KR20030094336A - Interferon gamma polypeptide variants - Google Patents

Abstract

The present invention relates to novel interferon gamma polypeptide variants having interferon gamma (IFNG) activity, methods for their preparation, pharmaceutical compositions comprising polypeptide variants and the treatment of diseases, in particular in interstitial lung diseases, for example idiopathic pulmonary fibrosis It is about the use of. These novel polypeptide variants all comprise S99T substitutions as compared to the amino acid sequence of huIFNG or fragments thereof. By making this modification, naturally occurring N-glycosylation sites at position 97 are significantly better utilized. Preferably, the variants include further modifications, for example to increase the AUC of such variants when administered subcutaneously.

Description

INTERFERON GAMMA POLYPEPTIDE VARIANTS}

Interferon-gamma (IFNG) is a cytokine produced by T-lymphocytes and natural killer cells and exists as a homodimer of two polypeptide subunits that are noncovalently bound. The complete form of each dimer consists of 143 amino acid residues (see SEQ ID NO: 17) and its precursor form comprises 166 amino acid residues (see SEQ ID NO: 18).

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

Gary 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), Rinderknecht et al. (J. Bol. Chem. 259: 6790-6797, 1984) and EP 0 077 670, EP 0 089 676, and EP 0 110 044 have been reported for the primary sequence of wild type human IFNG (huIFNG). The 3D structure of huIFNG has been reported by Ealick et al. (Science 252: 698-702, 1991).

Various naturally occurring or mutated IFNG subunit polypeptide types have also been reported, including the Cys-Tyr-Cys N-terminal amino acid sequence (positions (-3) to (-1) for SEQ ID NO: 17) ; Comprising an N-terminal methionine (position (-1) relative to SEQ ID NO 2); Various C-terminal truncations including 127-134 amino acid residues, and the like. It is known that 1-15 amino acid residues can be removed from the C-terminus without altering the activity of the IFNG molecule. In addition, the release of huIFNG C-terminus has been described in Pan et al. (Eur. J. Biochem. 166: 145-149, 1987).

Slodowski et al., (Eur. J. Biochem. 202: 1133-1140, 1991), Luk et al., (J. Biol. Chem. 265: 13314-13319, 1990), Seelig et al., (Biochemistry 27 : 1981-1987, 1988), Trousdale et al., (Invest.Ophthalmol. Vis. Sci. 26: 1244-1251, 1985), EP 146354, etc., reported on HuIFNG muteins. Nishi et al., (J. Biochem. 97: 153-159, 1985) reported on natural huIFNG variants.

US 6,046,034 describes thermostable recombinant huIFNG (rhuIFNG) variants by inserting up to four pairs of cysteines to form disulfide bonds, thereby stabilizing dimeric IFNG variants.

WO 92/08737 describes IFNG variants comprising methionine added at the N-terminus of all (residues 1-143) or part (1-132) amino acid sequences of wild type human IFNG. EP 219 781 describes a partial huIFNG sequence comprising amino acid residues 3-123 of SEQ ID NO: 17. US 4,832,959 describes a partial huIFNG sequence comprising amino acid sequences 1-127, 5-146, 5,127 residues having three additional N-terminal amino acid residues (Cys-Tyr-Tys) compared to SEQ ID NO: 17. It was. US 5,004,689 describes a DNA sequence encoding huIFNG without three N-terminal amino acid residues (Cys-Tyr-Tys), and also reported expression of it in E. coli . EP 0 446 582 describes that E. coli produces rhuIFNG free of N-terminal methionine. US 6,120,762 describes peptide fragments of huIFNG comprising amino acid residues 95-134 thereof for SEQ ID NO: 17.

Wang et al. (Sci. Sin. B 24: 1076-1084, 1994) reported on rhuIFNG expressed in large quantities.

Curling et al. (Biochem. J. 272: 333-337, 1990), Hooker et al., (J. of Interferon and Cytokine Research, 1998, 18: 287-295) report on glycosylation variation in rhuIFNG. I've done it.

Kita et al. (Drug Des. Deliv. 6: 157-167, 1990) and EP 236987, US 5109120 have reported on polymer-modification of rhuIFNG.

WO 92/22310 describes interalia huIFNG, an asialo glycoprotein conjugate derivative of interferon.

IFNG fusion proteins have also been described. For example, EP 0 237 019 describes a single chain polypeptide consisting of a moiety with interferon β activity and a moiety with IFNG activity. EP 0 158 198 describes a single chain polypeptide consisting of a portion showing IFNG activity and a portion showing IL-2 activity. Several documents (eg Landar et al. (J. Mol. Biol., 2000, 299: 169-179)) describe single chain dimer IFNG proteins.

WO 99/02710 describes IFNG single chain polypeptides as one example of these.

WO 99/03887 describes PEGylated polypeptide variants belonging to the growth hormone superfamily, where non-essential amino acid residues located in specific portions of the polypeptide have been replaced with cysteine residues. IFNG has been mentioned as an example of a growth hormone superfamily member, but no mention has been made of the modification of IFNG in detail.

