RU2268749C2 - Interferon-gamma conjugates - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: invention relates to conjugates having interferon-γ activity. Claimed conjugate contains at least one non-polypeptide group, covalent bonded to IFNG polypeptide, wherein polypeptide contains amino acid sequence differ from starting IFNG polypeptide sequence by at least one added or deleted amino acid residue containing group to add non-polypeptide group.

EFFECT: therapy of diseases of improved effectiveness.

34 cl, 1 ex

Description

FIELD OF THE INVENTION

The present invention relates to conjugates with γ-interferon-like activity, methods for their preparation, pharmaceutical compositions containing such molecules, and their use in the treatment of diseases.

State of the art

γ-Interferon (IFNG) is a cytokine produced by T cells and natural killer cells, and exists as a homodimer of two non-covalently linked polypeptide subunits. The mature form of each dimer contains 143 amino acid residues (shown in AFTER. No. 2), and the form of its predecessor includes a signal sequence of 166 amino acid residues (shown in AFTER. No. 1).

Each subunit has two potential N-glycosylation sites (Aggarwal et al .. Human Cytokines, Blackwell Scientific Publications, 1992) at positions 25 and 97. Depending on the degree of glycosylation, the molecular weight of the IFNG in dimeric form is 34-50 kDa (Farrar et al ., Ann. Rev. Immunol., 1993, 11: 571-611).

The primer sequence of wild-type human IFNG (hulFNG) has been reported by Gray et al. (Nature, 298: 859-863, 1982), Taya et al. (EMBO J., 1: 953-958, 1982), Devos et al. (Nucleic Acids Res., 10: 2487-2501, 1982) and Rinderknecht et al. (J. Biol. Chem., 259: 6790-6797, 1984), and in EP 77670, EP 89676 and EP 110044. The 3D structure of hulFNG is reported by Ealick et al. (Science, 252: 698-702, 1991).

There are reports of naturally occurring or mutated forms of polypeptides with IFNG subunits, including a form containing the N-terminal amino acid sequence of Cys-Tyr-Cys (positions (-3) - (- 1) relative to LAST. No. 2), about the form containing the N-terminal methionine (position -1 relative to LAST. No. 2) and various C-terminated forms containing 127-134 amino acid residues. It is known that 1-15 amino acid residues can be removed from the C-terminus without destroying the IFNG activity of the molecule. In addition, Pan et al. (Eur. J. Biochem., 166: 145-149, 1987) describe the heterogeneity of the C-terminus of hulFNG.

Hu IFNG muteins are reported by Slodowski et al. (Eur. J. Biochem., 202: 1133-1140, 1991), Luck et al. (J. Biol. Chem., 265: 13314-13319, 1990), Seelig et al. (Biochemistry, 27: 1981-1987, 1988), Trousdale et al. (Invest. Ophtalmol. Vis. Sci., 26: 1244-1251, 1985) and is reported in EP 146354. A natural variant of hulFNG is reported by Nishi et al. (J. Biochem., 97: 153-159, 1985).

No. 6,046,034 describes variants of thermostable recombinant hulFNG (rhuIFNG), which include up to 4 pairs of cysteine residues to allow the formation of a disulfide bond and, thus, stabilization of the IFNG variant in homodimeric form.

WO 92/08737 describes IFNG variants comprising additional methionine at the N-terminus of the complete (residues 1-143) or incomplete (residues 1-132) amino acid sequence of wild-type human IFNG. EP 219781 describes incomplete hulFNG sequences containing amino acid residues 3-124 (SEQ ID NO: 2). US Pat. No. 4,832,959 describes incomplete hulFNG sequences containing residues 1-127, 5-146 and 5-127 of the amino acid sequence, which, compared to the AFTER. No. 2, contain three additional N-terminal amino acid residues (CYC). US 5004689 describes a DNA sequence encoding hulFNG without the 3 N-terminal amino acid residues of CYC, and its expression in E. coli. EP 446582 describes an E. coli rhuIFNG produced free of N-terminal methionine. No. 6,120,762 describes a peptide fragment of rhuIFNG containing its residues 95-134 (relative to SEQ ID NO: 2).

High levels of rhuIFNG expression are reported by Wang et al. (Sci. Sin., B 24: 1076-1084, 1994).

The glycosylation variant in rhuIFNG is reported by Curling et al. (Biochim. J., 272: 333-337, 1990) and Hooker et al. (J. of Interferon and Cytokine Research, 1998, 18: 287-295).

Modifications of rhuIFNG polymers have been reported by Kita et al. (Drug Des. Deliv., 6: 157-167, 1990) and is reported in EP 236987 and US 5109120.

WO 92/22310 describes the azialoglycoprotein conjugate derivatives of interferons, in particular hulFNG.

Proteins fused to IFNG are described. For example, EP 237019 describes a single chain polypeptide having a region detecting interferon β activity and one region detecting IFNG activity.

EP 158198 describes a single chain polypeptide having a region that detects IFNG activity and a region that detects IL-2 activity. Some references describe single chain dimeric IFNG proteins, for example, Landar et al., J. Mol. Biol., 2000, 299: 169-179.

WO 99/02710 describes single chain polypeptides, one example of which, among many, is IFNG.

WO 99/03887 describes variants of polypeptides with attached PEG belonging to the superfamily of growth hormones, where non-essential amino acid residues located in a specific region of the polypeptide are replaced by a cysteine residue. IFNG is mentioned as an example of a member of the superfamily of growth hormones, but its modification is not discussed in any detail.

IFNG is proposed for the treatment of interstitial lung diseases (also known as interstitial pulmonary fibrosis (IPF) (Zeische et al. (N. Engl. J. Med., 341: 1264-1269, 1999, and Chest, 110: Suppl: 25S, 1996 ), and EP 795332), for which IFNG can be used in combination with prednisone. In addition to IPF, granulomatous diseases can also be treated with IFNG (Bolinger et al., Clinical Pharmacy, 1992, 11: 834-850), some mycobacterial infections ( N. Engl. J. Med., 330: 1348-1355, 1994), kidney cancer (J. Urol., 152: 841-845, 1994), osteoporosis (N. Engl. J. Med., 332: 1594- 1599, 1995), scleroderma (J. Rheumatol., 23: 654-658, 1996), hepatitis B (Hepatogastroentherology, 45: 2282-2294, 1998), hepatitis C (Int. Hepatol. Communic., 6: 264-273, 1997), septic shock (Nature Medicine, 3: 678-681, 1997) and rheumatoid arthritis.

As a pharmaceutical compound, rhuINFG is used with some success in all of the above cases, against certain viral infections and tumors. Typically, rhuINFG can be administered parenterally, preferably by subcutaneous injection. The maximum concentration in serum is detected after seven hours, the half-life in plasma is 30 minutes after iv administration. For this reason, effective treatment with rhuINFG involves frequent injections. The main side effects are fever, chills, sweating, myalgia, and drowsiness. This action is associated with rhuINFG injection and is observed during the first hours after injection. Rare side effects are local pain and erythema, elevated liver enzymes, reversible granulo- and thrombopenia, and cardiotoxicity.

It is necessary to create new molecules with INFG activity having improved properties compared to hulNFG or rhuINFG, in the sense of pharmacokinetics, homogeneity, immunogenicity and other side effects.

SUMMARY OF THE INVENTION

This application describes improved INFG-like molecules providing one or more of the above desired properties. In its first aspect, the invention relates to a conjugate exhibiting INFG activity and containing at least one first non-polypeptide group covalently attached to an INFG polypeptide, the polypeptide containing an amino acid sequence different from the original INFG polypeptide by at least one introduced and / or at least one deleted amino acid residue containing a group for joining a non-polypeptide group. The conjugates have an extended in vivo half-life compared to huINFG and rhuINFG and, optionally, cause a reduced immune response compared to rhuINFG. If necessary, this class of molecules also has even more improved properties in terms of the ability to form homogeneous molecules, improved resistance to proteolysis and / or increased bioavailability.

Consequently, the conjugate of the invention provides several advantages over currently available INFG compounds, including a longer period of time between injections or other forms of administration, weaker side effects and / or increased efficacy due to a decrease in the number of antibodies. In addition, using the conjugate of the invention, it is possible to obtain higher doses of the active protein and, thus, a more effective therapeutic response.

In another aspect, the invention relates to a conjugate exhibiting INFG activity comprising at least one IFNG polypeptide treated with a PEG at the N-terminus. The IFNG polypeptide may be huINFG or any of the IFNG polypeptides described herein.

In another aspect, the invention relates to means and methods for producing a conjugate of the invention, including nucleotide sequences and expression vectors, as well as methods for producing a polypeptide or conjugate.

In still other aspects, the invention relates to a therapeutic composition comprising a conjugate of the invention, to a conjugate or composition of the invention for use in treatment, to the use of a conjugate or composition in the treatment or for the manufacture of a medicament for treating diseases.

Finally, the invention relates to the use of certain INFG conjugates for the manufacture of a medicament, pharmaceutical composition or compound kit for the treatment of interstitial lung diseases, cancer, infectious diseases and / or inflammatory diseases, and in case of interstitial lung diseases, if necessary, also in combination with glucocorticoids .

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the context of this application and invention, the following terms apply.

The term “conjugate” (or, equivalently, “conjugated polypeptide”) is intended to define a heterogeneous (in terms of composition or chimera) molecule formed by covalently linking one or more polypeptides with one or more non-polypeptide groups. The term "covalent compound (attachment)" means that the polypeptide and non-polypeptide group either directly covalently connect to each other or, otherwise, indirectly covalently connect to each other through an intermediate group or groups, such as a bridging, spacer or linker group or groups. Preferably, the conjugate is dissolved under appropriate concentrations and conditions, i.e. soluble in body fluids, such as blood. Examples of conjugated polypeptides of the invention include glycosylated and / or PEG-treated polypeptides. The term "unconjugated polypeptide" can be used in relation to the polypeptide portion of the conjugate.

The term "non-polypeptide group" is intended to mean a molecule capable of conjugation with a group for attachment in an IFNG polypeptide. Preferred examples of such a molecule are polymer molecules, lipophilic compounds, sugar groups, or organic derivatizing agents. When used with respect to a conjugate of the invention, it should be borne in mind that the non-polypeptide group binds to the polypeptide portion of the conjugate via a group for attachment in the polypeptide.

The term "polymer molecule" means a molecule formed by covalent binding of two or more monomers, where none of the monomers is an amino acid residue, unless the polymer is human albumin or other common plasma protein. The term "polymer" can be used on an equal footing with the term "polymer molecule". The term “sugar group” is intended to mean a carbohydrate molecule attached by in vivo or in vitro glycosylation, for example, N- or O-glycosylation. Except where the number of non-polypeptide groups, such as polymer (s), molecule (s) in the conjugate is specifically indicated, each reference to the "non-polypeptide group" contained in the conjugate or otherwise used in the present invention should indicate one group or several non-polypeptide groups in the conjugate.

The term “attachment group” is intended to define an amino acid residue group capable of attaching a relevant non-polypeptide group, such as a polymer molecule or a sugar group. Useful groups for joining and their corresponding non-polypeptide groups are illustrated in the table below.

Group to join. Amino acid Examples of non-polypeptide. groups Conjugation Method / Asset Peg Link -NH ₂ N-conc. Lys Polymer e.g. PEG mPEG-SPA, transylated mPEG Shearwater Inc., Delgado et al., Critical rewiews in Therapeutic Drug Carrier Systems, 9 (3.4): 249-304 (1992) -COOH C-conc. Asp, Glu Polymer, e.g. PEG,
sugar group mPEG-Hz Comb. in vitro Shearwater Inc. -SH Cys Polymer e.g. PEG Peg vinyl sulfone peg maleimide Shearwater Inc., Delgado et al., Critical rewiews in Therapeutic Sys- sugar group Combines. in vitro Drug Carrier terns, 9 (3.4): 249-304 (1992) -IT Ser, Thr, OH-, Lys sugar group Glycosylir. O-bond. in vivo -CONH ₂ Asn as part of the N-glycosylation site sugar group In vivo glycosylation The aroma. balance Phe, Tug, Trp sugar group In vitro combination -CONH ₂ Gin sugar group In vitro combination Yan and Wold, Biochemistry, 1984, Jul 31: 23 (16): 3759-65 Aldehyde Ketone Oxidized carbohydrate polymer, e.g. PEG, PEG hydrazide PEG Processing Andresz et al., 1978, Macromol. Chem., 179: 301; WO 92/16555, WO 00/23114 Guanidino Arg sugar group In vitro combination Lundblad and Noyes, Chimical Reagents for Protein Modification, CRC Press Inc., Boca Raton, FI Imida ash cycle His sugar group In vitro combination As with guanidino

In the case of N-glycosylation in vivo, the term “attachment group” is used unusually to refer to the amino acid residues constituting the glycosylation site (with the sequence N-X′-S / T / CX ”, where X ′ represents any amino acid residue with the exception of proline, X ″ is any amino acid residue that may be identical to or different from X ′ and which is preferably different from proline, N is asparagine, and S / T / C is either serine or threonine or cysteine preferred a serine or threonine, and most preferably threonine). Although the asparagine residue of the N-glycosylation site is a residue to which a sugar group binds during glycosylation, such addition cannot be achieved unless other amino acid residues of the N-glycosylation site are present. Accordingly, when a non-polypeptide group is a sugar group and it is necessary to achieve conjugation by N-glycosylation, the term "amino acid residue containing a group for joining a non-polypeptide group" used in connection with changes in the amino acid sequence of the original polypeptide, it should be understood that one, two or all amino acid residues that make up the N-glycosylation site must be changed in such a way that functional ca is introduced into the amino acid sequence T N-glycosylation amino acid sequence or removed from said sequence.

In this application, the designations of amino acids and the designations of atoms (for example, CA, CB, CD, CG, SG, NZ, N, O, C, etc.) are used as indicated in the Protein Structure Database (PDB) (www .pdb.org), based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atoms names etc.), Eur. J. Biochem., 138, 9-37 (1984), together with corrections to him in Eur. J. Biochem., 152, 1 (1985). CA is sometimes referred to as Cα CB as Cβ. The term “amino acid residue” is intended to mean an amino acid residue in the group consisting of alanine residues (Ala or A), cysteine (Cys or C), and paraffinic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K) , leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y). The numbering of amino acid residues in the indicated document starts from the N-terminus of hulFNG without a signal peptide (i.e., SEQ. No. 2). The terminology used to identify amino acid positions / substitutions can be explained with examples N25 (shows the position # 25 occupied by asparagine in the amino acid sequence shown in SEQ. No. 2) and N25C (indicates that the Asp residue at position 25 is replaced by Cys). Multiple substitutions are indicated by "+", for example, QIN + P3T / S denotes an amino acid sequence comprising replacing the Gln residue at position 1 with Asn and replacing the Pro residue at position 3 with Thr or Ser, preferably Thr.

The term "nucleotide sequence" is intended to indicate a sequential region of two or more nucleotide molecules. The nucleotide sequence may be of genomic origin, derived from cDNA and RNA, semisynthetic and synthetic origin, or derived from any combination thereof.

The term "polymerase chain reaction" or "PCR" generally refers to a method for amplifying a desired nucleotide sequence in vitro, as described, for example, in US 4,683,195. In general, the PCR method includes repeated synthesis cycles for primer extension using oligonucleotide primers capable of to selective hybridization with a nucleic acid matrix.

The terms “cell”, “host cell”, “cell line” and “cell culture” are used interchangeably herein, and it should be borne in mind that all such terms include offspring resulting from cell growth or cultivation. The terms “transformation” and “transfection” are used interchangeably and refer to a method for introducing DNA into a cell.

