the addition of Bright GIo TM reagent (Promega) and the amount of accumulated luciferase was then counted with a luminometer (FLX 800, Bio-Tek Instrument). The crude activity in IFN ⁇ is expressed in RLU for "relative luciferase unit".
  • the calculation of the total activity (relative to the wild-type protein) of each variant (undenatured) is an average carried out on the basis of results obtained on 5 different manipulations (at least duplicates) and on culture supernatants coming from minimum of two independent transfections.
  • the error bars presented are calculated by the standard error formula on the average (sem).
  • One way to present the total activity results is to report the baseline activity of each variant as a percentage of the baseline activity of the non-mutated IFN ⁇ expressed under the same conditions for each transfection.
  • the amount of luciferase was measured after stimulation of the cells with 10 ⁇ l of supernatants of COS7 cells diluted to 1 / 100th containing IFN ⁇ which, depending on the case, were submitted. at a heat treatment of 10 minutes at 59 ° C or have not been treated.
  • One of the ways to present the fraction of activity of each conserved variant after thermal denaturation is to calculate the conserved residual activity of each variant defined by the percentage of the basal activity of the same variant before denaturation.
  • the calculation of the residual activity with respect to a total activity determined before denaturation is an average carried out on the basis of results obtained on 5 different manipulations (at least duplicates) and on culture supernatants coming, at least, from two independent transfections. .
  • the error bars presented are calculated on the standard error formula on the mean (s.e.m).
  • This half-life was obtained by controlling particularly all the following parameters: the same initial concentration of IFN ⁇ of 1000 g / ml, a concentration of serum SVF in the final sample to be denatured equivalent and adjusted to 0.15% and a temperature denaturation of 59 0 C for 30 minutes with sampling every 10 minutes.
  • the IFN ⁇ concentrations of the CHO cell supernatants were estimated by ELISA. These different lots of IFN ⁇ variants were then diluted to 1000 ⁇ g / ml in 0.15% IMDM SVF medium. These dilutions were then aliquoted to undergo thermal denaturation pretreatment at 59 ° C for 0, 10 and 30 minutes.
  • One of the ways of presenting the data is to postpone the gross activity corresponding to the specific stimulation of the transduction pathway by variant IFN ⁇ , a crude activity then expressed in RLU for "relative luciferase unit”.
  • Another way to present the fraction of activity conserved after thermal denaturation (also called residual activity) of each variant is to calculate, for each denaturation time, the residual percentage of activity retained relative to the basal activity of the even varying before denaturation. These calculations are done after subtraction of the signal share due to untransfected cells.
  • T 1/2 The half-lives of each variant thus determined are then compared with that of wild-type IFN ⁇ .
  • a (t) is the IFN gamma activity at time tps t and b is a constant.
  • thermostable variants of IFN ⁇ the biological half-life or terminal elimination half-life (hereinafter referred to as "half-life in vivo"), as well as the areas under the curve for different modes of administration (intravenous (iv) or subcutaneous (sc) injection).
  • iv biological half-life or terminal elimination half-life
  • sc subcutaneous injection
  • the measurement of the elimination half-life can be realized in multiple ways:
  • the half-life is then estimated by an anti-viral activity test directly on the murine sera (Hep-2 cells infected with a virus (vesicular stomatitis virus or VVS).
  • a virus vesicular stomatitis virus or VVS.
  • the ELISA is used as an alternative for detecting IFN ⁇ levels in murine serum.
  • Another way described in Croos and Roberts is to carry out experiments with radiolabelled isotope-labeled IFN ⁇ and to monitor the absorption of this labeled molecule in tissues and tissues. its passage into the serum of female Sprague-Dawley rats after subcutaneous injection. The tissue and blood samples are then analyzed for the amount of labeled IFN ⁇ they contain.
  • the use of ELISA method for determining the pharmacokinetic parameters of IFN has been described for IFN alpha by subcutaneous administration by Rostaing et al. (1998), J. Am. Soc. Nephrol.9 (12): 2344-48 and by intramuscular administration by Merimsky et al (1991) Cancer Chemother. Pharmacol. 27 (5).]
  • Wild-type IFN ⁇ and mutants were expressed from COS and CHO cells. These culture supernatants were centrifuged a second time at 4000 RPM, 10 min, and filtered through Millipore filter PES 0.22 .mu.m. The volumes of the filtered supernatants are then concentrated by centrifugation on Vivaspin filtration units with a cutoff of 5000 daltons (Sartorius). The IFN ⁇ concentrations are then estimated for these samples by applying the appropriate prior dilutions. The wild-type and variant IFN ⁇ lots are administered according to two modes:
  • mice 8 week old C57BL / 6J mice with an average weight between 20 and 30 grams are used. These animals are acclimatized one week in a room under a constant temperature of 24.1 0 C, a constant humidity of 55% and with rest / sleep cycles of 12h.
  • mice blood samples are collected at the following different times after administration of the molecule of interest. Retro-orbital samples are taken for the 3h, 6h, 24h, 48h, 72h, 96h, 12h, 144h, 168h, 192h and cardiac post-mortem samples for the final time at 216 hours.
  • the serum is prepared by allowing the blood to coagulate for 20 minutes at room temperature and recovering the fraction corresponding to the supernatant from a centrifugation at 5000 g, 20 min at 20 ° C.
  • the serum is then isolated and stored at -80 ° C until the activity of IFN ⁇ is measured using the ELISA assay described above.
  • the plasma concentration of IFN ⁇ is then evaluated over time by monitoring the amount of IFN ⁇ detected by ELISA in each mouse serum. For each sample taken in retro-orbital, the quantification of IFN ⁇ obtained results from the average of at least 3 different sera from 3 mice. These different pharmacokinetic experiments were reproduced for each variant at least twice from IFN ⁇ lots from different transfections. Finally, the concentration of IFN ⁇ in the serum is reported over time.
  • Another parameter described in Tables 4 and 5 are the ratio of improvement of the half-life or the area under the curve of each of the mutants with respect to the respective parameters of the unmutated wild-type molecule produced under the same conditions and relative to the respective parameters of bacterial recombinant human IFN ⁇ when data are available. The higher these ratios, the greater the improvement of the variant compared to that of the non-mutated human IFN ⁇ .
  • mutations S63C, G41S as well as their combination result in a marked improvement in the in vivo half-life of these variants compared to that of the recombinant human IFN ⁇ produced in CHO and that of recombinant human IFN ⁇ produced in vitro. bacterium. After subcutaneous administration, the double mutant still shows a marked improvement in these pharmacokinetic parameters compared to recombinant human IFN ⁇ produced in CHO.

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