In Ziesche et al. (N. Engl. J. Med. 341: 1264-1269, 1999 and Chest 110: Suppl: 255, 1996) and EP 795332, IFNG is known as interstitial lung disease (also known as interstitial pulmonary fibrosis (IPF). It has been suggested that it can be used for the treatment of)), which can be used with prednisolone for this purpose. In addition to IPF, granulomatosis (Bolinger et al, Clinical Pharmacy, 1992, 11: 834-850), certain 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. Rheurnatol. 23: 654-658, 1996), hepatitis B ( Hepatogastroenterology 45: 2282-2294, 1998), Hepatitis C (Int. Hopatol. Communic. 6: 264-273, 1997), Pneumonia Shock (Nature Medicine 3: 678-681, 1997), Rheumatoid Arthritis, etc. It can also be cured.

Above all, rhuIFNG has been successfully used as a pharmaceutical compound for some viral infections and tumors. rhuIFNG can be used parenterally, via subcutaneous injection, as appropriate. After several hours, the maximum plasma concentration is reached. The half-life in plasma is about 30 minutes after iv administration. For this reason, frequent injections are required for effective treatment with rhuIFNG. Major side effects include fever, chills, sweating, headache, muscle pain and drowsiness. This side effect is associated with rhuIFNG injections, which are observed within the first few hours after injection. Rare symptoms include local pain, erythema, elevated liver enzymes, reversible granulocytopenia, thrombin reduction and cardiac poisoning.

WO 01/36001 discloses a novel IFNG conjugate comprising, for example, a non-polypeptide moiety attached to a modified IFNG polypeptide by the introduction and / or removal of an attachment site to such non-polypeptide moiety of PEG and glycosylation sites. have.

When N-glycosylation molecules, such as IFNG, are produced in glycosylation hosts, it is known that not all potential Nglycosylation moieties are utilized. Quite often this means that a mixture of proteins with in vivo N-glycosylation strains is obtained, which eventually leads to the conclusion that subsequent purification is required. Moreover, isolating the same protein with varying degrees of glycosylation is time consuming and cumbersome. Surprisingly, by substitution of one or more amino acid residues adjacent to the in vivo N-glycosylation site (as described in WO 01/36001, whether or not the in vivo N-glycosylation site has been obtained or the in vivo N-glycosylation site) It has recently been found that it is possible to obtain an increased portion of all glycosylated IFNG molecules, whether the site is naturally occurring in IFNG). In particular, changing the naturally occurring N-glycosylation site N-Y-S to N-Y-T at the 97, 98, and 99 positions of hIFNG increases the portion of the dramatically glycosylated IFNG molecule which is dramatically increased.

Brief description of the invention

Thus, in a first aspect, the present invention relates to an interferon gamma (IFNG) polypeptide variant having an amino acid sequence exhibiting IFNG activity and having an amino acid sequence represented by SEQ ID NO: 1 ([S99T] huIFNG) or a fragment thereof exhibiting IFNG activity. will be.

In another aspect, the invention relates to a variant of SEQ ID NO: 1 comprising a variant of a fragment of SEQ ID NO: 1 (eg, SEQ ID NOS: 2-16), wherein the variant is at least one Other modifications, including IFNG activity.

In another aspect, the invention relates to a nucleotide sequence that encodes a polypeptide variant of the invention.

In another aspect, the invention relates to an expression vector comprising the nucleotide sequence of the invention, and to a glycosylated host cell comprising the expression vector of the invention or the nucleotide sequence of the invention.

The invention also relates to a pharmaceutical composition comprising the polypeptide variant of the invention, a polypeptide variant of the invention, or a pharmaceutical composition of the invention for a drug.

In another aspect, the present invention relates to the use of polypeptide variants of the invention and to the use of the pharmaceutical compositions of the invention for the manufacture of a medicament for the treatment of interstitial lung disease.

Similarly, the present invention also relates to a method of treating or preventing interstitial lung disease, which method comprises administering to a mammal, in particular a human in need thereof, an effective amount of a polypeptide variant of the invention, or a pharmaceutical composition of the invention. It includes.

In another aspect, the invention relates to a composition comprising a population of IFNG polypeptide variants or a population of IFNG polypeptide variants, wherein said population comprises at least 70% of the IFNG polypeptide variants of the invention.

In another aspect, the invention relates to a method for vaporizing in vivo N-glycosylation of a parental IFNG polypeptide comprising at least one in vivo N-glycosylation site having the amino acid sequence of NXS, wherein X is any proline Any amino acid residue except for the above method, wherein the method includes substituting a seronine residue with a threonine residue in the NXS amino acid sequence to obtain an IFNG variant.

In another aspect, the invention relates to a method for producing an IFNG polypeptide variant of the invention, wherein the method

(a) culturing a glycosylated host cell comprising a nucleotide sequence encoding the INFG polypeptide variant of the invention under favorable conditions for expression of the polypeptide variant;

(b) optionally reacting said polypeptide variant in vitro with a non-polypeptide moiety under conditions favorable for occurrence of conjugation;

(c) recovering the polypeptide variants

It includes.

In another aspect, the present invention will become apparent from the following description.

The present invention relates to novel interferon gamma polypeptide variants having interferon gamma (IFNG) activity, methods of making the same, pharmaceutical compositions comprising the polypeptide variants and the treatment of diseases, in particular interstitial such as idiopathic pulmonary fibrosis And their use in the treatment of lung disease.