The term "operably linked" refers to the covalent joining of two or more nucleotide sequences by enzymatic ligation or otherwise in such a configuration one relative to another that normal function of the sequence can be performed. For example, a nucleotide sequence encoding a presequence or secretory leader sequence operably binds to the nucleotide sequence of a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide: a promoter or enhancer operably binds to the coding sequence if it affects the transcription of the sequence; the ribosome binding site operably binds to the coding sequence, if positioned so that translation is facilitated. Generally, “randomly linked” means that the nucleotide binding sequences are adjacent and, in the case of a secretory leader sequence, adjacent and in the reading phase. Binding is accomplished by ligation at normal restriction saigas. If there are no such sites, then synthetic oligonucleotide adapters or linkers are used, in conjunction with conventional recombinant DNA methods.

The term "introduction" is mainly intended to mean replacement of an existing amino acid residue, but may also mean the incorporation of an additional amino acid residue. The term "deletion" is primarily intended to mean the replacement of an amino acid residue that is deleted with another amino acid residue, but may also indicate a deletion (without replacement) of the deleted amino acid residue.

The term "amino acid residue containing a group for joining a non-polypeptide group" is intended to show that the amino acid residue is a residue to which a non-polypeptide group binds (in the case of an introduced amino acid residue) or could bind (in the case of a deleted amino acid residue).

The term “one difference” or “different”, as applied to the amino acid sequence of the IFNG polypeptide described herein, is intended to indicate the possibility of the presence of other differences. Accordingly, in addition to differences in a particular amino acid, other amino acid residues other than those indicated can be mutated.

The term "in vivo functional half-life" is used in its usual meaning, i.e., means the time during which 50% of the conjugate molecules circulate in the plasma or bloodstream before excretion (also called the "half-life in the bloodstream"), or time during which 50% of the functionality of the conjugate is maintained. The polypeptide or conjugate is usually excreted by the action of one or more reticuloendothelial systems (RES), kidneys, spleen or liver, or by specific or non-specific proteolysis. Usually, excretion depends on size (relative to the glomerular filtration segment), load, attached hydrocarbon chains, and the presence of receptors for the protein. Typically, retained functionality is selected among antiviral, antiproliferative, immunomodulatory, or IFNG receptor-binding activities. The in vivo functional half-life can be determined by any suitable method known in the art, as described further in the Methods section.

The term “increased functional half-life” is used to show that the functional half-life of an in vivo conjugate has statistically significantly increased relative to the half-life of a molecule for comparison, such as hulFNG, optionally in glycosylated form, for example, hulFNG or rhuIFNG, when determined under comparable conditions .

The term "immunogenicity", used in connection with this substance, is intended to indicate the ability of a substance to induce a response of the human immune system. The immune response may be a cell or antibody mediated reaction (for further definition of immunogenicity, see, e.g., Roitt:. Essential Immunology (8 ^th Edition, Blackwell)).

The term "reduced immunogenicity" is intended to show that the conjugate of the present invention elicits a measurably weaker immune response than a molecule for comparison, such as hulFNG or rhuIFNG, when determined under comparable conditions.

The term "exhibiting IFNG activity" is intended to show that the polypeptide has one or more functions of native IFNG, in particular, hulFNG or rhuIFNG, including the ability to bind to the IFNG receptor and cause transduction of the signal transduced after huIFNG binding its receptor as determined in vitro or in vivo (i.e., biological activity in vitro or in vivo). IFNG receptor is described by Aguet et al. (Cell, 55: 273-280, 1988) and Calderon et al. (Proc. Natl. Acad. Sci. USA, 85: 4837-4841, 1988). An “IFNG polypeptide” is a lolipeptide exhibiting IFNG activity, and such a term is used herein for a polypeptide in monomeric or dimeric form, respectively. For example, when specific substitutions are indicated, they are usually indicated relative to the IFNG polypeptide monomer. When reference is made to the IFNG portion of the conjugate of the invention, it is typically a dimeric form (and thus, for example, contains two IFNG polypeptide monomers modified as described). The dimeric form of IFNG polypeptides can be formed by the usual combination of two monomers, or it can be a single chain dimeric IFNG polypeptide.

The IFNG polypeptide described herein may have an in vivo or in vitro biological activity of the same magnitude as huIFNG or rhuIFNG, or lower or higher, for example, in vivo or in vitro biological activity in 1-100% of the biological activity of hulFNG or rhuIFNG when measured under comparable conditions, for example 1-25% or 1-50% or 25-100% or 50-100% of the biological activity of huIFNG or rhuIFNG.

The term "source IFNG" is intended to mean a molecule modified according to the invention. Typically, the source IFNG is encoded by a nucleotide sequence that is modified according to the present invention so as to encode the polypeptide portion of the conjugate of the invention. The source IFNG is usually huIFNG or rhuIFNG or their variants or fragments. "Variant" is a polypeptide that differs from its original polypeptide, usually 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues. nt represents a part of the full-length sequence of huIFNG, exhibiting IFNG-activity, for example its version, the processed C-terminal or N-end.

The term "functional site" is intended to define one or more amino acid residues that are indispensable or otherwise involved in the function or action of IFNG. Such amino acid residues are “localized” in the functional site. A functional site can be determined by methods known in the art, and is preferably identified by analysis of the structure of a polypeptide complexed with a relevant receptor, such as an IFNG receptor.

Conjugate of the invention

As indicated above, in its first aspect, the invention relates to a conjugate exhibiting IFNG activity and containing at least one non-polypeptide group covalently linked to an IFNG polypeptide, wherein the polypeptide contains an amino acid sequence different from the amino acid sequence of an IFNG polypeptide, according to at least one introduced and / or at least one deleted amino acid residue containing a group for joining a non-polypeptide group.

By removing or introducing an amino acid residue containing a group to attach a non-polypeptide group, the polypeptide can be specifically adapted so as to obtain a molecule more conjugated to the selected non-polypeptide group in order to optimize the conjugation pattern (for example, to ensure optimal distribution of non-polypeptide groups on the surface of the IFNG polypeptide ) and, thus, to obtain a new conjugate molecule exhibiting IFNG activity and, in addition, one or more improved properties compared to currently available molecules based on huIFNG and rhuIFNG. For example, by introducing groups for attachment, the IFNG polypeptide is enhanced or otherwise changed in terms of the content of specific amino acid residues to which the relevant non-polypeptide group binds, whereby a more effective and / or expanded conjugation is achieved. By removing one or more groups for attachment, conjugation with a non-polypeptide group in parts of the polypeptide where such conjugation is disadvantageous, for example, with an amino acid residue located at or near the functional site of the polypeptide (since conjugation at such a site can lead to inactivation or reduction of IFNG, can be avoided). -activity of the resulting conjugate due to impaired receptor recognition). In addition, it may be advantageous to remove the attachment group located adjacent to another attachment group in order to avoid heterogeneous conjugation of such groups. In preferred embodiments, more than one amino acid residue of the IFNG polypeptide is altered, for example, alteration involves the removal as well as the introduction of amino acid residues containing attachment sites for the selected non-polypeptide group. It is believed that this option is particularly interesting in that it is possible to specifically create an IFNG polypeptide in such a way as to obtain optimal conjugation with a non-polypeptide group.

In addition to the deleted and / or introduced amino acid residue, the polypeptide may contain other substitutions that are not related to the introduction and / or removal of amino acid residues containing a group for joining a non-polypeptide group.

Although the starting polypeptide modified by the present invention can be any IFNG-active polypeptide, and thus can be of any origin, for example, not from a human, but from another mammal, it is preferred that the starting polypeptide is an amino acid hulFNG the sequence presented in the LAST. No. 2 or its variant or fragment. Examples of huIFNG variants are described above with respect to the background of the invention and include, for example, N-terminally huIFNG CYC and cysteine-modified variants described in US 6046034. Specific examples of fragments are the fragments described above in the Background section and to them huIFNGs processed at the C-terminus of 1-15 amino acid residues, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, and / or processed at the N-ends of 1-3 amino acid residues.

It should be borne in mind that when the original IFNG polypeptide is a variant or fragment of huIFNG, the modified IFNG polypeptide obtained from such a source contains mutations or truncations of the original molecule.

The starting IFNG polypeptide may also be a hybrid molecule of a monomer of an IFNG polypeptide and another homologous polypeptide, optionally containing one or more additional substitutions introduced into the hybrid molecule. Such hybrids are described above in the "Background" section. Such a hybrid molecule may contain an amino acid sequence that differs, within 15, for example, by 10 amino acid residues, from the amino acid sequence shown AFTER. No. 2. In order to be useful for the present invention, the hybrid molecule must exhibit IFNG activity.

Initial non-human IFNGs can be modified in the same way as described herein, for example, by modifying the corresponding non-human IFNG position (for example, determined from a comparative analysis of the amino acid sequence or 3D structure of said IFNG and huIFNG), position described here.

It should be borne in mind that the amino acid residue containing a group for joining a non-polypeptide group, either deleted or introduced, is selected based on the nature of the selected non-polypeptide group and, in most cases, on the basis of the conjugation method used. For example, when a non-polypetidic group is a polymer molecule, such as a polyethylene glycol or a molecule derived from polyalkylene oxide, amino acid residues capable of functioning as a group for addition can be selected from the group consisting of cysteine, lysine, aspartic acid, glutamic acid and arginine. When the non-polypeptide group is a sugar group, the attachment group is, for example, an in vivo glycosylation site, preferably an N-glycosylation site.

Whenever it is necessary to introduce a group into the IFNG polypeptide to attach a non-polypeptide group or to remove said group according to the present invention, the position of the modified polypeptide is usually selected as follows.

The position is preferably located on the surface of the IFNG polypeptide and is preferably occupied by an amino acid residue in which more than 25% of its side chain is accessible to the solvent, preferably more than 50% of its side chain is accessible to the solvent, which is determined based on the 3D structure or IFNG model in its dimeric form, the structure or model optionally also containing one or two molecules of the IFNG receptor. Such positions (for example, representing over 25% or over 50% of the available surface on a model with or without receptor molecules) are listed in this description in the Materials and Methods section.

It should also be of interest to modify any of the 23 C-terminal amino acid residues of the original IFNG (by introducing and / or removing amino acid residues containing a group to attach a non-polypeptide group), since such residues are believed to be localized on the surface of the IFNG polypeptide.

In addition, in the IFNG polypeptide portion of the conjugate of the invention, the attachment group located at the IFNG receptor binding site is preferably removed, preferably by replacing an amino acid residue containing such a group. The amino acid residues of the IFNG receptor binding site are identified below in the Materials and Methods section. In the case of a single chain IFNG polypeptide, it may be sufficient to remove groups to attach only one of the monomers at the receptor binding site and thereby produce a conjugate of a single chain IFNG polypeptide with one active and one inactive receptor binding site.

In order to determine the optimal distribution of groups for attachment, the distance between amino acid residues localized on the surface of the IFNG polypeptide is calculated based on the 3D structure of the IFNG dimeric polypeptide. More specifically, the distance between the CB of amino acid residues containing such groups for attachment, or the distance between the functional group (NZ in the case of lysine, CG in the case of aspartic acid, CD in the case of glutamic acid, SG in the case of cysteine) of one and the CB of the other amino acid residue, is determined containing the group to join. In the case of glycine, CA is used instead of CB. In the IFNG polypeptide portion of the conjugate of the invention, any of these distances is preferably more than 8 Å, in particular more than 10 Å, in order to avoid or reduce heterogeneous conjugation.

Also, the amino acid sequence of an IFNG polypeptide may differ from the amino acid sequence of the parent polypeptide in that one or more amino acid residues that are part of the epitope are removed, preferably by replacing it with an amino acid residue containing a group to attach a non-polypeptide group in order to destroy or inactivate epitope. Epitopes huIFNG or rhuIFNG can be identified using methods known in the art, also known as epitope mapping, see, for example, Romagnoli et al., Biol. Chem., 1999, 380 (5): 553-9, DeLisser H. M., Methods Mol. Biol., 1999, 96: 11-20; Van de Water et al., Clin. Immunol. Immunopathol., 1997, 85 (3): 229-35, Saint-Remy J.M., Toxicology, 1997, 119 (1): 77-81 and Lane D.P. and Stephen C.W., Curr. Opin. Immunol., 1993, 5 (2): 268-71. According to one of the methods, it is necessary to establish a phage imaging library expressing randomized oligopeptides from, for example, 9 amino acid residues. Anti-IgGl antibodies from a specific anti-huIFNG or rhuIFNG antiserum are isolated by immunoprecipitation in its pure form, and reactive phages are identified by immunoblotting. DNA sequencing of purified reactive phages can determine the sequence of the oligopeptide, followed by localization of the sequence on the 3D structure of IFNG. Through this, the identified region on the structure constitutes an epitope, which can then be selected as the target region for introducing a group for joining a non-polypeptide group.

In order to avoid too much disruption of the structure and function of the molecule of the starting IFNG, the total number of altered amino acid residues according to the present invention (when compared with the amino acid sequence represented by FOLLOW-UP No. 2) should generally not exceed 15. Preferably, IFNG- the polypeptide contains an amino acid sequence that differs by 1-15 amino acid residues from the amino acid sequence presented after. No. 2, for example, differing by 1-8 or 2-8 amino acid residues, for example by 1-5 or 2-5 amino acid residues, from the amino acid sequence represented by AFTER. No. 2. Thus, as a rule, an IFNG polypeptide contains an amino acid sequence that is different from the mature portion of the amino acid sequence represented by AFTER. No. 2, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues. Preferably, the above numbers represent either the total number of introduced or the total number of deleted amino acid residues containing the group for joining the relevant non-polypeptide group / s, or the total number of introduced and deleted amino acid residues containing the group.

The exact number of attachment groups available for conjugation and present in the IFNG polypeptide in dimeric form depends on the action to be achieved by conjugation. The effect obtained, for example, depends on the nature and degree of conjugation (e.g., the identity of a non-polypeptide group, the number of non-polypeptide groups required for conjugation, or which can conjugate to a polypeptide, where they should be conjugated and where conjugation should be avoided, etc.).

The IFNG polypeptide portion of the conjugate of the invention may be in processed form (for example, with processed 1-15 C-terminal amino acid residues, as described above in connection with the original IFNG polypeptide, or processed 1-3 N-terminal amino acid residues).

The in vivo functional half-life depends, for example, on the molecular weight of the conjugate, and the number of attachment groups required to obtain an increased half-life, thus, depends on the molecular weight of the non-polypeptide group being discussed. In one embodiment, the conjugate of the invention has a molecular weight of at least 67 kDa, in particular at least 70 kDa, as measured by SDS-PAGE according to Laemmli UK, Nature, Vol.227 (1970), p.680- 85. IFNG has a molecular weight in the range of 34-50 kDa, and therefore, approximately 20-40 kDa is still required to obtain the desired effect. This can be achieved, for example, using 2-4 PEG molecules of 10 kDa or otherwise, as described in this description.

Preferably, in the conjugate of the invention, at least about 50% of all conjugated groups for addition, for example, at least 80%, and preferably all such groups, are occupied by relevant non-polypeptide groups. Accordingly, in a preferred embodiment, the conjugate of the invention contains, for example, 1-10 non-polypeptide groups, for example 2-8 or 3-6.

As indicated above, under physiological conditions, IFNG exists as a dimeric polypeptide. According to the invention, the IFNG polypeptide portion of the conjugate of the invention is usually in homodimeric form (for example, obtained by association of two molecules of the IFNG polypeptide obtained as described above). However, if necessary, the IFNG polypeptide portion of the conjugate of the invention can be obtained in the form of a single chain, where two monomers of the IFNG polypeptide are linked via a peptide bond or peptide linker. The preparation of the IFNG polypeptide in single chain form has the advantage that the two constituents of the IFNG polypeptide can be different when it can be beneficial, for example, in order to provide asymmetric mutagenesis of the polypeptides. For example, from the receptor binding site of one of the monomers, you can remove the PEG attachment sites, but save them in another. By means of this, after PEG treatment, one monomer has an intact receptor-binding site, while in the other the complete PEG attachment can occur (and thus provide a substantial increase in molecular weight).