1 is a western blot of optimized glycosylation variants of rhuIFNG.

Left western blot: 1 lane; Standard, Lane 2: Actimmune, Lane 3: rhuIFNG, Lane 4: [E38N] rhuIFNG. Middle western blot: 1 lane; Standard, 2 lanes: rhuIFNG, 3 lanes: [E38N + S40T] rhuIFNG. Right western blot: 1 lane; Standard, 2 lanes: rhuIFNG, 3 lanes: [S99T] rhuIFNG, 4 lanes: [E38N + S40T + S99T] rhuIFNG.

2 shows IFNG activity in serum-time curves after subcutaneous administration to rats. ●: Actimmune, ■: rhuIFNG, ▲: [E38N + S40T + S99T] rhuIFNG.

3 shows IFNG activity in serum-time curves after subcutaneous administration to rats. ●: [N16S + S99T] rhuIFNG (with 5kDa mPGA), ■: [N16S + S99T] rhuIFNG (with 10kDa mPGA), ▲: [E38N + S40T + S99T] rhuIFNG.

The [E38N + S40T + S99T] rhuIFNG variant was administered at a dose of 1.15 × 10 ⁷ AU / kg, while the two PEGylated variants were administered at a dose of 4.6 × 10 ⁶ AU / kg.

Justice

In the context of the present application and invention the following definitions may apply;

A conjugate (or interchangeably "conjugated polypeptide" or "conjugated variant") refers to a heterogeneous (in composition and chimeric sense) molecule formed by covalently attaching one or more polypeptides to one or more non-polypeptide moieties. It is intended to be. The term covalent attachment means that the polypeptide and non-polypeptide moieties are directly covalently bonded to each other or indirectly covalently bonded through an intermediary moiety such as a linking bridge, a space or a linking moiety. Preferably the conjugate polypeptide variant is soluble under relevant concentrations and conditions, for example in physiological fluids such as blood. Examples of conjugated polypeptide variants of the invention include glycosylated and / or PEGylated polypeptides. The term "non-conjugated polypeptide portion" refers to the polypeptide portion of the conjugated polypeptide variant.

"Non-polypeptide moiety" is intended to denote a molecule capable of binding to the attachment group of an IFNG polypeptide variant. Such molecules may include, for example, polymer molecules, lipophilic molecules, sugar moieties or organic inducing agents. It is to be understood that the non-polypeptide moiety is linked to the polypeptide of the conjugate via the attachment group of the polypeptide variant. Except where expressly expressed the number of nonpolypeptides, such as polymer molecules attached to an IFNG polypeptide moiety, all criteria for “non-polypeptide moieties” attached to IFNG polypeptide variants or otherwise used in the present invention refer to IFNG polypeptide variants. Reference will be made to the attached nonpolypeptide moiety.

A "polymer molecule" refers to a molecule formed by two or more monomers covalently bonded, wherein the monomers are not amino acid residues. "Polymer" can be used interchangeably with "polymer molecule".

"Sugar part" refers to a carbohydrate molecule attached by in vivo and in vitro glycosylation reactions such as N- or O-glycosylation.

"N-glycosylation site" has an NXS / T / C sequence, where X is any amino acid residue except proline, N is asparagine, and S / T / C is serine, threonine, or cysteine, Preferably serine or threonine, and most preferably threonine. "O-glycosylation site" is the OH- group of a serine or threonine residue.

An "adherent" is intended to refer to a group of amino acid residues capable of binding to a related non-polypeptide moiety, such as a polymeric molecule or sugar moiety. Useful attachment groups and paired non-polypeptide moieties will be apparent from the following table.

In the case of in vivo glycosylation, an "adhesive group" has an N-glycosylated site (SEQ ID NO: S / T / C sequence, where X represents any amino acid residue except proline, and N represents asparagine, S / T / C represents serine, threonine or cysteine and is preferably used in a free manner to indicate the amino acid residues that make up serine or threonine, most preferably threonine). Asparagine residues in the N-glycosylation moiety are where the sugar moiety is attached during the glycosylation reaction, but such attachment can only occur when other amino acid residues in the N-glycosylation reaction are present. Thus, when the non-polypeptide moiety is a sugar moiety and the conjugation is obtained by N-glycosylation, an "amino acid residue comprising an attachment group of the non-polypeptide moiety" when used in connection with amino acid sequence modification of an IFNG polypeptide Is a serine in which a functional N-glycosylation site is introduced into or removed from the sequence, or a functional N-glycosylation site is maintained in the amino acid sequence (eg, already part of an N-glycosylation site). It is to be understood that by substituting with a residue threonine residue and vice versa, one, two or both of the amino acid residues making up the N-glycosylation moiety are altered.

In the present invention, amino acid names and atomic names (eg, CA, CB, CD, CG, SG, NZ, N, O, C, etc.) use those defined by Protein DataBank (PDB) (www.pdb.org). IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atom narnes etc.) and Eur. J. Biochem. 138, 9-37 (1984) and their revisions (Eur. J. Biochem. 152, 1 (1985)) CA is sometimes referred to as Cα and CB as Cβ "amino acid residues" are intended to refer to amino acid residues belonging to: alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), cystidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Glu or Q), arginine (Arg) Or R) , Serine (Ser or S), threonine (Thr or T), valine (Vla or V), tryptophan (Trp or W), tyrosine (Tyr or Y).