Preferably, the conjugate of the invention has one or more of the following improved properties: 1) increased in vivo half-life compared to hulFNG or rhuIFNG, for example, increased at least about 5 times, for example at least 10 times or even more; 2) reduced immunogenicity compared to hulFNG or rhuIFNG, for example, reduced by at least 25%, for example, by at least 50%, and more preferably by at least 75%.

The conjugate of the invention, where the non-polypeptide group is a sugar group

In a preferred embodiment of the conjugate of the invention, the first non-polypeptide group is a sugar group, for example an O-linked or N-linked sugar group, and the IFNG polypeptide contains at least one deleted and / or at least one introduced in vivo glycosylation site.

For example, an in vivo glycosylation site is introduced at the position of the starting IFNG polypeptide occupied by an amino acid residue facing the surface of the polypeptide, preferably with more than 25% of the side chain available for the solvent, in particular with more than 50% of the side chain available for the solvent (indicated positions are identified in this description in the "Methods" section). The N-glycosylation site is introduced in such a way that the N residue of the specified site is localized at the indicated position. Similarly, the O-glycosylation site is introduced in such a way that the residue S or T constituting such a site is localized at the indicated position. In addition, in order to provide effective glycosylation, it is preferable that the in vivo glycosylation site, in particular the N residue of the N-glycosylation site or the S or T residue of the O-glycosylation site, is located within the 118 N-terminal amino acid residues of the IFNG polypeptide, preferably within 93 N-terminal amino acid residues. Even more preferably, the in vivo glycosylation site is introduced at a position where only one mutation is required to create the site (i.e., where any other amino acid residues required to create the functional glycosylation site are already present in the molecule).

For example, substitutions that lead to the introduction of an additional N-glycosylation site at positions facing the surface of the IFNG polypeptide and occupied by amino acid residues with over 25% of the side chain facing the surface (in the structure with the receptor molecule) include

Q1N + P3S / T, P3N + V5S / T, K6N + A8S / T, E9N + L11S / T, K12S / T, K13N + F15S / T, Y14N + N16S / T, G18S / T, G18N, G18N + S20T, H19N + D21S / T, D21N + A23S / T, G26N + L28S / T, G31N + L33S / T, K34N + W36S / T, K37S / T, K37N + E39S / T, E38N, E38N + S40T, E39N + D41S / T, S40N + R42S / T, K55N + F57S / T, K58N + F60S / T, K61S / T, K61N + D63S / T, D62N + Q64S / T, D63N, D63N + S65T, Q64N + I66S / T, S65N + Q67S / T, Q67N, Q67N + S69T, K68N + V70S / T, E71N + I73S / T, T72N + K74S / T, K74N + D76S / T, E75N + M77S / T, K80S / T, V79N + F81S / T, K80N + F82S / T, N85S / T, S84N + K86S / T, K87S / T, K86N + K88S / T, K87N + R89S / T, D90N + F92S / T, E93N + L95S / T, K94N, K94N + T96S, S99N, S99N + T101S, T101N + L103S / T, D102N + N104S / T, L103N + V105S / T, Q106S / T, E119N, E119N + S121T, P122N + A124S / T, A123N + K125S / A, A4 T126S, K125N + G127S / T, T126N + K128S / T, G127N + R129S / T, K128N + K130S / T, R129N + R131S / T, K130N, K130N + S132T, R131N + Q133S / T, S132N + M Q133N + L135S / T, M134N + F136S / T, L135N + R137S / T, F136N + G138S / T, R137N + R139S / T, G138N + R140S / T, R139N + A141S / T, R140N and R140N + S142T rel huIFNG with respect to the amino acid sequence represented by SEQ. No. 2. S / T shows a substitution for a serine or threonine residue, preferably a threonine residue.

Substitutions that lead to the introduction of an additional N-glycosylation site at positions facing the surface of the IFNG polypeptide with over 50% of the side chain facing the surface (in the structure with the receptor molecule) include P3N + V5S / T, K6N + A8S / T, K12S / T, K13N + F15S / T, G18S / T, D21N + A23S / T, G26N + L28S / T, G31N + L33S / T, K34N + W36S / T, K37N + E39S / T, E38N, E38N + S40S / T, E39N + D41S / T, K55N + F57S / T, K58N + P60S / T, K61S / T, D62N + Q64S / T, Q64N + I66S / T, S65N + Q67S / T, K68N + V70S / T, E71N + I73S / T, E75N + M77S / T, N85S / T, S84N + K86S / T, K86N + K88S / T, K87N + R89S / T, K94N, K94N + T96S, S99N, S99N + T101S, T101N + L103S / T, D102N + N104S / T, L103N + V105S / T, Q106S / T, P122N + A124S / T, A123N + K125S / T, A124N, A124N + T126S, K125N + G127S / T, T126N + K128S / T, G R129S / T, K128N + K130S / T, R129N + R131S / T, K130N, K130N + S132T, R131N + Q133S / T, S132N + M134S / T, Q133N + L1 35S / T, M134N + F136S / T, L135N + R137S / T, F136N + G138S / T, R137N + R139S / T, G138N + R140S / T, R139N + A141S / T, R140N and R140N + S142T, with replacements indicated relative to huIFNG with the amino acid sequence represented by AFTER. No. 2.

Substitutions where only one mutation is required in order to introduce an N-glycosylation site include K12S / T, G18S / T, G18N, K37S / T, E38N, M45N, I49N, K61S / T, D63N, Q67N, V70N, K80S / T, F82N, N85S / T, K87S / T, K94N, S99N, Q106S / T, E119N, A124N, K130N and R140N, in particular K12S / T, G18N, G18S / T, K37S / T, E38N, K61S / T, D63N, Q67N, K80S / T, N85S / T, K94N, S99N, Q106S / T, A124N, K130N and R140N (positions with over 25% of their side chain facing the surface (in the structure with no receptor molecule) or, more preferably, G18N, E38N, D63N, Q67N, K94N, S99N, A124N, K130N and R140N (with over 50% of its side chain facing the surface in a structure with no receptor molecule).

From the above list of substitutions, it is preferable to choose substitutions localized within 118 N-terminal amino acid residues, in particular within 93 N-terminal amino acid residues.

As indicated above, in addition to one or more of the introduced glycosylation sites, existing glycosylation sites can be deleted from the IFNG polypeptide. For example, any of the above substitutions for introducing a glycosylation site can be combined with a substitution to remove either of the two natural IFNG N-glycosylation sites. For example, an IFNG polypeptide may comprise an N25 and / or N97 substitution, for example, one of the N25K / C / D / E and / or N97K / C / D / E substitutions if the conjugate of the invention contains a non-polypeptide group, wherein the polypeptide contains as a group for attaching relevant K, C, D, E.

The IFNG polypeptide portion of the conjugate of the invention may contain one in vivo glycosylation site per monomer. However, in order to become large enough to increase the functional half-life, it is often desirable that the polypeptide contains more than one in vivo glycosylation site, in particular 2-7 in vivo glycosylation sites, for example 2, 3, 4, 5, 6, or 7 in vivo glycosylation sites. Thus, the IFNG polypeptide may contain one additional glycosylation site per monomer or may contain two, three, four, five, six, seven or more introduced in vivo glycosylation sites, preferably introduced using one or more substitutions listed in any of the above listings.

Removal and / or administration of an in vitro glycosylation site can be achieved as described in the following sections in connection with the modification of the IFNG polypeptide for introduction and / or removal of polymer attachment sites.

Any of the glycosylated IFNG polypeptides described in this section having at least one glycosylation site inserted and / or deleted can then be conjugated to another non-polypeptide group. For example, another non-polypeptide group is a polymer molecule, such as PEG / or any other non-polypeptide group. For this purpose, conjugation can be achieved using the attachment groups already in the IFNG polypeptide, or the attachment groups can be introduced and / or removed, in particular so that 1-6 are generally available for conjugation, in particular 3-4 or 1, 2, 3, 4, 5 or 6 groups to join. Preferably, in the conjugate of the invention, where the IFNG polypeptide contains two glycosylation sites, the number and molecular weight of the non-polypeptide groups are chosen so that the total molecular weight added by the non-polypeptide group is in the range of 20-40 kDa, in particular about 20 kDa or 30 kDa .

In particular, a glycosylated IFNG polypeptide can be conjugated to a polymer having cysteine as an attachment group. For this purpose, one or more cysteine residues are inserted into an IFNG polypeptide, for example, as described in the section entitled “Conjugate of the invention, where the non-polypeptide group is a molecule containing cysteine as an attachment group”.

Alternatively or in addition, the glycosylated IFNG polypeptide can be conjugated to a polymer having lysine as an attachment group. For this purpose, one or more lysine residues of the parent polypeptide can be removed, for example, by any of the substitutions indicated in the section entitled “Conjugate of the invention, where the non-polypeptide group is a molecule containing lysine as an attachment group”. On the other hand, or in addition, the lysine residue can be introduced, for example, using one or more substitutions mentioned in this section.

As an alternative to conjugation with a polymer through a cysteine or lysine group, conjugation can be achieved through an acid group, as described in the section entitled “Conjugate of the invention, where a non-polypeptide group binds to an acid group”, or via any other suitable group.

The conjugate of the invention, where the first non-polypeptide group is a polymer

Alternatively, the first non-polypeptide group is a polymer, for example, any of those described in the section entitled "Conjugation with a polymer molecule", in particular a linear or branched PEG molecule, for example, with cysteine, lysine, aspartic acid and glutamic acid as an attachment group . The introduction and / or removal of a group for addition of such a polymer is illustrated in the following sections. The IFNG polypeptide portion of the conjugate of this embodiment may be a glycosylated polypeptide, for example, where one or both of the natural huIFNG N-glycosylation sites or the introduced glycosylation site are used, as described in the previous section.

The conjugate of the invention, where the non-polypeptide group is a molecule with cysteine as a group to join

In a preferred embodiment, the first non-polypeptide group is a polymer for which cysteine acts as an attachment group, and at least one cysteine residue is introduced at the position of an IFNG polypeptide, which in the wild-type human IFNG is a surface-facing amino acid residue. Preferably, the cysteine residue is administered according to the general concepts of introducing and / or removing groups for joining non-polypeptide groups described in the section entitled "Conjugate of the invention." For example, an IFNG polypeptide may contain at least one substitution selected from the group consisting of P3C, K6C, N10C, K13C, N16C, D21C, N25C, G26C, G31C, K34C, K37C, E38C, E39C, K55C, K58C , N59C, D62C, Q64C, S65C, K68C, E71C, E75C, N83C, S84C, K86C, K87C, K94C, N97C, S99C, T101C, D102C, L103C and N104C (introduction of a cysteine residue in the position occupied by an amino acid residue that 50% of its side chain faces the surface in a receptor structure). Substitutions of N25C and N97C are of particular interest, especially N25C + N97C, when the IFNG polypeptide is expressed in a non-glycosylating host cell such as E. coli, since N25C and N97C form part of the inherent huIFNG glycosylation site.

Also, or on the other hand, the IFNG polypeptide of this embodiment of the invention may contain at least one cysteine residue inserted at the position occupied by any of amino acid residues 121-143 of hulFNG.

Preferably, the conjugate IFNG polypeptide according to this aspect contains a total of 1-8, for example 2-6 Cys residues, for example 1-3 Cys residues, per monomer.

The conjugation between the polypeptide and the polymer can be achieved by any suitable method, for example, as described in the section entitled "Conjugation with the polymer molecule", for example, using the single-stage method or the two-stage method described in this section. When the conjugate contains two or more of the first non-polypeptide groups, as a rule, each of them has a molecular weight of 5 or 10 kDa. A suitable polymer is VS-PEG.

The conjugate of the invention, where the non-polypeptide group is a molecule with lysine as a group to join

According to this embodiment, the non-polypeptide group is a polymer with lysine as an attachment group, and the IFNG polypeptide is modified so that at least one lysine residue is removed, and the lysine residue is selected from the group consisting of K6, K12 , K13, K34, K37, K43, K55, K61, K68, K74, K80, K86, K87, K88, K94, K108, K125, K128 and K130, and the numbering is relative to LAST. No. 2. More preferably, at least one lysine residue selected from the group consisting of K12, K34, K37, K108, K128 and K130 is removed. By this, conjugation of such / such residues can be avoided. The lysine (s) residue (s) can be replaced with any other amino acid residue, but it is preferable to replace it with arginine or glutamine.

In addition, the IFNG polypeptide can be modified to introduce one or more lysine residues, in particular, at the position of the IFNG polypeptide occupied by the surface-facing amino acid residue. Preferably, the lysine residue is introduced according to the general concepts of the introduction and / or removal of groups for joining a non-polypeptide group given in the section entitled "Conjugate of the invention", in particular, in the position occupied by an amino acid residue with at least 25%, for example at least 50% of its side chain facing the surface (moreover, such positions are identified in the "Materials and Methods" section of this description). Also, at least one lysine residue can be introduced by replacing any of amino acid residues 121-143 AFTER. No. 2. Alternatively, the IFNG polypeptide may contain lysine in at least one position selected from the group consisting of D2, E7, E9, H19, D21, D24, N25, E38, E39, D41, R42, D62, D63 , E71, E75, D76, R89, D90, D91, E93, N97, R107, H111, E112, E119, R129, R131, R137, R139 and R140 FOLLOW. No. 2 (positions taken by the remainder of N, R, D, E or H in huIFNG).

According to this embodiment, the IFNG polypeptide comprises a substitution in one or more of the above positions, in particular 1-15, for example 1-8 or 2-8, preferably in 1-5 or 2-5 positions (removal and / or introduction of residues lysine) in the monomer. For example, an IFNG polypeptide may comprise a substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the above positions. Substitutions of N25K and N97K are of particular interest, especially N25K + N97K, when the IFNG polypeptide is expressed in a non-glycosylating host cell such as E. coli, since N25K and N97K form part of the inherent hulFNG glycosylation site.

For example, the conjugate IFNG polypeptide of the invention according to this embodiment may contain at least one of the above substitutions for introducing a lysine residue in combination with at least one substitution that removes the lysine residue as described above (preferably, a substitution for R or Q), for example, an IFNG polypeptide contains at least one of the substitutions N25K and N97K in combination with at least one substitution K128R, K128Q, K130R or K130Q. More specifically, the IFNG polypeptide contains the substitution N25K + K128R, N25K + K130R, N25K + K128R + K130R, N97K + K128R, N97K + K130R, N97K + K128R + K130R, N25K + N97K + K128R + K130R, N25K N25K + N97K + K130R.

Despite the fact that the non-polypeptide group of the conjugate according to this aspect of the invention can be any molecule for which, using this conjugation method, lysine acts as a group for attachment (such as a sugar group, lipophilic group or an organic derivatizing agent), preferably, the non-polypeptide group is a polymer molecule. The polymer molecule may be any of the molecules indicated in the section entitled "Conjugation with the polymer molecule", but preferably selected from the group consisting of linear or branched polyethylene glycol or polyalkylene oxide. Most preferably, the polymer molecule is SS-PEG, NPC-PEG, aldehyde-PEG, mPEG-SPA, mPEG-SCM or mPEG-BTC from Shearwater Polymers Inc., SC-PEG from Enzon Inc., trisylated mPEG described in US 5880255, or hydroxycarbonyloxy-N-dicarboximide-PEG (US 5122614). Typically, for conjugation to a lysine residue, the non-polypeptide group has an MM of about 5 or 10 kDa.

Conjugate of the invention wherein the non-polypeptide group binds to an acid group

In yet another embodiment, the non-polypeptide group of the conjugate of the invention is a molecule for which an acid group acts as an attachment group, and the IFNG polypeptide contains an amino acid sequence different from the amino acid sequence shown in SEQ. No. 2, in that at least one surface-facing amino acid residue is replaced by an aspartic acid residue or a glutamic acid residue, preferably according to the general concepts given in the section entitled “Conjugate of the invention”.

Alternatively, an Asp or Glu residue can be introduced at the position of the original IFNG polypeptide occupied by K, R, Q or N. For example, N25, N97, K125, K128, R129, K130 and / or R131 can be replaced with an Asp or Glu residue. more preferably N25 and / or N97, most preferably N25 + N97.

As described in the previous sections, one or more Asp or Glu residues can be removed, for example, from the receptor-binding site in the case where the non-polypeptide group is a group that binds to these residues.

Despite the fact that the non-polypeptide group of the conjugate, according to the specified aspect of the invention, which has an acid group as a group for attachment, can be any non-polypeptide group with this property, it is currently preferred that the non-polypeptide group is a polymer molecule or an organic derivatizing agent , in particular a polymer molecule, and a conjugate are prepared, for example, as described in Sakane and Pardridge, Pharmaceutical Research, Vol.14, No.8, 1997, pp.1085-1091.

Non-polypeptide group of the conjugate of the invention

As mentioned above, the non-polypeptide group of the conjugate of the invention is preferably selected from the group consisting of a polymer molecule, a lipophilic compound, a sugar group (for example, by in vivo glycosylation) and an organic derivatizing agent. All of these agents can confer the desired properties to the polypeptide portion of the conjugate, in particular, increased in vivo functional lifetime and / or reduced immunogenicity. The polypeptide portion of the conjugate is usually conjugated to only one type of non-polypeptide group, but it can also be conjugated to two or more different types of non-polypeptide groups, for example, a polymer molecule and a sugar group, a lipophilic group and a sugar group, with an organic derivatizing agent and a sugar group, with a lipophilic group and a polymer molecule, and the like. Conjugation with two or more different non-polypeptide groups can be performed simultaneously or sequentially.

Methods for producing the conjugate of the invention

The following sections, Conjugation with a Lipophilic Compound, Conjugation with a Polymer Molecule, Conjugation with a Sugar Group, and Conjugation with an Organic Derivatizing Agent, describe conjugation with specific groups of non-polypeptide groups.

Lipophilic conjugation

The polypeptide and lipophilic compound can be conjugated to each other either directly or using a linker. The lipophilic compound may be a malfunction of a natural compound such as saturated or unsaturated fatty acid, fatty acid diketone, terpene, prostaglandin, vitamin, carotenoid or steroid, or a synthetic compound such as carboxylic acid, alcohol, amine and sulfonic acid, with one or more alkyl, aryl, alkenyl or other compounds with several unsaturations. The conjugation between the polypeptide and the lipophilic compound, optionally via a linker, can be carried out according to methods known in the art, for example, as described in Bodanzky, Peptide Synthesis, John Wiley, New York, 1976, and in WO 96/12505.

Conjugation with a polymer molecule

The polymer molecule to be combined with the polypeptide can be any suitable polymer molecule, such as a natural or synthetic homopolymer or heteropolymer, typically with a molecular weight in the range of 300-100000 Yes, for example 300-20000 Yes, more preferably in the range of 500-10000 Yes and even more preferably in the range of 500-5000 Yes.

Examples of homopolymers are a polyol (i.e., a poly-OH compound), a polyamine (i.e., a poly-NH ₂ compound) and a polycarboxylic acid (i.e., a poly-COOH compound). A heteropolymer is a polymer containing one or more different combining groups, for example a hydroxyl group and an amino group.

Examples of suitable polymer molecules are polymer molecules selected from the group consisting of polyalkylene oxide (PAO), including polyalkylene glycol (PAG) such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEG, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolide polyethylene with maleic anhydride, polystyrene with maleic anhydride, dextran, including carboxymethyldextran, or any other biopolymer suitable to reduce immunogenicity and / or increase the functional half-life or in vivo and / or half-life in the bloodstream. Another example of a polymer molecule is human albumin or another common plasma protein. As a rule, polymers derived from polyalkylene glycols are biocompatible, non-toxic, non-antigenic, non-immunogenic, have different solubility in water and are easily excreted from living organisms.

PEG is the preferred polymer molecule used, since it has only a few reactive groups capable of crosslinking, compared, for example, with polysaccharides such as dextran and the like. In particular, monofunctional PEG is of interest, i.e. monomethoxypolyethylene glycol (mPEG), since its combination chemistry is relatively simple (only one reactive group is available for conjugation with groups for attachment in the polypeptide). Therefore, the danger of crosslinking is eliminated, the resulting polypeptide conjugates are more uniform, and it is easier to control the interaction of polymer molecules with the polypeptide.

In order to covalently attach the polymer molecule (s) to the polypeptide, the hydroxyl end groups of the polymer molecules must have an activated form, i.e. have reactive functional groups (examples of which are primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl propionate (SC), succinimidylcarbonate (SC), hydroxysuccinimide (NHS), aldehyde, nitrophenyl carbonate (NPC) and trasylate (TRES) groups). Suitably activated polymer molecules are commercially available, for example, from Shearwater Polymers Inc., Huntsville, Alabama, USA. On the other hand, polymer molecules can be activated by conventional methods known in the art, for example, as described in WO 90/13540. Specific examples of activated linear or branched polymer molecules for use in the present invention are provided in the Shearwater Polymers Inc. catalogs. for 1997 and 2000 (Functionalized Biocompatible Polymers for Research and Pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers are the following linear PEGs: NHS-PEG (e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG and SCM-PEG) and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG and MAL-PEG, and branched PEGs such as PEG2-NHS, and PEG, described in US 5932462 and US 5643575, which are both incorporated into this description by reference. In addition, the publications listed below, incorporated by reference in this description, describe useful polymer molecules and / or PEG processing chemistry: US 5824778, US 5476653, WO 97/32607, EP 229108, EP 402378, US 4902502, US 5281698 , US 5122614, US 5219564, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, W095 / 13090 , WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466 , WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, US 5736625, WO 98/05363, EP 809 996, US 5629384, WO 96/41813, WO 96 / 07670, US 5473034, US 5516673, EP 605963, US 5382657, EP 510356, EP 400472, EP 183503 and EP 154316.

The conjugation of polypeptides and activated polymer molecules is carried out using a conventional method, i.e. as described in the following references (which are also incorporated herein by reference): Harris and Zaiipsky, eds., Poly (ethylene glycol) Chemistry and Biological Applications, AZC, Washington; R.F. Taylor (1991), "Protein immobilization. Fundamenyal and applications". Marcel Dekker, N.Y .; S. S. Wong (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; G. T. Hermanson et al. (1993), "Immobilized Affinity Ligand Techniques", Academic Press. N.Y.). It is known to those skilled in the art that the activation method and / or conjugation chemistry used depends on the group (s) to be attached to the IFNG polypeptide, as well as on the functional groups of the polymer (for example, amino, hydroxyl, aldehyde group or sulfhydryl). PEG treatment may be directed to conjugation to all available groups for attachment to the polypeptide (i.e., such attachment groups that face the surface of the polypeptide) or may be directed to specific groups for attachment, for example, the N-terminal amino group (US 5985265 ) In addition, conjugation can be achieved in one stage or in stages (for example, as described in WO 99/55377).

It should be borne in mind that the addition of PEG is planned in such a way as to obtain the optimal molecule in terms of the number of attached PEG molecules, size and shape (for example, whether they will be linear or branched) of such molecules and where such molecules are attached to the polypeptide. For example, the molecular weight of the polymer used can be selected based on the desired achieved effect. For example, if the main purpose of conjugation is to obtain a conjugate with a high molecular weight (for example, to reduce renal clearance), it is generally desirable to attach as few polymer molecules with high MM as possible to obtain the desired molecular weight. When a high degree of epitope screening is desired, this effect can be obtained by using a sufficiently large number of low molecular weight polymers (e.g., with a molecular weight of about 5000 Da) to effectively screen all or most of the epitopes of the polypeptide. For example, 2-8, for example 3-6, of such polymers can be used.

Due to conjugation of only one group for attachment to a protein (as described in US Pat. No. 5,985,265), it may be advantageous for the polymer molecule of a linear or branched polymer to have a high molecular weight, for example about 20 kDa.

Typically, the conjugation of the polymer is carried out under conditions conducive to the interaction of all possible groups for the addition of the polymer with polymer molecules. As a rule, the molecular ratio of activated polymer molecules to the polypeptide is 1000-1, in particular 200-1, preferably 100-1, for example 10-1 or 5-1, in order to obtain the optimal interaction. However, equimolecular ratios can also be used.

According to the invention, it is also contemplated to combine polymer molecules with a polypeptide via a linker. Suitable linkers are well known in the art. A preferred example is cyanuric acid chloride (Abuchowski et al. (1977), J. Biol. Chem., 252, 3578-3581; US 4179337; Shafer et al. (1986), J. Polym. Sci. Chem., Ed., 24, 375-378).

After conjugation, the remaining activated polymer molecules are blocked according to methods known in the art, for example by adding a primary amine to the reaction mixture, and the resulting inactivated polymer molecules are removed in a suitable manner.

Sugar group

The addition of a sugar group can occur in vivo or in vitro. In order to achieve glycosylation of an in vivo polypeptide with IFNG activity modified to introduce one or more in vivo glycosylation sites (see section "Conjugates of the invention where the non-polypeptide group is a sugar group"), the nucleotide sequence encoding the polypeptide part conjugate should be integrated into a glycosylating eukaryotic expressing host. The expressing host cell can be selected among fungal cells (hyphomycetes or yeast), insects or animals, or among transgenic plant cells. In addition, glycosylation can be achieved in humans using the nucleotide sequence encoding the polypeptide portion of the conjugate of the invention or the polypeptide of the invention in gene therapy. In one embodiment, the host cell is a mammalian cell, such as a CHO cell, a BHK or HEK cell, for example HEK293, or an insect cell, such as an SF9 cell, or a yeast cell, for example a Saccharomyces cerevisae cell, Pichia pastoris , or any other suitable glycosylation host, for example, from those also described below. Optionally, sugar groups attached to the IFNG polypeptide via in vivo glycosylation are also modified using glycosyl transferases, for example, using glycoAdvanct ™ technology sold by Neose, Horsman, PA, USA. By this means, for example, sialylation of the glycosylated IFNG polypeptide after expression and glycosylation in vivo by CHO cells can be increased.

In vitro covalent interaction of glycosides with amino acid residues of IFNG can be used to modify or increase the number or profile of carbohydrate substituents. Depending on the coupling method used, sugar (a) may (gut) attach to a) arginine and histidine, b) free carboxyl groups, c) free sulfhydryl groups, such as cysteine groups, d) free hydroxyl groups, such as serine groups , threonine, tyrosine or hydroxyproline, e) aromatic residues such as phenylalanine or tryptophan residues, or f) the amide group of glutamine. These amino acid residues are examples of groups for addition of a sugar group that can be introduced and / or removed from the IFNG polypeptide of the conjugate of the invention. Suitable in vitro coupling methods are described, for example, in WO 87/05330 and in Aplin et al., CRC Crit. Rev. Biochem., Pp. 259-306, 1981. The in vitro combination of sugar groups or PEG with protein and peptide-linked GLn residues can also be carried out using transglutaminases (TGases), for example, as described in Sato et al., 1996, Biochemistry, 35, 13072-13080, or in EP 725145.

Combination with an Organic Derivative Agent

Covalent modification of an IFNG polypeptide can be accomplished by reacting a group (s) of a polypeptide to attach to an organic derivatizing agent. Suitable derivatizing agents and methods are well known in the art. For example, cysteinyl residues are most often reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to form carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β- (4-imidosoyl) propionic acid, chloroacetyl phosphate, N-alkyl maleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-2-pyridyl disulfide, p-2 chloro-mercury-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-di-azoll. Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0, since this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the interaction is preferably carried out in a 0.1 M solution of sodium cacodylate at pH 6.0. Lysinyl and amino-terminal residues are reacted with succinic anhydride or other carboxylic acid anhydrides. Derivatization by these agents has the effect of reversing the load of lysinyl residues. Other suitable reagents for the derivatization of α-amino residues are imido esters such as methyl picolinimidate; pyridoxalphosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentane-dione and glyoxylate in a transaminase-catalyzed reaction. Arginyl residues are modified by reaction with one or more conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residues requires that the interaction be carried out in an alkaline environment due to the high pKa of the guanidine functional group. In addition, such reagents can interact with lysine groups as well as an arginine guanidino group. Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (RN = C = N = R ′), where R and R ′ are different alkyl groups such as 1-cyclohexyl-3- (2-morpholinyl-4- ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylphenyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to aspartic and glutaminyl residues by reaction with ammonium ions.

Functional site blocking

There are reports that excessive conjugation with the polymer can lead to a loss of activity of the polypeptide with which the polymer is conjugated. This problem can be eliminated, for example, by deleting groups for joining, located in the functional site, or by blocking the functional site before conjugation. These latter strategies constitute other embodiments of the invention: examples of the first strategy are given above, for example, the removal of lysine residues that may be located near a functional site. According to a second strategy, conjugation between a polypeptide and a non-polypeptide group is performed under conditions where the functional site of the IFNG polypeptide is blocked by a helper molecule capable of binding to the functional site of the polypeptide. Preferably, the helper molecule is a molecule that specifically recognizes a functional site of a polypeptide, such as a receptor. Alternatively, the helper molecule may be an antibody, in particular a monoclonal antibody recognizing a polypeptide exhibiting IFNG activity. In particular, the helper molecule may be a neutralizing monoclonal antibody.

The polypeptide is introduced into interaction with a helper molecule prior to conjugation. This ensures that the functional site of the polypeptide is shielded or protected and therefore not accessible for derivatization by a non-polypeptide group such as a polymer. After being released from the helper molecule, the conjugate between the non-polypeptide group and the polypeptide can be removed with at least partially preserved functional site.

Subsequent conjugation of the polypeptide with a functional site blocked for the polymer, lipophilic compound, sugar group, organic derivatizing agent or any other compound is carried out in the usual way, for example, as described above in the sections "Conjugation with ...".

In another embodiment, the helper molecule is first covalently attached to a solid phase, such as column packing materials, for example, Sephadex or agarose granules, or the surface of, for example, a reactor. Then, a polypeptide is loaded into the column material containing the helper molecule, and conjugation is carried out according to methods known in the art, for example, as described above in the sections "Conjugation with ...". This procedure allows you to separate the conjugate of the polypeptide from the helper molecule by elution. The polypeptide conjugate is eluted by conventional methods under physicochemical conditions that do not substantially degrade the polypeptide conjugate. The liquid phase containing the conjugate of the polypeptide is separated from the solid phase on which the helper molecule remains, bound covalently to it. Separation can be achieved in many ways. For example, a helper molecule can be derivatized with a second molecule (e.g., biotin) that a specific binding agent (e.g., streptavidin) can recognize. A specific binding agent can be attached to the solid phase, whereby it is possible to separate the conjugate of the polypeptide from the complex of the helper molecule with the second molecule by passing through the second column with the helper on the solid phase, where the complex of the helper molecule with the second molecule will be retained during subsequent elution, but the conjugate of the polypeptide will not be retained. The conjugate of the polypeptide can be freed from the helper molecule in any suitable manner. Removal of the protective group can be achieved by creating conditions under which the helper molecule is separated from the functional site of the IFNG with which it is associated. For example, the complex of the antibody with which the polymer conjugates and the anti-idiotypic antibody can be decomposed by adjusting the pH to acidic or alkaline.

Conjugation of labeled polypeptide

In another embodiment, the IFNG polypeptide is expressed as a tagged fusion protein, i.e. amino acid sequence or peptide chain, composed, as a rule, of 1-30, for example 1-20, amino acid residues. Besides the possibility of quick and easy purification, the label is a convenient tool for achieving conjugation between the labeled IFNG polypeptide and the non-polypeptide group. In particular, the label can be used to achieve conjugation on microtiter plates or other carriers, such as paramagnetic beads, on which the labeled polypeptide can be immobilized using the label. Conjugation with a labeled IFNG polypeptide has the advantage that the labeled polypeptide can be immobilized on microplates directly from the culture broth (in principle, without any purification) and conjugated. Through this, the total number of process steps (from expression to conjugation) can be reduced. In addition, the label can function as a spacer molecule, providing improved accessibility to immobilized polypeptide for conjugation. Conjugation using a labeled polypeptide can be carried out with any of the non-polypeptide groups described herein, for example, with a polymer molecule such as PEG.