The number of amino acid residues in this document is from the N-terminus of [S99T] huIFNG without the signal peptide (ie SEQ ID N: 1), or from the N-terminus of huIFNG without the signal peptide (ie SEQ ID NO: 17), if relevant. Count it.

The terms used to identify amino acid positions and substituents may be described as follows; G18 represents position 18 occupying glycine in the amino acid sequence found in SEQ ID NO: 1 or SEQ ID NO: 17. G18N indicates that the Gly residue at position 18 is substituted with Asn. Multiple substituents are represented by "+", for example G18B + S20T refers to an amino acid sequence comprising substitution of Gly residues for Asn at position 18 and Ser for Thr at position 20. Optionally, the substitution may be expressed as "/". For example, G18S / T covers the following individual substitutions: G18S and G18T. Deletion is represented by an asterix. For example G18 * indicates that the Gly residue was deleted at position 18. The insertion is expressed in the following manner: The insertion of additional Ser residues after the Gly residue located at position 18 is expressed as G18GS. Combined substitutions and insertions are expressed in the following manner: substitution of the Gly residues with the Ser residues at position 18 and insertion of Ala after the 18 position amino acid residues is represented with G18SA.

A "nucleotide sequence" refers to the contiguous extension of two or more nucleotide molecules. The nucleotide sequence may be a genome, cDNA, RNA, semisynthetic or synthetic origin or any complex portion thereof.

"Polymerase chain reaction" or "PCR" generally refers to a method used to amplify a desired nucleotide sequence in vitro and is also described in US Pat. No. 4,683,195. In general, the PCR method is to repeat the primer extension synthesis process using oligonucleotide primers that can preferentially hybridize to the template nucleic acid.

"Cells", "host cells", "cell lines", "cell cultures" may be interchanged with each other, and these terms should be understood to include progeny generated due to the growth or culture of the cells.

"Transformation" or "transfection" refers to a process of introducing DNA into a cell and is used interchangeably.

“Operably linked” refers to the covalent binding of two or more nucleotide sequences in one shape with respect to each other by enzymatic ligation or other means, such that the sequences are linked so that they can function normally. For example, a nucleotide encoding a pre-sequence or secretory leader is linked so that it can act on the nucleotide sequence that encodes the polypeptide to be expressed as a pre-protein that participates in polypeptide secretion. To say that it can; That the promoter or enhancer is linked to act on the coding sequence means that it can affect the transcription of the sequence; Coordination of the coding sequence to the ribosomal binding site means that it is positioned to perform translation. In general, “operably linked” refers to the contact of a linked nucleotide sequence, and in the case of a secretory leader, to contact on a reading. Linkage is achieved by ligation in the usual restriction enzyme moiety. If such sites do not exist, the synthesized oligonucleotide adapters or linkers can be used in parallel with standard recombinant DNA methods.

"Altered", as used herein, covers substitutions, insertions and removals.

"Modification" and "substitution" are used interchangeably herein.

"Introduce" means replacing an existing amino acid residue, but may also mean inserting additional amino acid residues.

"Remove" means substitution of another amino acid residue with an amino acid residue to be removed, but can also mean a deletion (without substitution) of an amino acid residue to be removed.

"Amino acid residues comprising an attachment group for non-polypeptide moieties" refers to cases where an amino acid residue binds to a non-polypeptide moiety (in the case of an introduced amino acid residue) or is bound (in the case of an removed amino acid residue).

As used in connection with a particular modification, the meaning of "one difference" or "different" means to allow for additional differences that exist separately from the specific amino acid difference. Thus, in addition to amino acid residue modifications disclosed herein aimed at optimizing the use of glycosylation sites or for introducing or removing amino acid residues comprising attachment groups for nonpolypeptide moieties, IFNG polypeptide variants are not related to such modifications. Changes can be included if necessary. For example, they are cleavage of the C-terminus by one or more amino acid residues, addition at the N- and / or C-terminus of at least one extra residue, for example addition at the N-terminus of the Met residue, amino acid sequence Cys At the N-terminus of -Tyr-Cys, and "conservative amino acid residue substitutions", that is to say that have similar properties, for example small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and Substitutions are performed within a group of amino acids, such as aromatic amino acids. Examples of conservative substitutions in the present invention are specifically meant to be selected from the conservative substituents described in the table below.

"At least one" as used for nonpolypeptide moieties, amino acid residues, substitutions, etc., is intended to mean one or more.

"AUC _sc " or "area under the curve when injected subcutaneously" is used in its normal sense, that is, the area under IFNG activity in the serum-time curve, where the IFNG polypeptide variant can be injected subcutaneously, Especially when injected subcutaneously in rats. Once the experimental IFNG activity-time point is determined, the AUC _sc can typically be calculated by a computer program such as GraphPad Prism 3.01.

" In vivo functional half-life" is the time at which 50% of the biological activity of the polypeptide is still present in the body / target organ, or at which time the activity of the polypeptide is 50% of its initial value.