The features of the particular label used are not critical as long as the label is capable of being expressed with the polypeptide and is capable of immobilizing on a suitable surface or carrier material. A number of suitable labels are commercially available, for example, from Unizyme Laboratories, Denmark. For example, a label may be any sequence of sequences

His-His-His-His-His-His-His;

Met-Lys-His-His-His-His-His-His-His;

Met-Lys-His-His-Ala-His-His-Gln-His-His;

Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln

(all available from Unizyme Laboratories, Denmark)

or may be the following:

EQKLI SEEDL (C-terminal label described in Mol. Cell. Biol., 5: 3610-16, 1985);

DYKDDDDK (C- or N-terminal label);

YPYDVPDYA.

Antibodies against the above labels are commercially available, for example, from ADI, Aves Lab and Research Diagnostics.

A convenient method using a labeled polypeptide for processing PEG is given below in the Materials and Methods section.

Subsequent cleavage of the label from the polypeptide can be achieved using commercially available enzymes.

Polypeptides of the Invention

In another aspect, the invention relates to the generally novel IFNG polypeptides described herein. New polypeptides are important intermediates for the preparation of a conjugate of the invention. In addition, the polypeptides themselves may have interesting properties.

For example, a new IFNG polypeptide contains at least one substitution for K, R, D, E, C, S, T or N of an amino acid residue facing the surface, which is described in more detail in the previous part of the application.

Methods for producing IFNG polypeptide

The IFNG polypeptide, optionally, in glycosylated form can be obtained by any suitable method known in the art. Such methods include constructing a nucleotide sequence encoding a polypeptide and expressing the sequence in a suitable transformed or transfected host. However, the polypeptide of the invention can be obtained, although less efficiently, by chemical synthesis or a combination of chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.

The nucleotide sequence of the invention encoding an IFNG polypeptide (in monomeric or single-stranded form) can be constructed by isolating or synthesizing a nucleotide sequence encoding the original IFNG, such as huIFNG with the amino acid sequence SEQUENTIAL. No. 2, and then changing the nucleotide sequence in such a way as to implement the introduction (i.e., insertion or replacement) or deletion (i.e., removal or replacement) of the relevant amino acid (s) residue (s).

The nucleotide sequence is conveniently modified by site-directed mutagenesis according to well-known methods, see, for example, Mark et al., "Site-specific Mutagenesis of the Human Fibroblast Interferon Gene", Proc. Natl. Acad. Sci. USA 81, pp. 5662-66 (1984); and US 4,588,585.

On the other hand, the nucleotide sequence is obtained by chemical synthesis, for example, using an oligonucleotide synthesizer, where oligonucleotides are created based on the amino acid sequence of the desired polypeptide and, preferably, selecting those codons that are preferred in the host cell where the recombinant polypeptide will be produced. For example, you can synthesize several small oligonucleotides encoding parts of the desired polypeptide, and assemble by PCR, ligation or ligation chain reaction (LCR). Individual nucleotides typically contain 5'- or 3'-protruding ends for complementary assembly.

Immediately after assembly (by synthesis, site-directed mutagenesis or another method), the nucleotide sequence encoding the polypeptide is inserted into a recombinant vector and operably linked to the regulatory sequences necessary for the expression of IFNG in the desired transformed host cell.

Of course, it should be borne in mind that not all vectors and expression control sequences function equally well for the expression of a nucleotide sequence encoding an IFNG polypeptide described herein. And not all hosts will function equally well with the same expression systems. However, a person skilled in the art can make a choice among these vectors that regulate the expression of sequences and hosts without undue experimentation. For example, when choosing a vector, the host should be evaluated, since the vector must replicate in it or be able to integrate into the chromosome. The number of copies of the vector, the ability to regulate this number of copies, and the expression of any other proteins encoded by the vector, such as antibiotic markers, must also be taken into account. When choosing an expression control sequence, various factors should also be considered. These include, for example, the relative stability of the sequence, its controllability and its compatibility with the nucleotide sequence encoding the polypeptide, especially when considering potential secondary structures. Hosts should be selected based on their compatibility with the selected vector, the toxicity of the product encoded by the nucleotide sequence, their secretion characteristics, their ability to fold the polypeptide correctly, their fermentation, or culture requirements and ease of purification of products encoded by the nucleotide sequence.

The recombinant vector may be an autonomously replicating vector, i.e. a vector existing as an extrachromosomal entity whose replication is independent of chromosomal replication, for example, a plasmid. On the other hand, said vector is a vector which, when introduced into the host cell, integrates into the genome of the host cell and replicates together with the chromosome (s) into which it is integrated.

The vector is preferably an expression vector in which the nucleotide sequence encoding the IFNG polypeptide is operably linked to additional segments necessary for transcription of the nucleotide sequence. A vector is usually derived from a plasmid or viral DNA. Many suitable expression vectors for expression in the host cells described herein are commercially available or are described in the literature. Useful expression vectors for eukaryotic hosts are, for example, vectors containing expression regulatory sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Specific vectors are, for example, pCDNA3.1 (+) \ Hyg (Invitrogen, Carlsbad, California, USA) and pCI-neo (Stratagene, La Jola, California, USA). Useful expression vectors for bacterial hosts are known bacterial plasmids, such as those from E. coli, including pBR322, pET3a and pET12a (both from Novagen Inc., Wisconsin, USA), plasmids for a wider host range, such as RP4, phage DNAs, for example, numerous derivatives of lambda phage, for example NM989, and other phages with DNA, such as M13, and filamentous phages with single-stranded DNA. Useful expression vectors for yeast cells are plasmid 2μ and its derivatives, vector POT1 (US 4931373), vector pJS037 described in Okkels, Ann. New York Acad. Sci., 782, 202-207 (1996), and pPICZ A, B or C (Invitrogen). Useful vectors for insect cells are pVL941, pBG311 (Cate et al., "Isolation of the Bovine and Human Genes for Mullerian Inhibiting Substance and Expression of the Human Gene In Animal Cells", Cell, 45, pp. 585-998 (1986) , pBluebac 4.5 and pMelbac (both available from Invitrogen).

Other vectors for use in this invention include vectors that allow amplification of the nucleotide sequence encoding an IFNG polypeptide, in number of copies. Such amplifiable vectors are known in the art. These include, for example, vectors capable of amplification by amplification with DHFR (see, for example, Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression ", Mol. Cell. Biol., 2, pp. 1304-19 (1982)) and glutamine synthetase amplification method (" GS ") (see, for example, US 5122464 and EP 338841).

The recombinant vector may also contain a DNA sequence that allows the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of replication. When the host cell is a yeast cell, suitable sequences allowing the vector to replicate are the replication genes of the yeast plasmid 2μ REP 1-3 and the origin of replication.

The vector may also contain a selectable marker, for example a gene whose product corresponds to a defect in the host cell, such as a gene encoding dihydrofolate reductase (DHFR) or the TPI gene Schizosaccharomyces pombe (described by PRRussell, Gene, 40, 1985, pp. 125-130 ), or a gene that confers resistance to a drug, for example, ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate. In the case of hyphomycetes, selectable markers include amds , pyrG, arcB, naiD , sC .

The term "regulatory sequence" in this description is defined as including all components necessary or beneficial for the expression of the IFNG polypeptide. Each regulatory sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. Such regulatory sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, an enhancer or an activating sequence, a signal peptide sequence and a transcription terminator. At a minimum, regulatory sequences include a promoter.

A variety of expression regulatory sequences can be used in the present invention. Such useful regulatory expression sequences include expression regulatory sequences associated with the structural genes of the above expression vectors, as well as any sequence known as regulating the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

Examples of suitable regulatory expression sequences for controlling transcription in mammalian cells are the early and late promoters of SV40 and adenovirus, for example, the main late promoter of adenovirus 2, the MT-1 promoter (metallothionein gene), the immediate early promoter of human cytomegalovirus (CMV), the elongation factor promoter 1α (EF-1α) human, Drosophila minimal heat shock protein promoter 70, Routh sarcoma virus (RSV) promoter, human ubiquitin C promoter (UdC), human growth hormone terminator, polyad region signals the generation of SV40 or Elb adenovirus and the Kozak consensus sequence (Kozak M., J. Mol. Biol., 1987, Aug. 20; 196 (4): 947-50).

In order to improve expression in mammalian cells, a synthetic intron can be inserted into the 5'-untranslated region of the nucleotide sequence encoding the IFNG polypeptide. An example of a synthetic intron is the synthetic intron from pCI-Neo plasmid (available from Promega Corporation, Wisconsin, USA).

Examples of suitable regulatory sequences for controlling transcription in insect cells are the polyhydrin promoter, the P10 promoter, the Autographa californica polyhedrosis virus main protein promoter, the baculovirus immediate 1 gene promoter, and the baculovirus 39K delayed early gene promoter and the SV40 polyadenylation sequence.

Examples of suitable regulatory sequences for use in yeast host cells are the α-cross yeast system, the yeast triose phosphate isomerase (TPI) promoter, the promoters from yeast glycolytic or alcohol dehydrogenase genes, the ADH2-4c promoter, and the inducible GAL promoter.

Examples of suitable regulatory sequences for use in hyphomycete host cells are the ADH3 promoter and terminator, a promoter derived from genes encoding TAKA amylazotriose phosphate isomerase or Aspergillus oryzae alkaline protease, A. niger α-amylase, A. niger, A. nidulans glucoamylase A. nidulans acetamidase, Rhizomucor miehei aspartic proteinase or lipase, TPI1 terminator and ADH3 terminator.

Examples of suitable regulatory sequences for use in bacterial host cells are the promoters of the lac system, trp system, TAC or TRC system, and the main promoter regions of the lambda phage.

The nucleotide sequence of the invention, whether obtained by site-directed mutagenesis, synthesis or other methods, may also contain, or may not contain, a nucleotide sequence encoding a signal peptide. A signal peptide is present when the polypeptide must be secreted from the cells in which it is expressed. Such a signal peptide, if present, must be recognized by the cell selected for expression of the polypeptide. The signal peptide may be homologous (e.g., such as is commonly associated with huIFNG) or heterologous (i.e., originating from a source other than huIFNG) with respect to the polypeptide, may be homologous or heterologous with respect to the host cell, i.e. represent a signal peptide, usually expressed from the host cell, or a signal peptide that is usually not expressed from the host cell. Accordingly, the signal peptide may be prokaryotic, for example, obtained from a bacterium, such as E. coli, or eukaryotic, for example, obtained from a mammalian, insect or yeast cell.

The presence or absence of a signal peptide will, for example, depend on the expression of the host cell used to produce the polypeptide, the expressed protein (whether it is an intracellular or intercellular protein) and whether secretion is desired. When used in hyphomycetes, the signal peptide can usually be obtained from a gene encoding amylase or glucoamylase of the species Aspergillus, a gene encoding a Rhizomucor meihei lipase or protease or Humicola lanuginosa lipase. The signal peptide is preferably obtained from a gene encoding TAKA amylase A. oryzae, neutral A. niger α-amylase, acid resistant A. niger amylase or A. niger glucoamylase. When used in insect cells, the signal peptide can usually be obtained from an insect gene (cf. WO 90/05783), for example, the gene for the adipokinetic hormone of the lepidopteran Manduca sexta (cf. US 5023328), mellitin of the honey bee (Invitrogen), ecdysteroid-UDP-glucosyltrans (egt) (Murphy et al .. Protein Expression and Purification, 4, 349-357 (1993) or the human pancreatic lipase gene (hpl) (Methods in Enzymology, 284, pp. 262-272, 1997).

A preferred signal peptide for use in mammalian cells is the hulFNG signal peptide or murine Ig kappa light chain signal peptide (Coloma M. (1992), J. Imm. Methods, 152: 89-104). When used in yeast cells, suitable signal peptides have been found to be the α-factor signal peptide from S. cereviciae (cf. US 487008), mouse saliva amylase signal peptide (cf. O. Hagenbuchle et al., Nature, 289, 1981, pp.643-646), a modified carboxypeptidase signal peptide (cf. LAValls et al., Cell, 48, 1987, pp.887-897), BAR1 yeast signal peptide (cf. WO 87/02670) and aspartic protease 3 signal peptide yeast (YAP3) (cf. M. Egel-Mitani et al., Yeast, 6, 1990, pp. 127-137).

Any suitable host can be used to produce the IFNG polypeptide, including cells or cell lines of bacteria, fungi (including yeast), plants, insects, mammals or any other suitable animals, as well as transgenic animals or plants. Examples of bacterial host cells are gram-positive bacteria, such as Bacillus strains, for example B. brevis or B. subtilis, Pseudomonas or Streptomyces, or gram-negative bacteria, such as E. coli strains. The introduction of the vector into the bacterial host cell can be accomplished, for example, by protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular General Genetics, 168: 111-115) using competent cells (see, for example, Young and Spizizen , 1961, Journal of Bacteriology, 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology, 56: 209-221), by electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques, 6: 742-751) or conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology, 169: 5271-5278).

Examples of suitable hyphomycete host cells are strains of Aspergillus, for example A. oryzae, A. niger or A. nidulans, Fusarium or Trichoderma. Fungal cells can be transformed by a method including the formation of protoplasts, transformation of protoplasts and cell wall regeneration in a manner known per se. Suitable transformation procedures for Aspergillus host cells are described in EP 238023 and US 5679543. Suitable transformation procedures for Fusarium species are described by Malardier et al., 1989, Gene, 78: 147-156, and in WO 96/00787. Yeast can be transformed using the procedures described by Becker and Guarente, in Abelson J.N. and Simon M.I., editors. Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology, 153: 163; and in Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA, 75: 1920.

Examples of suitable yeast host cells are Saccaromyces strains, for example S. cerevisiae, Schizosaccharomyces, Kluyveromyces, Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H. Polymorpha, or Yarrowia. Methods for transforming yeast cells of heterologous DNA and preparing heterologous polypeptides therein are described by Clontech Laboratories, Inc., Palo Alto, California, USA (in the product protocol for Yeastmaker ™ Yeast Transformation System Kit), and Reeves et al., FEMS Microbiology Letters, 99 (1992), 193-198, Manivasakam and Schiestl, Nucleic Acids Research, 1993, Vol.21, No.18, pp. 4414-4415, and in Ganeva et al., FEMS Microbiology Letters, 121 (1994), 159 -164.

Examples of suitable insect host cells are Lepidoptora cell lines, such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni (High Five) cells (US 5077214). Transformation of insect cells and obtaining heterologous polypeptides in them can be carried out as described by Invitrogen.

Examples of suitable mammalian cells are Chinese Hamster Ovary (CHO) cell lines (e.g. CHO-K1; ATCC CCL-61), green monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL -1651)); mouse cells (e.g., NS / O), hamster cub (KNK) kidney cell lines (e.g., ATCC CRL-1632 or ATCC CCL-10) and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well as growing cells in tissue culture. Other suitable cell lines available from repositories, such as the American Type Culture Collection, Rockville, Maryland, are known in the art. Also, mammalian cells, such as CHO cell, can be modified to express sialyl transferase, for example 1,6-sialyl transferase, for example, as described in US 5047335, in order to provide improved glycosylation of the IFNG polypeptide.

Methods for introducing exogenous DNA into mammalian host cells include phosphate-mediated transfection, electroporation, DEAE-dextran-mediated transfection, liposome-mediated transfection, viral vectors and the transfection method described by Life Technologies Ltd., Paisley, United Kingdom, using lipofectamine 2000. These methods well known in the art and described, for example, by Ausbel et al. (eds.). Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. The cultivation of mammalian cells is carried out according to established methods, for example, as described in Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc., Totowa, New Jersey, USA, and Harrison M.A. and Rae I.F., General Techniques of Cell Culture, Cambridge University Press, 1997.