As an alternative to measuring functional in vivo half-life, a "serum half-life" can be determined, ie the time to circulate in the serum or blood stream before 50% of the polypeptide is removed. Determination of serum half-life is simpler than determining functional in vivo half-life, and the size of serum half-life is typically a good indicator of the size of functional in vivo half-life. Optional terms for serum half-life include “plasma half-life”, “circulating half-life”, serum clearance ”,“ plasma clearance ”and“ clearance half-life. ”Serum half-life can typically be determined in rats, where materials and methods See section It should be noted that when "serum half-life" is used for the IFNG polypeptide variants given herein (iv).

"Serum" as used herein in its usual sense, ie, in the absence of fibrinogen and other coagulation factors.

Polypeptides are normally removed by one or more of the actions of the reticular endothelial cell system (RES), kidney, spleen or liver, or by specific or nonspecific proteolysis. The term "renal clearance" refers to renal, for example glomerular filtration, coronary excretion. Or as its normal meaning to indicate any clearance caused by coronary removal. Typically, renal clearance depends on the physical properties of the polypeptide and includes the presence of cellular receptors for molecular weight, size (relative filtering by glomerular filtration), symmetry, shape / strength, charge, attached carbohydrate chains and proteins. A molecular weight of about 67 kDa is recognized as the cutoff value of elongation clearance. Renal clearance can be measured by any suitable measurement, eg, established in vivo measurements. For example, renal clearance can be determined by introducing a labeled (eg, radiolabeled or fluorescently labeled) conjugated polypeptide into a patient and measuring the labeling activity from urine recruited from the patient. Reduced kidney clearance is measured in relation to the reference molecule, for example huIFNG, [S99T] huIFNG or Actimmune. The functionality retained is selected as antiviral, antiproliferative, immunomodulatory or IFNG receptor binding activity.

"Increase" used for functional half-life or serum half-life in vivo means that the relevant half-life of huIFNG is a reference molecule, eg, glycosylated huIFNG (SEQ ID NO: 17), glycosylated [S99T] huIFNG (SEQ ID NO: 1) or functional half-life at a statistically significant level compared to Actimmune (produced by SEQ ID NO: 34-E. Coil), administered intravenously and increased in comparable conditions. A, Lesa. Interesting IFNG polypeptide variants are those with increased in vivo functional half-life or increased serum half-life compared to any of the reference molecules mentioned above.

More particularly, the IFNG variants of interest have a ratio between the serum half-life (or functional in vivo half-life) of the variant and the serum half-life (or functional in vivo half-life) of huIFNG or [S99T] huIFNG in glycosylated form, especially When administered intravenously to rats, it is a variant that is at least 1.25, more preferably 1.75, for example at least 2, even more preferably at least 3, for example 4, for example 5.

Other examples of IFNG of interest include the serum half-life (or functional in vivo half-life) of the variant in glycosylated form and the serum half-life (or functional in vivo half-life) of Actimmune (generated from SEQ ID NO: 34-E.Coli). When the ratio between them is administered intravenously, in particular intravenously to rats, at least 2 more preferably 3, eg at least 4, even more preferably at least 6, eg at least 7, eg 8, most preferred Preferably at least 9, for example at least 10 variants.

The term “increased” as used for AUC _SC is glycosylated huIFNG (SEQ ID NO: 17), when administered substantially, when the area under the curve for the IFNG variant of the invention is determined under comparable conditions, Compared to any reference molecule, such as glycosylated [S99T] huIFNG (SEQ ID NO: 1), or Actimmune (produced in SEQ ID NO: 34-E. Coil). Thus, preferred variants are those variants with an increased AUC _sc compared to any of the reference molecules mentioned above. Obviously, the same amount of IFNG activity should be administered to the IFNG variants and reference molecules of the invention. As a result, to make direct comparisons between different IFNG molecules, AUC _sc values can be normalized, ie they can be expressed in AUC _sc / administered doses.

Particularly preferred IFNG variants have at least 1.25, more preferably at least 1.5 when the ratio between the AUC _sc of the variant and the AUC _sc of glycosylated huIFNG or AUC _sc of glycosylated [S99T] huIFNG is administered (particularly subcutaneously). , For example 2, even more preferably at least 3, for example at least 4, for example at least 5 or at least 6, even more preferably at least 7, for example at least 8, for example at least 9 or at least 10, most Preferably at least 12, for example at least 14, for example at least 16, at least 18, or at least 20 variants.

Another example of particularly preferred IFNG is that the ratio between the AUC _sc of the variant and the AUC _sc of Actimmune is at least 100, more preferably 150, for example at least 200, for example at least, when administered (subcutaneously) to rats. 250, even more preferably at least 300, for example at least 400, for example 500, most preferably at least 750, for example at least 1000, for example at least 1500 or at least 2000.

The term "Tmax, sc" is used for time in IFNG activity when the highest IFNG activity is observed in the bloodstream in the serum-time curve. Preferred IFNG variants of the invention are those variants having an increased Tmax, sc "for Actimmune and / or glycosylated huIFNG. More particularly, such preferred variants are at least 200 minutes, for example at least 250 minutes, for example at least Tmax, sc (as measured after subcutaneous administration to rats) of 300 minutes, more preferably at least 350 minutes, for example at least 400 minutes.

"Reduced immunogenicity" refers to an IFNG polypeptide variant which, when measured under this comparable condition, results in a significantly lower immune response than reference molecules such as huIFNG and Actimmune. The immune response is by cell or antibody mediated response (see Roitt: Essential Immunology (8 ^th Edition, Blackwell) for further definition of immunogenicity). Typically, reduced antibody activity is indicative of reduced immunogenicity. Reduced immunogenicity can be measured using appropriate methods known in vivo, or in vitro.

In this context, the terms "increased glycosylation", "increased in vivo N-glycosylation", or "increased N-glycosylation" are commonly obtained as a result of increased use of glycosylation sites. It is intended to show an increased level of hydrocarbon molecules. When huIFNG is expressed in CHO cells, only about 50% of IFNG molecules use both glycosylation moieties, about 40% use one glycosylation site (1N), and about 10% are not glycosylated (0 N) (Hooker et al., 1998, J. Interferon and Cytokine Res. 18, 287-295 and Sarenva et al., 1995, Biochem J., 308, 9-14). Increased in vivo N-glycosylation can be measured by any suitable method known in the art, such as SDS-PAGE. One convenient assay for measuring increased glycosylation is found here in the Materials and Methods section in the manner described in the section "Measurement of Increased Glycosylation".

Wherein the term "community of IFNG polypeptide variants" or "composition comprising a population of IFNG polypeptide variants" covers a composition comprising at least two IFNG polypeptide variants glycosylated to different degrees, wherein the increase present in the population Amount of IFNG molecules are all glycosylated.

Thus, the invention also relates to a composition comprising a homogeneous population of the IFNG polypeptides of the invention (eg, a population in which most IFNG polypeptides are fully glycosylated) or a homogeneous population of IFNG polypeptides of the invention. For example, the population of IFNG polypeptides may comprise at least 70% of the IFNG polypeptides of the invention, preferably at least 75%, for example 80%, for example at least 85%, more preferably at least 90%, In one example at least 95%, for example at least 96%, even more preferably at least 97%, in one example at least 98%, for example 99%.

"IFNG shows the activity" it is to say that the polypeptide variant has one or more unique huIFNG or rhuIFNG function of IFNG, in particular the ability to be bonded to the IFNG receptor and in vitro or in vivo (in vitro or in vivo bioactivity When measured at), functions including the signal causing conversion of the receptor upon conversion to huIFNG are included. IFNG receptors are described in Agues. et. (Cell 55: 273-280, 1988) and Calderon et al. (Proc. Natl. Acad. Sci. USA 85: 4837-4841, 1988). When using an IFNG activated “primary assay”, polypeptide variants “having IFNG activity” have a specific activity of at least 5% relative to rhuIFNG. It is understood that as certain modifications are made, for example, depending on whether the variant is PEGylated, a wide range of activities can be achieved. Thus, an example of specific activity is at least 10% (eg 10-125%), for example at least 15% (eg 15-125%), for example at least 20% (relative to the specific activity of rhuIFNG) For example 20-125%), at least 20% (eg 25-125%), at least 30% (eg 30-125%), at least 35% (eg 35-125%), at least 40% (eg 40-125%), at least 45% (eg 45-125%), at least 50% (eg 50-125%), at least 55% (eg 55-125%), at least 60% (eg 60- 125%), at least 65% (eg 65-125%), at least 70% (eg 70-125%), at least 75% (eg 75-125%), at least 80% (eg 80-125%) ), At least 90% (eg 90-110%).

An "IFNG polypeptide" is a polypeptide that exhibits IFNG activity, that is, the term "IFNG polypeptide" refers to any IFNG molecule (the molecule is huIFNG, its truncated form, or a combination thereof) so long as the IFNG polypeptide molecule exhibits IFNG activity as defined herein. Whether or not variants). The term "IFNG polypeptide" refers to a monomer or dimeric polypeptide. For example, where there are certain substitutions, they normally relate to IFNG polypeptide monomers. When referring to the IFNG portion of the conjugate of the present invention, it is usually a dimeric form (and thus consists of two modified IFNG polypeptide monomers described herein). Dimeric IFNG polypeptides refer to cases where two monomers are commonly associated or make a single chain dimeric IFNG polypeptide.

"Mother" refers to an IFNG polypeptide having a glycosylation moiety to be modified in accordance with the present invention. Although the parent polypeptide to be modified in accordance with the present invention may be any polypeptide having IFNG activity, and thus may be derived from any origin, such as a non-human mammalian origin, the parent polypeptide has the amino acid sequence shown in SEQ ID NO; It is preferable that it is huIFNG which has the fragment.