In order to obtain a glycosylated polypeptide, it is preferable to use a eukaryotic host cell, for example, of the type indicated above.

In the methods for producing the present invention, cells are cultured in a nutrient medium suitable for producing a polypeptide using methods known in the art. For example, a cell can be cultured by culturing in a vial with shaking, fermentation on a small or large scale (including continuous, batch, batch fed or solid phase fermentation) in laboratory or industrial fermenters, carried out in a suitable medium and under conditions allowing synthesize and / or isolate the polypeptide. Cultivation takes place in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers, or can be obtained in accordance with the compositions given in the publications (for example, in catalogs of the American type culture collection). If the polypeptide is secreted into the culture medium, it can be extracted directly from the medium. If the polypeptide is not secreted, it can be extracted from cell lysates.

The resulting polypeptide can be extracted by methods known in the art. For example, a polypeptide can be recovered from a culture medium using conventional procedures, including centrifugation, filtration, extraction, spray drying, evaporation or precipitation, and other methods.

The polypeptide can be purified by many methods known in the art, including chromatography (e.g., ion exchange, affinity, hydrophobic chromatography, chromatographic focusing and gel chromatography), electrophoresis (e.g. preparative isoelectric focusing) using various solubilities (e.g., precipitation ammonium sulfate), SDS-PAGE or extraction (see, for example, Protein Purification, J.-C. Jonson and Lars Ryden, editors, VCH Publishers, New York, 1989). Specific methods for purifying polypeptides exhibiting IFNG activity are described in EP 110004 and Japanese Unexamined Patent Application No. 186995/84.

The biological activity of the IFNG polypeptide can be analyzed by any suitable method known in the art. Such assays include antibody neutralization of antiviral activity, induction of protein kinase, oligoadenylate-2,5-A synthetase or phosphodiesterase activity, as described in EP 41313 B1. Such assays also include immunomodulatory assays (see, for example, US 4,753,795), growth inhibition assays, and measurement of binding to cells expressing interferon receptors. Specific analyzes are described herein in the Materials and Methods section.

The invention further relates to improved methods of treating, in particular, interstitial lung diseases, as well as granulomatous diseases, cancer, infectious diseases, bone disorders (for example, bone metabolism disorders such as malignant osteoporosis) and autoimmune diseases such as rheumatoid arthritis the key advantage being a less frequent and / or less invasive administration of a more effective therapy and, optionally, a lower risk of immune responses to therapeutically active (s) ) compound (s).

The conjugate of the invention is preferably incorporated into a composition comprising a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable" means that the carrier or excipient does not have any adverse effects on the patients to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (Remington's Pharmaceutical Sciences, 18th edition, ARGennaro, 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]).

The conjugate of the invention can be used "as is" and / or in the form of its salt. Suitable salts are, but are not limited to, salts with alkali metals or alkaline earth metals such as sodium, potassium, calcium and magnesium, as well as, for example, zinc salts. Such salts or complexes may be present in the form of a crystalline and / or amorphous structure.

The conjugate of the invention is administered at a dose approximately equivalent to that used in the treatment of well-known commercial IFNG preparations, such as Actimmune or described in EP 795332. The exact dose administered depends on the circumstances. Typically, the dose should be capable of preventing or reducing the severity or spread of the condition or indications for treatment. It will be apparent to those skilled in the art that the effective amount of the conjugate or composition of the invention depends, inter alia, on the disease, dose, administration schedule, whether the polypeptide, conjugate or composition is administered alone or in combination with other therapeutic agents, the half-life of the composition in the blood mainstream and general health of the patient.

The invention also relates to the use of a) a conjugate containing at least one non-polypeptide group covalently linked to an IFNG polypeptide, wherein the IFNG polypeptide is selected from the group consisting of huIFNG, rhuIFNG or IFNG polypeptide described herein (t ie, a conjugate constituting the conjugate of the invention), or b) a pharmaceutical composition of the invention for the manufacture of a medicament, pharmaceutical composition or composite kit for the treatment of interstitial lung diseases, cancer, infectious diseases, naru eny bone (e.g., bone metabolism disorders, such as malignant osteopetrosis) and / or inflammatory diseases, in particular interstitial pulmonary diseases, most often idiopathic pulmonary fibrosis. A glucocorticoid, such as prednisone, may also be included. The preferred dosage is 1-4, preferably 2-3, μg of the polypeptide component per kg of patient weight per dose. The preferred dosage is 100-350, more preferably 100-150, μg glucocorticoid per kg of patient weight per dose.

Improved delivery vehicles for molecules or preparations, optionally additionally containing glucocorticoids, are also described.

The invention also relates to a composite kit suitable for the treatment of interstitial lung diseases, comprising a first pharmaceutical composition containing the active components a) or b) above and a second pharmaceutical composition containing at least one glucocorticoid, each optionally together with a pharmaceutically acceptable carrier and / or excipient.

The conjugate of the invention can be incorporated into pharmaceutical compositions by well-known methods. Suitable formulations are described in Remington's Pharmaceutical Sciences, E.B. Martin, and in US 5183746.

The pharmaceutical composition can be obtained in many forms, including liquid, gel, lyophilized powder, compressed solid, or in any other suitable form. The preferred form will depend on the specific indications for which the treatment is carried out, and will be clear to those skilled in the art.

The pharmaceutical composition can be administered orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, into the lungs, vaginally, rectally, into the eyes, or by any other suitable method, for example, using PowderJect or ProLease technology. The compositions can be administered continuously by infusion, although a bolus injection is acceptable using methods well known in the art, such as pumps or implantation. In some cases, the composition can be applied directly in the form of a solution or spray. The preferred route of administration will depend on the particular indication for treatment and will be apparent to one skilled in the art.

The pharmaceutical composition of the invention can be administered in combination with other therapeutic agents. Such agents may be included in the same pharmaceutical composition as part thereof, or may be administered separately from the polypeptide or conjugate of the invention, or simultaneously or according to any other suitable treatment regimen. In addition, the polypeptide, conjugate or pharmaceutical composition of the invention can be used as an adjuvant for other treatments. In particular, combinations with glucocoricoids are considered, as described in EP 795332.

Parenteral drugs

An example of a pharmaceutical composition is a solution formulated for parenteral administration. Although in many cases, pharmaceutical solution compositions are provided in liquid form suitable for immediate use, such compositions for parenteral administration may also be provided in frozen or lyophilized form. In the first case, the composition should be thawed before use. The latter form is often used to increase the stability of the active compound contained in the composition over a wide range of storage conditions, as it is known to those skilled in the art that lyophilized preparations are generally more stable than their liquid counterparts. Such lyophilized preparations are reconstituted before use by adding one or more pharmaceutically acceptable diluents, such as sterile water for injection or sterile saline.

In the case of parenteral preparations, they are prepared for storage in the form of lyophilized compositions or aqueous solutions, mixing, accordingly, a polypeptide of the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers commonly used in the art (all called “excipients”), for example, buffering agents, stabilizers, preservatives, isotonicity agents, nonionic detergents, antioxidants and / or various other additives .

Buffering agents help maintain the pH in a range approaching physiological conditions. Typically, they are present in a concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present invention are both organic and inorganic acids and their salts, such as citrate buffers (e.g., a mixture of monosodium citrate and disodium citrate, a mixture of citric acid and trisodium citrate, a mixture of citric acid and monosodium citrate, etc.), succinate buffers (e.g., a mixture of succinic acid and monosodium succinate, a mixture of succinic acid and sodium hydroxide, a mixture of succinic acid and disodium succinate, etc.), tartrate buffers (e.g., a mixture of tartaric acid and sodium arthrate, a mixture of tartaric acid and potassium tartrate, a mixture of tartaric acid and sodium hydroxide, etc.), fumarate buffers (e.g., a mixture of fumaric acid and monosodium fumarate, a mixture of fumaric acid and disodium fumarate, a mixture of monosodium fumarate and disodium fumarate, etc.) gluconate buffers (e.g., a mixture of gluconic acid and sodium gluconate, a mixture of gluconic acid and sodium hydroxide, a mixture of gluconic acid and potassium gluconate, etc.), oxalate buffers (e.g., a mixture of oxalic acid and sodium oxalate, a mixture of oxalic acid and sodium hydroxide, a mixture of oxalic acid and potassium oxalate, etc.), lactate buffers (for example, a mixture of lactic acid and sodium lactate, a mixture of lactic acid and sodium hydroxide, a mixture of lactic acid and potassium lactate, etc.) and acetate buffers (for example, a mixture of acetic acid and sodium acetate, a mixture of acetic acid and sodium hydroxide, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts such as tris can also be used.

Preservatives are added to slow the growth of microbes, and, as a rule, they are added in amounts of about 0.2% -1% (w / v). Suitable preservatives for use in the present invention are phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g. benzalkonium chloride, bromide or iodide), hexamethonium parabenyl chloride, alkyl benzene resorcinol, cyclohexanol and 3-pentanol.

Isotonicity agents are added to provide isotonicity in the liquid compositions and include polybasic sugar alcohols, preferably tribasic or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polybasic alcohols may be present in an amount of from 0.1 wt.% To 25 wt.%, Typically 1% -5%, taking into account the relative amount of other ingredients.

Stabilizers belong to a wide category of excipients having functions from a filler to an additive that dissolves the therapeutic agent or helps to prevent denaturation or adherence to the walls of the container. Typical stabilizers may be polybasic sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine and the like, organic sugars or sugar alcohols such as lactose, trehalose, stachyose , mannitol, sorbitol, xylitol, ribite, myoinisite, galactite, glycerin and the like, including cyclites such as inositol; polyethylene glycol; polyamino acids; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerin and sodium thiosulfate; low molecular weight polypeptides (i.e., with a residual number <10); proteins, such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides, such as raffinose, and polysaccharides, such as dextran. Stabilizers, as a rule, are present in an amount of from 0.1 to 10,000 wt. parts relative to the weight of the active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) may be present in order to facilitate dissolution of the therapeutic agent, as well as to protect the therapeutic polypeptide from aggregation caused by mixing, which also prevents denaturation of the polypeptide in the composition subjected to surface shear stress . Suitable non-ionic surfactants are polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), pluronic® polyols, polyoxyethylene sorbitan monoesters (tween®-20, tween®-80, etc.). P.).

Various other excipients include bulking agents or fillers (e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g. ascorbic acid, methionine, vitamin E) and cosolvents. The active ingredient can also be captured by microcapsules obtained, for example, by coacervation or interfacial polymerization, for example, microcapsules from hydroxymethyl cellulose, gelatin or polymethyl methacrylate, colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules). Such methods are described in Remington's Pharmaceutical Sciences, cit. above.

Parenteral compositions used in vivo must be sterile. This is easily accomplished, for example, by filtration through membranes for filter sterilization.

Delayed release formulations

Suitable examples of delayed release formulations include semi-permeable matrices of solid hydrophobic polymers containing a conjugate, the matrices having a suitable shape, such as for example a film or microcapsules. Examples of delayed release matrices are polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or polyvinyl alcohol), polyacids, copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable copolymer of ethylene and vinyl acetate, degradable copolymers of lactic acid acids such as ProLease® or Lupron Depot® (injection microspheres consisting of a copolymer of lactic acid and glycolic acid and leuprolide acetate), and poly-D - (-) - 3-hydroxybutyl acid. While polymers, such as a copolymer of ethylene and vinyl acetate and lactic and glycolic acids, are able to release molecules for a long time - over 100 days, some hydrogels release proteins for a shorter period. When the encapsulated polypeptides remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37 ° C, and as a result, biological activity and possible changes in immunogenicity are lost. A smart strategy can be designed to stabilize, depending on the mechanism it contains. For example, if it is found that the aggregation mechanism is the formation of an S-S intermolecular bond through the thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling moisture content, using appropriate additives and developing certain polymer matrix formulations.

Oral administration

In the case of oral administration, the pharmaceutical composition may be in solid or liquid form, for example, in the form of a capsule, tablet, suspension, emulsion or solution. The pharmaceutical composition is preferably obtained in the form of a dosage unit containing a predetermined amount of the active ingredient. A suitable daily dose for a human or other mammal can vary widely depending on the condition of the patient and other factors, but can be determined by those skilled in the art using conventional methods.

Solid dosage forms for oral administration may include capsules, tablets, suppositories, powders and granules. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose or starch. Such dosage forms may also contain, as is usual practice, additional substances, for example, lubricants, such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents. Tablets and pills can also be obtained with enteric coatings.

The conjugates can be mixed with adjuvants such as lactose, sucrose, starch powder, cellulose and alkanoic acid esters, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gum arabic, gelatin, sodium alginyl polynyl, and / or polyvinyl alcohol, and tableted or encapsulated for ease of administration. Alternatively, they can be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth resin and / or various buffers. Other adjuvants and methods of administration are well known in pharmacy. The carrier or diluent may contain a dissolution retarding material, such as glyceryl monostearate or glyceryl distearate, alone or with wax or other substances well known in the art.

The pharmaceutical compositions may be subjected to conventional pharmaceutical operations, such as sterilization, and / or they may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffering agents, excipients and the like, for example, which are mentioned in this description.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also contain adjuvants, such as wetting agents, sweeteners, flavoring agents and perfumes.

Local introduction

Compositions suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniment, lotions, ointments, creams or pastes), and drops suitable for administration to the eyes, ears or nose.

Delivery to the lungs

Compositions suitable for use with a nebulizer or liquid or ultrasound will typically contain a polypeptide or conjugate dissolved in water at a concentration of, for example, about 0.01-25 mg of the conjugate per ml of solution, preferably about 0.1-10 mg / ml The composition may also contain a buffer and simple sugar (for example, to stabilize and regulate the osmotic pressure) and / or human serum albumin in the concentration range from 0.1 to 10 mg / ml. Examples of buffers that can be used are buffers with sodium acetate, citrate, and glycine. Preferably, the buffer will have a composition and molarity suitable for adjusting the pH of the solution to a value in the range of 3-9. Typically, a buffer molarity of 1 mM to 50 mM is suitable for this purpose. Examples of sugars that can be used are lactose, maltose, mannitol, sorbitol, trehalose and xylose, typically in amounts ranging from 1 wt.% To 50 wt.% Of the composition.

The nebulizer composition may also contain a surfactant to reduce or prevent protein aggregation on the surface caused by the conversion of the solution into an aerosol. Various conventional surfactants can be used, such as polyoxyethylene fatty acid esters of alcohols and polyoxyethylene sorbitan fatty acid esters. Amounts will typically be from 0.001 wt.% To 4 wt.% Of the composition. A particularly preferred surfactant for the purposes of this invention is polyoxyethylene sorbitan monooleate.

Specific compositions and methods for preparing suitable dispersions of liquid particles according to the invention are described in WO 94/20069, US 5915378, US 5960792, US 5957124, US 5934272, US 5915378, US 5855564, US 5826570 and US 5522385, incorporated herein by reference.

Compositions for use with a metering device for inhalation, as a rule, will contain fine powder. Such a powder can be obtained by lyophilization of a liquid composition with a conjugate and subsequent grinding, and it may contain a stabilizer, such as human serum albumin (HSA). Typically, HSA add more than 0.5% (wt./mass.). In addition, one or more sugars can be added to the preparation if necessary. Examples are lactose, maltose, mannitol, sorbitol, sorbitol, trehalose, xylitol and xylose. The amount added to the composition may be in the range of about 0.01-200% (May./mass.), Preferably about 1-50%, of the conjugate present. Such compositions are then lyophilized and pulverized to the desired particle size.