"Fragment" means a portion of a full-length IFNG polypeptide sequence exhibiting IFNG activity, eg, a C-terminal or N-terminal truncation thereof (eg, a portion of a full-length huIFNG polypeptide as shown in SEQ ID NO: 17 or Full length [S99T] huIFNG fragment shown in SEQ ID NO: 1). Specific examples of IFNG polypeptide variant fragments include those C-terminally truncated to 1-15 amino acid residues with [S99T] huIFNG, for example one amino acid residue (SEQ ID NO: 2), two amino acid residues (SEQ ID NO: 3), 3 amino acid residues (SEQ ID NO: 4), 4 amino acid residues (SEQ ID NO: 5), 5 amino acid residues (SEQ ID NO: 6), 6 amino acid residues (SEQ ID NO: 7), 7 amino acid residues (SEQ ID NO: 8), 8 amino acid residues (SEQ ID NO: 9), 9 amino acid residues (SEQ ID NO: 10), 10 amino acid residues (SEQ ID NO: 11), 11 amino acid residues (SEQ ID NO: 12 ), 12 amino acid residues (SEQ ID NO: 13), 13 amino acid residues (SEQ ID NO: 14), 14 amino acid residues (SEQ ID NO: 15), 15 amino acid residues (SEQ ID NO: 16), and / or 1 N-terminal cleavage with -3 amino acid residues. Specific examples of huIFNG fragments include huIFNG, which are C-terminally truncated to 1-15 amino acid residues, for example one amino acid residue (SEQ ID NO: 19), two amino acid residues (SEQ ID NO: 10), 3 amino acid residues (SEQ ID NO: 21), 4 amino acid residues (SEQ ID NO: 22), 5 amino acid residues (SEQ ID NO: 23), 6 amino acid residues (SEQ ID NO: 24), 7 amino acid residues (SEQ ID NO: 25), 8 amino acid residues (SEQ ID NO: 26), 9 amino acid residues (SEQ ID NO: 27), 10 amino acid residues (SEQ ID NO: 28), 11 amino acid residues (SEQ ID NO: 29), 12 Amino acid residue (SEQ ID NO: 30), 13 amino acid residue (SEQ ID NO: 31), 14 amino acid residue (SEQ IDNO: 32), or 15 amino acid residue (SEQ ID NO: 33), and / or 1-3 amino acids N-terminal cleavage with residues.

As above, an IFNG polypeptide variant may comprise at least one additional modification other than an S99T substitution, as long as the variant exhibits IFNG activity. That is, the variant may be a [S99T] huIFNG variant, or a variant of the [S99T] huIFNG fragment. Specific examples of such variants are those having introduced and / or removed amino acid residues comprising attachment groups for nonpolypeptide moieties. Other examples of variants of [S99T] huIFNG (and fragments thereof) are those described in the section “Background of the Invention”, for example [S99T] having Met having an N-terminal insertion Cys-Tyr-Cys. huIFNG and includes cysteine modified variants as disclosed in US Pat. No. 6,046,034.

Typically, variants according to the invention are encoded by nucleotide sequences, which have been modified in accordance with the invention as compared to the nucleotide sequences encoding the parent IFNG polypeptide.

This is not the case, however, because variant polypeptides may undergo C- or N-terminal cleavage during post-translational processes, e.g., purification of C- or N-terminal cleavage by proteases in intracellular expression media. And the resulting variant polypeptide is a truncated form of the originally produced variant polypeptide (e.g., even if the full-length variant is produced early, the C-terminal truncated variant polypeptide is subjected to the post-translational process of the full-length variant polypeptide. Can be obtained). In this case, the term "parent" should be construed as a cut form modified according to the invention.

A “variant” refers to a polypeptide that differs from one or more amino acid residues in the parent polypeptide (usually any truncation of those shown in SEQ ID NO: 17 or SEQ ID NOS: 19-33), usually typically 1-15 amino acid residues. (Eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues), for example 1-10 amino acid residues, 1-5 Amino acid residues, or 1-3 amino acid residues, are different.

"Function site" refers to one or more amino acid residues that are essential to be involved in the function or operation of IFNG, or otherwise related to the function or performance of IFNG. Such amino acid residues are "located" at the site of function. Sites that function can be found by methods known in the art, preferably by analyzing the structure of a polypeptide complexed with a related receptor, such as an IFNG receptor.

"huIFNG" is intended to cover the mature form of wild-type human IFNG with the amino acids shown in SEQ ID NO: 17.

"rhuIFNG" is intended to cover the mature form of wild-type human IFNG with the amino acids shown in SEQ ID NO: 17, which is produced by recombinant means.

"[S99T] huIFNG" is intended to cover the mature form of wild-type human IFNG with a serine residue at position 99 substituted with a threonine residue (published at SEQ ID NO: 1).

As used herein, “glycosylated huIFNG” is produced in a cell in which a huIFNG polypeptide can glycosylate the polypeptide, and therefore, the huIFNG polypeptide is at its original N-glycosylation site (positions 25 and 29 of SEQ ID NO: 17). Points to glycosylation.

In a similar manner, the term “glycosylated huIFNG variant” indicates that IFNG polypeptide variants are produced in cells capable of glycosylation of polypeptide variants.

As used herein, the term "Actimmune" is used to determine the 140 amino acid type of IFNG (SEQ ID NO: 34), which is usually achieved by fermentation of a processed E. Coil bacterium (Actimmune is cleaved C-terminally to four amino acid residues). To mention. Additional information on Actimmune is available at www.actimmune.com.

Interferon Gamma Polypeptide Variants of the Invention

IFNG variants of the invention optimized at in vivo glycosylation sites

As mentioned above, surprisingly, glycosylation of naturally occurring N-glycosylation sites located at position 97 of huIFNG can be increased, i.e., an increased fraction of all or substantially all glycosylated IFNG molecules is huIFNG. It has been found that it can be obtained by substituting a threonine residue for a serine residue located at position 99 of the (or fragment thereof). A review of FIG. 1 shows that a significant increase in the degree of all glycosylated IFNG polypeptide can be obtained. For the [S99T] huIFNG (SEQ ID NO: 1) polypeptide, about 90% of the polypeptide variants present in the harvested media utilized both N-glycosylation sites, whereas about 60 rhuIFNG polypeptides present in the harvested media Only% was all glycosylated.