Then the particles of the desired size are suspended in the propellant using a surfactant. The propellant may be a common substance used for such a purpose, such as fully chlorofluorinated hydrocarbon, partially chlorofluorinated hydrocarbon, fully fluorinated hydrocarbon or partially fluorinated hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,2,2-tetrafluoroethane or their combinations. Suitable surfactants are sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant. Then this mixture is loaded into the device for delivery. An example of a commercially available metered dose inhaler suitable for use in the present invention is a Ventolin metered dose inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina.

Compositions for powder inhalers will contain finely divided dry powder containing a conjugate, and may also contain a filler, such as lactose, sorbitol, sucrose or mannitol, in quantities that facilitate the flow of powder from the device, for example 50 wt.% 90 wt.% Of the composition. The powder particles should have aerodynamic properties in the lung corresponding to particles with a density of about 1 g / cm ² with an average diameter of less than 10 microns, preferably from 0.5 to 5 microns, most preferably from 1.5 to 3.5 microns. An example of a powder inhaler suitable for use as described herein is a Spinhaler powder inhaler manufactured by Fisons Corp., Bedford, Mass.

Powders for these devices can be obtained and / or delivered by the methods described in US 5997848, US 5993873, US 5985248, US 5976574, US 5922354, US 5785049 and US 5654007.

Mechanical devices designed to deliver therapeutic products to the lungs include nebulizers, metered-dose inhalers and powder inhalers, and other devices that are familiar to those skilled in the art. Specific examples of commercially available devices suitable for the practice of this invention are an Ultravent atomizer manufactured by Mallinckrodt, Inc., St. Louis, Missouri; Acorn II nebulizer manufactured by Marquest Medical Products, Inglewood, Colorado; Ventolin volumetric inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina; Spinhaler Powder Inhaler manufactured by Fisons Corp., Bedford, Massachusetts; standing cloud device from Inhale Therapeutic Systems, Inc., San Carlos, California; AIR inhaler manufactured by Alkermes, Cambridge, Massachusetts; and an AERx lung drug delivery system manufactured by Aradigm Corporation, Hayward, CA.

The invention relates to compositions and methods for treating bacterial and viral infectious diseases, cancers or tumors, interstitial lung diseases, such as idiopathic pulmonary fibrosis, granulomatous diseases, bone disorders (e.g., bone metabolism disorders such as malignant osteoporosis) and autoimmune diseases, such as rheumatoid arthritis.

In another aspect, the invention relates to a method for treating a mammal with circulating antibodies against huIFNG or rhuIFNG, comprising administering a compound having IFNG biological activity not interacting with said antibodies. The compound is preferably a conjugate described herein, wherein the mammal is preferably a human. Treated mammals may suffer from any of the diseases listed above, in which case IFNG is a useful therapeutic agent. In addition, the invention relates to a method for producing a pharmaceutical product for use in the treatment of mammals having circulating anti-huIFNG or rhuIFNG antibodies, wherein a compound having IFNG biological activity and not interacting with said antibodies is formulated for injection or other suitable compositions. The term "circulating antibodies" is intended to refer to antibodies formed in the body of a mammal in response to treatment with any commercially available IFNG drugs.

The use of a nucleotide sequence encoding an IFNG polypeptide of the invention in gene therapy is also contemplated. In particular, it may be of interest to use the nucleotide sequence encoding the IFNG polypeptide described above in the section "Glycosylated polypeptides of the invention modified to introduce additional glycosylation sites". Glycosylation of the polypeptide is thus achieved during the course of gene therapy, i.e. after expression of the nucleotide sequence in the human body.

Considerable uses in gene therapy include the treatment of diseases in which the polypeptide is expected to provide effective treatment.

Local delivery of IFNG using gene therapy can provide the targeted area with the drug, while avoiding potential toxicity problems associated with non-specific administration.

The methodology of gene therapy both in vitro and in vivo is considered.

Several methods are known for transferring potentially therapeutic genes to specific cells. For more information, see, for example, Mulligan, "The Basic Science Of Gene Therapy", Science, 260, pp. 926-31 (1993). These methods include

direct gene transfer, for example, as described in Wolff et al., "Direct Gene transfer Into Mouse Muscle In vivo", Science, 247, pp. 1465-68 (1990);

Liposome-mediated DNA transfer, for example, as described in Caplen et al., "Liposome-mediated CFTR Gene Transfer to the Nasal Epithelium Of Patients With Cystic Fibrosis", Nature Med., 3, pp. 39-46 (1995); Crystal, "The Gene As A Drug", Nature Med., 1, pp. 15-17 (1995); Gao and Huang, "A Novel Cationic Liposome Reagent For Efficient Transfection of Mammalian Cells", Biochem. Biophys. Res. Comm., 179, pp. 280-85 (1991);

Retroviral mediated DNA transfer, for example, as described in Kau et al., "In vivo Gene Therapy of Hemophilia B: Sustained Partial Correction In Factor IX-Deficient Dogs", Science, 262, pp. 117-19 (1993); Anderson, "Human Gene Therapy", Science, 256, pp. 808-13 (1992);

DNA virus-mediated DNA transfer. Such DNA viruses include adenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses (preferably herpes simplex vectors) and parvoviruses (preferably defective or non-autonomous parvoviruses, more preferably vectors based on adeno-associated viruses, most preferably vectors based on AAV-2). See, for example, Ali et al., "The Use Of DNA Viruses as Vectors for Gene Therapy", Gene Therapy, 1, pp. 367-84 (1994); US 4797368 and US 5139941.

The invention is also described in the following examples. The examples should in no way be understood as limiting the general statements of the present description and claims.

Materials and methods

Analyzes

Interferon Assay Scheme

It was previously reported that IFNG interacts and activates IFNG receptors on HeLa cells. Therefore, transcription is activated by promoters containing an interferon stimulated response element (ISRE). Thus, it is possible to screen interferon receptor agonists using ISRE in combination with the luciferase receptor gene (ISRE-luc) placed on HeLa cells.

Primary analysis

HeLa cells are cotransfected with ISRE-Luc and create pCDNA 3.1 / hygro and foci (cell clones) by selection in DMEM containing hygromycin B. Cell clones are screened for luciferase activity in the presence or absence of IFNG. These clones, showing the highest ratio of stimulated luciferase activity to unstimulated, are used in further analyzes.

In order to screen muteins, 15,000 cells / well were seeded in 96-well culture plates and incubated overnight in DMEM. The next day, muteins, as well as the well-known standard, are added to the cells in different concentrations. The plates are incubated for 6 hours at 37 ° C in an atmosphere with 5% CO ₂ . Then, LucLite substrate (Packard Bioscience, Groningen, Netherlands) was added to each well. The plates are sealed and luminescence is measured on a TopCount (Packard) luminometer using the SPC method (counting single photons). Each individual plate contains wells incubated with IFNG as a stimulated control, and other wells containing normal medium as an unstimulated control. The relationship between stimulated and unstimulated luciferase activity serves as an internal standard both for mutein activity and for changes from experiment to experiment.

In vivo functional half-life of IFNG conjugate

The measurement of biological half-life can be carried out in many ways described in the literature. In one of the methods described by Rutenfranz et al. (J. Interferon Res., 1990, vol. 10, p.337-341), using intravenous and intramuscular injection of IFNG to 8-week-old C57BL / 6 mice. Biological half-life is measured using a biological assay determining the titer of IFNG in murine serum using Hep-2 cells and vesicular stomatitis virus (WS). As an alternative, the authors also used ELISA to determine serum IFNG levels.

As another method, radioactive isotopes of IFNG can be used to study subcutaneous absorption and local distribution of IFNG. Croos and Roberts (J. Pharm., 1993, vol. 45, p. 606-609) conducted studies with ¹²⁵ IFNG on anesthetized female Spraque-Dawley rats. After subcutaneous administration, blood and tissue samples were collected and the amount of IFNG was determined by counting gamma rays.

IFNG PEG Processing

The treated PEG ruIFNG can be obtained as described in Example 2 in US 5109120. Similarly, the PEG IFNG polypeptides described herein, for example, containing the N25K mutation, can be treated. The resulting PEG-IFNG-N25K conjugate contains an additional attached PEG molecule compared to the ruIFNG conjugate.

Obtaining a pharmaceutical composition

A pharmaceutical composition, for example, for the treatment of interstitial lung diseases, can be prepared by incorporating the relevant purified conjugate of the invention into an injection composition according to procedures well known to those skilled in the art, so that in each ampoule the conjugate is contained in an amount corresponding to 50, 100, 200, 300, 400 or 500 μg, for example ruIFNG or IFNG-N25K.

Identification of surface-facing amino acid residues

Structures

Experimental 3D structures of ruIFNGs determined by X-ray crystallography are reported in Ealick et al .. Science, 252: 698-702 (1991), where a C-alpha trace of the IFNG homodimer is reported. Walter et al., Nature, 376: 230-235 (1995) report the structure of an IFNG homodimer in complex with two soluble molecules of the IFNG receptor. The coordinates of such a structure have not been published anywhere before. Thiel et al., Structure, 8: 927-936 (2000) report the structure of an IFNG homodimer in complex with two molecules of a soluble form of an IFNG receptor containing a third receptor molecule in a structure that does not interact with the IFNG homodimer.

Methods

Available Surface Area (ASA)

To calculate the accessible surface area (ASA) of individual atoms in the structure, the Access computer program is used (B. Lee and FV Richards, J. Mol. Biol., 55: 379-400 (1971)), version 2 (Copyright (c) Yale University ) With this method, a 1.4 Å probe is typically used and the available surface area (ASA) is determined as the area formed with the probe in the center. Before such a calculation, all water molecules, hydrogen atoms and other atoms not directly associated with the protein are removed from the coordinate system.

Fractional ASA Side Chain

The fractional ASA of the side chain atoms is calculated by dividing the sum of the ASA atoms in the side chain by an amount representing the ASA of the side chain atoms of this type of residue in the extended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thronton (1991), J. Mol. Biol., 220: 507-530. In the case of this example, the CA atom is considered as part of the side chain of glycine residues, but not for the remaining residues. The following table is used as the standard 100% ASA for the side chain.

Ala 69.23 Å ²

Arg 200.35 Å ²

Asn 106.25 Å ²

Asp 102.06 Å ²

Cys 96.69 Å ²

Gin 140.58 Å ²

Glu 134.61 Å ²

Gly 32.28 Å ²

His 147.00 Å ²

De 137.91 Å ²

Leu 140.76 Å ²

Lys 162.50 Å ²

Met 156.08 Å ²

Phe 163.90 Å ²

Pro 119.65 Å ²

Ser 78.16 Å ²

Thr 101.67 Å ²

Tip 210.89 Å ²

Tyr 176.61 Å ²

Val 114.14 Å ²

Residues not found in the structure are defined as having 100% availability, since it is believed that the residue is located in flexible areas.

Determination of distances between atoms

The distance between atoms is determined using molecular computer graphics software, for example, InsightII v.98.0, MSI INC.

Receptor binding site definition

A receptor binding site is defined as containing all residues that change the surface area after binding to the receptor. This is determined by at least two ASA calculations: one on the isolated ligand (s) in the ligand (s) / receptor (s) complex and the other on the full ligand (s) / receptor (s) complex.

results

The x-ray structure used corresponds to the IFNG homodimer in complex with two molecules of the soluble form of the IFNG receptor containing a third IFNG receptor molecule in the structure that does not interact with the IFNG homodimer, as reported by Thiel et al .. Structure, 8: 927-936 (2000). The structure consists of an IFNG homodimer, where two molecules are called A and B. For design purposes, there is an additional methionine located in front of the IFNG sequence called M0, and the sequence is processed at the C-terminus with ten residues (Q133 being the last residue in the constructed molecules) . M0 is removed from the structure for all calculations in this example. The structure of the two IFNG monomers has a very low electron density after residue 120, and the residues are modeled only up to residue T126. Therefore, residuals S121-T126 are removed from the structure before calculations in this example. Two fragments of the receptor, named C and D, directly interact with the IFNG homodimer, and a third receptor molecule, called E, does not contact the IFNG homodimer and is not involved in these calculations.

Surface accessibility

By calculating fractional ASA on a homodimer from molecules A and B, excluding MO and S121-T126 in both molecules, the following residues are obtained, in which more than 25% of their side chains face the surface in at least one of the monomers: Q1, D2, P3, K6, E9, N10, K12, K13, Y14, N16, G18, H19, S20, D21, A23, D24, N25, G26, T27, G31, K34, N35, K37, E38, E39, S40, K55, K58, N59, K61, D62, D63, Q64, S65, Q67, K68, E71, T72, K74, E75, N78, V79, K80, N83, S84, N85, K86, K87, D90, E93, K94, N97, S99, T101, D102, L103, N104, H111, Q115, A118 and E119.

In the following residues, more than 50% of their side chains are facing the surface in at least one of the monomers: Q1, D2, P3, K6, E9, N10, K13, N16, G18, H19, S20, D21, A23, D24 , N25, G26, T27, G31, K34, K37, E38, E39, K55, K58, N59, D62, Q64, S65, K68, E71, E75, N83, S84, K86, K87, K94, N97, S99, T101 , D102, L103, N104, Q115, A118, E119.

By calculating the fractional ASA on the homodimer from molecules A and B, excluding M0 and S121-T126 in both molecules and including the receptor molecules C and D, the following residues are obtained, in which more than 25% of their side chains face at least , one of the monomers: Q1, D2, P3, K6, E9, N10, K13, Y14, N16, G18, H19, D21, N25, G26, G31, K34, N35, K37, E38, E39, S40, K55, K58 , N59, K61, D62, D63, Q64, S65, Q67, K68, E71, T72, K74, E75, N78, V79, K80, N83, S84, N85, K86, K87, D90, E93, K94, N97, S99 , T101, D102, L103, N104, E119. In the following residues, more than 50% of their side chains are facing the surface in at least one of the monomers: P3, K6, N10, K13, N16, D21, N25, G26, G31, K34, K37, E38, E39, K55 , K58, N59, D62, Q64, S65, K68, E71, E75, N83, S84, K86, K87, K94, N97, S99, T101, L103 and N104.

All of the above positions are targets for modification according to the present invention.

Comparison of the two lists leads to the fact that K12, S20, A23, D24, T27, H111, Q115 and A118 are removed from the list with 25% ASA side chains after binding of the receptor, a Q1, D2, E9, G18, H19, S20, A23 , D24, T27, Q115, A118 and E119 are removed from the list with 50% ASA side chains after receptor binding.

Residues not defined in the structure are treated as with a completely accessible surface, i.e., residues S121, P122, A123, A124, K125, T126, G127, K128, R129, K130, R131, S132, Q133, M134, L135, F136, R137, G138, R139, R140, A141, S142, Q143. These residues also constitute separate targets for the introduction of groups for joining according to the present invention (or they can be considered as belonging to the group facing the surface of amino acid residues, for example, in which the available side chains of more than 25% or more than 50%).

Receptor binding site

Carrying out the ASA calculations as described above, the following residues of the IFNG molecule with a reduced ASA are obtained, compared with the calculation for an isolated dimer in at least one of the monomers in the complex: Q1, D2, Y4, V5, E9, K12 , G18, H19, S20, D21, V22, A23, D24, N25, G26, T27, L30, K34, K37, K108, H111, E112, I114, Q115, A118, E119.

Examples

Example 1

Creation of an expression cassette for IFNG expression in

yeast and CHO cells

The DNA sequence, GenBank, accession number X13274, including a full-sized cDNA encoding mature huIFNG without its native signal peptide, is modified in order to facilitate high expression in yeast cells. First, the MATa signal peptide is introduced instead of the IFNG signal peptide in order to facilitate secretion into yeast media. Secondly, the codons of the hulFNG nucleotide sequence are modified by shifting the use of codons towards codons often used in yeast. Then, some nucleotides in the sequence are replaced by others in order to introduce a restriction site for endonucleases for DNA. Create such primers so that the gene can be synthesized.

Primers are assembled on a synthetic gene using a one-step PCR method using a Platinum Pfx polymerase kit (Life Technologies) and three-step PCR cyclization parameters. The assembled gene is amplified by PCR using the same conditions, and the sequence shown in AFTER is obtained. Number 3.

The synthesized gene is cloned into pJS037-lip (Okkels JS (1966), Rec. DNA Biotech. III, vol. 782, 202-207) between the HindIII site at the 5'-end and XbaI at the 3'-end, and receive pIGY-1 .

In order to create a single chain construct containing covalently linked monomeric IFNG polypeptides, the constructs described below are created.

I) A construct containing two mature full-length huIFNG polypeptides linked via a 19-dimensional linker peptide modeled after the hinge regions of human IgAl (Lunn, C.A. et al., J. Biol. Chem., 17920-17924, 1992). This is carried out by PCR using the following primers:

ADJ002 5'-GGTTTGATATCGATGGCCAA-3 '(LAST. No. 4);

ADJ003 5'-GCGGCCCTCTAGATTACT-3 '(LAST. No. 5);

ADJ004 5'-CATCTCCGTCCACTCCGACTCCATAGCATGCAAGATCCATATGTGA AAGAA-3 '(LAST No. 6);

ADJ007 5'-ATCTTGCATGCTATGGAGTCGGAGTGGACGGAGATGGAGTTGGCGG AGTAGAAGGAACCGCTGTTTTAGCAGCTGGAGACAATT-3 (LAST. No. 7).

PCR fragments were cloned into pIGY-1 between ClaI at the 5'-end and XbaI at the 3'-end, collecting two monomers at SphI (introduced into the linker region). This design is called pIGY-2.

II) A construct containing two monomeric mature full-length huIFNG polypeptides without a linker. For this, the following primers are used for PCR:

ADJ002 5'-GGTTTGATATCGATGGCCAA-3 '(LAST. No. 8);

ADJ003 5'-GCGGCCCTCTAGATTACT-3 '(LAST. No. 9);

ADJ006 5'-TTTAGAGGTAGAAGAGCTTCTCAGCAAGATCCATATGTGAAAGAA

ST-3 '(LAST. No. 10);

ADJ009 5'-AGCTTCTTTCACATATGGATCTTGCTGAGAAGCTCTTCTACGTCT

AAA-3 '(LAST. No. 11).

A PCR fragment was collected by a two-stage PCR and cloned between ClaI at the 5'-end and XbaI at the 3'-end and called pIGY-3.

III) A construct containing two C-terminally processed (last 11 amino acids) monomeric IFNG polypeptides covalently linked via the 19-mer peptide linker as defined above. The following primers are used:

ADJ001 5'-TGCTCTAGACATCTGAGATCGTTTTCTCTTTCC-3 '(LAST. No. 12);

ADJ002 5'-GGTTTGATATCGATGGCCAA-3 '(LAST. No. 13);

ADJ004 5 '-CATCTTCCGTCCACTCCGACTCCATAGCATGCAAGATCCATATGT GAAAGAA-3' (LAST No. 14);

ADJ005 5 ^' -ATCTTGCATGCTATGGAGTCGGAGTGGACGGAGATGGAGTTGGCG GAGTAGAAGGAACCGGCATCTGAGATCTTTTTCTCC-3' (LAST. No. 15).

PCR fragments were cloned into pIGY-1 between ClaI at the 5'-end and XbaI at the 3'-end, collecting two monomers at SphI (introduced into the linker region). This design is called pIGY-4.

Designs for expression in CHO cells

In order to express IFNG in CHO cells, the following oligonucleotides are synthesized to enable the cloning of IFNG, including its signal peptide, into pcDNA3.1 / hygro (Invitrogen).

ADJ012 5'-CGCGGATCCATGAAATATACAAGTTATATCTTGGCTTTTTCAGCTCTGCATCGTTTT GGGTTCTCTTGGCTGTTACTGCCAAGATCCATATGTGAAAGAAGCT-3 '(LAST. No. 16).

In order to clone IFNG in such an expression vector, PCR amplification using ADJ012 and ADJ003 and pIGY-1 as a template yields a 450 bp fragment that can be cloned between BamHI at the 5'-end and Xbal at 3 ' end of pcDNA3.1 / hygro, whereby come to pIGY-5.

In order to introduce glycosylation sites into IFNGs, oligonucleotides are created so that PCR-generated changes can be introduced into the expression plasmid (pIGY-5) of the classic two-step PCR followed by cloning of the PCR fragment between BamHI at the 5'-end and XbaI at 3 '-end.

Therefore, two vector primers are created for use with specific mutational primers.

ADJ013 5'-GATGGCTGGCAACTGAGAAG-3 '(LAST. No. 17)

(antisense subsequent vector primer) and

ADJ014 5'-TGTACGGTGGGAGGTCTAT-3 '(LAST. No. 18)

(semantic previous vector primer).

For various muteins create the following primers:

K12T

ADJ015 5'-AGCATTAAAATACTTCGTCAAGTTTTCAGC-3 '(LAST No. 19)

ADJ016 5'-GCTGAAAACTTGACGAAGTATTTTAATGCT-3 '(LAST. No. 20)

G18t

ADJ017 5'-CACATCAGAATGAGTAGCATTAAAATA-3 '(LAST No. 21)

ADJ018 5'-TATTTTAATGCTACTCATTCTGATGTG-3 '(LAST No. 22)

E38n

ADJ019 5'-CATAATTTTTCGATCGGATTCGTTTTTCCAATTCTT-3 '(LAST No. 23)

ADJ020 5'-AAGAATTGGAAAAACGAATCCGATCGAAAAATTATG-3 '(LAST No. 24)

K61T

ADJ021 5'-AATAGACTGATCGTCTGTAAAGTTTTTAAA-3 '(LAST. No. 25)

ADJ022 5'-TTTAAAAACTTTACAGACGATCAGTCTATT-3 '(LAST No. 26)

N85T

ADJ023 5'-TSTTTTSTTTTTASTASTATTSTAAAAAASTT-3 '(LAST. No. 27)

ADJ024 5'-AAGTTTTTCAATAGTACTAAAAAGAAAAGA-3 '(LAST. No. 28)

K94N

ADJ025 5'-ATAATTAGTCAAATTTTCGAAGTCATG-3 '(LAST No. 29)

ADJ026 5'-GATGACTTCGAAAATTTGACTAATTAT-3 '(LAST. No. 30)

S99n

ADJ027 5'-AATCAAGTCAGTAACGTTATAATTAGTCAA-3 '(LAST No. 31)

ADJ028 5'-TTGACTAATTATAACGTTACTGACTTGAAT-3 '(LAST. No. 32)

Q106T

ADJ029 5'-ATGAATAGCTTTACTAGTCACATTCAAGTC-3 '(LAST No. 33)

ADJ030 5'-GACTTGAATGTGACTAGTAAAGCTATTCAT-3 '(LAST. No. 34)

After two-step PCR and hydrolysis with BamHI and XbaI, each of these pairs of primers gives a 447 bp fragment that is cloned into pIGY-5.

Expression of interferon γ in CHO cells

The above constructs are ready for transfection into the CHO K1 cell line (ATCC # CCL-61) by using Lipofectamine 2000 (Life Technologies, USA) as a transfection agent. After 24 hours, the culture medium is ready for collection and analysis for the activity of interferon γ and its content.

Claims (35)

1. The conjugate exhibiting the activity of γ-interferon (IFNG activity) containing a) a polypeptide comprising an amino acid sequence that differs by 1-15 amino acid residues from wild-type human γ-interferon (huIFNG) shown in SEQ ID NO: 2, or a fragment thereof shortened from the C-terminus by 1-15 amino acid residues , and b) at least one N-attachable carbohydrate group covalently attached to the introduced N-glycosylation site, wherein said introduced N-glycosylation site is located inside the N-terminal portion with a length of 118 amino acid residues of the IFNG polypeptide. 2. The conjugate according to claim 1, where the specified entered N-glycosylation site is located inside the N-terminal portion with a length of 93 amino acid residues of the IFNG polypeptide. 3. The conjugate of claim 1 or 2, wherein said polypeptide comprises an amino acid sequence that differs by 1-8 amino acid residues from huIFNG, or a fragment thereof. 4. The conjugate of claim 3, wherein said polypeptide comprises an amino acid sequence that differs by 1-5 amino acid residues from huIFNG, or a fragment thereof. 5. The conjugate according to claim 1, where the specified N-glycosylation site is introduced at a position that is occupied by an amino acid residue, in which at least 25% of its side chain faces the surface. 6. The conjugate according to claim 5, where the specified N-glycosylation site is introduced at a position that is occupied by an amino acid residue, in which at least 50% of its side chain faces the surface. 7. The conjugate according to claim 1, wherein said N-glycosylation site is introduced by substitution selected from the group consisting of Q1N + P3S / T, P3N + V5S / T, K6N + A8S / T, E9N + L11S / T, K12S / T, K13N + F15S / T, Y14N + N16S / T, G18S / T, G18N, G18N + S20T, H19N + D21S / T, D21N + A23S / T, G26N + L28S / T, G31N + L33S / T, K34N + W36S / T, K37S / T, K37N + E39S / T, E38N, E38N + S40T, E39N + D41S / T, S40N + R42S / T, K55N + F57S / T, K58N + F60S / T, K61S / T, K61N + D63S / T, D62N + Q64S / T, D63N, D63N + S65T, Q64N + I66S / T, S65N + Q67S / T, Q67N, Q67N + S69T, K68N + V70S / T, E71N + I73S / T, T72N + K74S / T, K74N + D76S / T, E75N + M77S / T, K80S / T, V79N + F81S / T, K80N + F82S / T, N85S / T, S84N + K86S / T, K87S / T, K86N + K88S / T, K87N + R89S / T, D90N + F92S / T, E93N + L95S / T, K94N, K94N + T96S, S99N, S99N + T101S, T101N + L103S / T, D102N + N104S / T, L103N + V105S / T and Q106S / T T. 8. The conjugate according to claim 7, wherein said N-glycosylation site is introduced by substitution selected from the group consisting of P3N + V5S / T, K6N + A8S / T, K12S / T, K13N + F15S / T, G18S / T, D21N + A23S / T, G26N + L28S / T, G31N + L33S / T, K34N + W36S / T, K37N + E39S / T, E38N, E38N + S40S / T, E39N + D41S / T, K55N + F57S / T, K58N + F60S / T, K61S / T, D62N + Q64S / T, Q64N + I66S / T, S65N + Q67S / T, K68N + V70S / T, E71N + I73S / T, E75N + M77S / T, N85S / T, S84N + K86S / T, K86N + K88S / T, K87N + R89S / T, K94N, K94N + T96S, S99N, S99N + T101S, T101N + L103S / T, D102N + N104S / T, L103N + V105S / T and Q106S / T T. 9. The conjugate according to claim 1, wherein said N-glycosylation site is introduced by substitution selected from the group consisting of K12S / T, G18S / T, G18N, K37S / T, E38N, M45N, I49N, K61S / T, D63N, Q67N, V70N, K80S / T, F82N, N85S / T and K87S / T. 10. The conjugate of claim 9, wherein said N-glycosylation site is introduced by substitution selected from the group consisting of K12T, G18N, G18T, K37T, E38N, K61T, D63N, Q67N, K80T, and N85T. 11. The conjugate of claim 10, wherein said N-glycosylation site is introduced by substitution selected from the group consisting of G18N, E38N, D63N and Q67N. 12. The conjugate of claim 8, where the specified N-glycosylation site is introduced by replacing E38N + S40T. 13. The conjugate according to claim 1, where the specified amino acid sequence further comprises at least one introduced cysteine residue. 14. The conjugate according to item 13, where the cysteine residue is introduced into a position that is occupied by an amino acid residue, in which at least 25% of its side chain is facing the surface. 15. The conjugate according to 14, where the cysteine residue is introduced into a position that is occupied by an amino acid residue, in which at least 50% of its side chain is facing the surface. 16. The conjugate of claim 15, wherein the cysteine residue is administered by substitution selected from the group consisting of N10C, N16C, E38C, N59C, N83C, K94C, and N104C. 17. The conjugate according to item 13, where the cysteine residue is introduced into the position occupied by any of amino acid residues 121-143. 18. The conjugate of claim 13, wherein the non-polypeptide moiety is covalently linked to the introduced cysteine residue. 19. The conjugate of claim 18, wherein the non-polypeptide group is a polymer molecule. 20. The conjugate according to claim 19, where the polymer is a linear or branched polyethylene glycol. 21. The conjugate according to any one of claims 1 and 2 and 4-20, where the conjugate is shortened from the C-terminus to 11 amino acid residues. 22. The conjugate according to any one of claims 1 to 2 and 4-20 for use as a therapeutic agent. 23. The conjugate according to claim 3 for use as a therapeutic agent. 24. The conjugate according to item 21 for use as a therapeutic agent. 25. The nucleotide sequence encoding the polypeptide portion of the conjugate according to any one of claims 1 to 24. 26. An expression vector containing the nucleotide sequence of claim 25. 27. A method of obtaining a host cell producing the polypeptide portion of the conjugate according to any one of claims 1 to 24, which comprises introducing a nucleotide sequence according to claim 25 or an expression vector according to claim 26. 28. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 24, and a pharmaceutically acceptable diluent, carrier or adjuvant. 29. The pharmaceutical composition according to p. 28 for use as a therapeutic agent. 30. A therapeutic agent for the treatment of interstitial lung diseases, containing the conjugate according to any one of claims 1 to 24 as an active principle. 31. The medicine according to claim 30, wherein the interstitial lung disease is idiopathic pulmonary fibrosis. 32. The therapeutic agent according to p. 31, which is administered subcutaneously. 33. A method of treating interstitial lung diseases, comprising administering to a patient in need thereof a conjugate according to any one of claims. 1-24 or the pharmaceutical composition according to p. 34. The method according to p, where the interstitial lung disease is idiopathic pulmonary fibrosis. 35. The method according to clause 34, where the therapeutic agent is administered subcutaneously. The following priorities have been established for the claims: According to claim 1 - November 12, 1999. on the basis of priority application RA 1999 01631 filed with the Patent Office of Denmark (with the exception of the clause “whereby the indicated N-glycosylation site is located inside the N-terminal portion with a length of 118 amino acid residues of the IFNG polypeptide”); November 13, 2000 - in relation to the said reservation. According to claim 2 - November 13, 2000 According to paragraphs 3, 4 - 12 November 1999, on the basis of priority application RA 1999 01631, filed with the Patent Office of Denmark. However, these clauses are dependent on clause 1, in which the above reservation has priority on November 13, 2000. According to paragraphs 5, 6 - November 13, 2000 According to paragraphs 7-12 - November 12, 1999 on the basis of priority application RA 1999 01631, filed with the Patent Office of Denmark, regarding a part of the listed substitutions, November 13, 2000 regarding a part of the listed substitutions. According to paragraph 13 - November 12, 1999 on the basis of priority application RA 1999 01631 filed with the Patent Office of Denmark. However, this paragraph is dependent on paragraph 1, in which the above reservation has priority on November 13, 2000. According to paragraphs 14, 15 - 13 November 2000 According to clause 16 - November 12, 1999, on the basis of the priority application of RA 1999 01631, filed with the Patent Office of Denmark, regarding part of the above substitutions, November 13, 2000. in relation to part of the listed replacements. On p. 17 - November 13, 2000 According to paragraphs 18-20 - November 12, 1999 on the basis of priority application RA 1999 01631, filed with the Patent Office of Denmark. However, these clauses are dependent on clause 1, in which the above reservation has priority on November 13, 2000. According to item 21 - November 13, 2000 According to paragraphs 22-35 - November 12, 1999 on the basis of priority application RA 1999 01631, filed with the Patent Office of Denmark, regarding a part of the listed substitutions, November 13, 2000 regarding a part of the listed substitutions.