Thus, in one aspect of the invention the invention relates to an IFNG polypeptide variant which exhibits IFNG activity and has an amino acid sequence as shown in SEQ ID NO: 1 (ie [S99T] huIFNG activity), or a fragment thereof which exhibits IFNG activity. .

As mentioned above, the C-terminal truncated form of huIFNG retains its activity, and in some cases increases its activity relative to huIFNG. Thus, in a preferred embodiment of the invention, the IFNG polypeptide variant of the invention is a fragment of SEQ ID NO: 1, which is C-terminally truncated to 1-15 amino acid residues, and typically C-terminally to 1-10 amino acid residues. Is cut. Details of such C-terminal truncation forms of SEQ ID NO: 1 are disclosed in SEQ ID NO: 2-16. IFNG polypeptide fragments according to embodiments of the invention exhibit IFNG activity.

Glycosylated IFNG polypeptide variants according to this aspect are recombinantly expressed in glycosylated host cells, preferably mammalian host cells, such as those mentioned in the “Conjugation to Part” title section.

As always described, only 50-60% of the total population of expressed IFNG polypeptides are fully glycosylated when rhuIFNG is expressed in CHO cells. Thus, one of the major advantages of the IFNG polypeptide variants of the present invention is the higher utilization of position 97 in in vivo N-glycosylation, which in turn results in a more homogeneous community than huIFNG. Due to the more homogeneous populations, compositions comprising such populations of IFNG polypeptide variants (eg, harvest medium) do not require a cumbersome and time consuming purification process as rhuIFNG.

Thus, in another aspect the invention relates to a population of IFNG polypeptide variants, or to a composition comprising a population of IFNG polypeptide variants, wherein said population comprises at least 70% of the IFNG polypeptide variants of the invention, preferably, at least 75%, more preferably at least 80%, for example about 90% of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 , An IFNG polypeptide variant fragment having an amino acid sequence selected from the group consisting of SEQ ID NO: 16.

In addition to the previously mentioned S99T modifications required for optimization of the in vivo glycosylation site at position 97 of rhuIFNG, introduced with SEQ ID NO: 1 or a fragment thereof (e.g., increases serum half-life and / or increases AUCsc). Other in vivo glycosylation sites can be optimized. Typically, in vivo glycosylation sites are N-glycosylation sites, but also O-glycosylation sites are contemplated in the context of the present invention. Such optimization can be achieved through performance of the modification, preferably substitution, at a position adjacent to the glycosylation site, in particular close to the in vivo N-glycosylation. Typically, such in vivo N-glycosylation sites are introduced in vivo N-glycosylation sites. Details of suitable locations for introducing in vivo N-glycosylation sites are further disclosed in WO 01/36001 and below.

Amino acid residues "closely located" at the glycosylation site are at the -4, -3, -2, -1, +1, +2, +3 or +4 position relative to the amino acid residue of the glycosylation site to which the hydrocarbon is attached. It is usually located, preferably -1, +1, or +3, in particular position +1 or +3. Thus, amino acid residues located close to the in vivo N-glycosylation site (SEQ ID NOS / T / C) are -4, -3, -2, -1, +1, +2, +3 with respect to the N-residue , Or +4.

When the +2 position is modified in relation to the N-residue, only a limited number of modifications are possible since the amino acid residues at that position must be Ser, Thr or Cys in order to maintain / introduce the glycosylation site in vivo. I understand.

In a particularly preferred embodiment of the invention, the modification of the amino acid residue at the + 2 position with respect to the in vivo N-glycosylation site is a substitution wherein the amino acid residue in question is replaced by a Thr residue. On the other hand, if the amino acid residue is already a Thr residue, it is not necessary or desirable to perform any substitution at that position. When X is modified, X should not be Pro and preferably Trp, Asp, Glu, and Leu. Also introduced amino acid residues are from Phe, Asn, Gln, Tyr, Val, Ala, Met, Ile, Lys, Gly, Arg, Thr, His, Cys and Ser, preferably Ala, Met, Ile, Lys, Gly, Arg, Thr, His, Cys, and Ser, in particular Ala, or Ser.

When the + 3 position is changed in relation to the N-residue, the amino acid residues introduced are composed of His, Asp, Ala, Met, Asn, Thr, Arg, Ser and Cys, more preferably Thr, Arg, Ser and Cys It is preferably selected from the group.

Thus, in view of naturally existing in vivo N-glycosylation, it is expected that the N-glycosylation site at position 97 can be further optimized by performing modifications, E93, K94, L95, T96, Y98, V100 and T101 ( Ie, at position -4, -3, -2, -1, +1, +3 or +4 relative to N97). Specific examples of the substitutions performed at position 98 of SEQ ID NO: 1 (or a fragment thereof) include Y98F, Y98N, Y98Q, Y98V, Y98A, Y98M, Y98I, Y98K, Y98G, Y98R, Y98T, Y98H, Y98C and Y98S, Preferably Y98A, Y98M, Y98I, Y98K, Y98G, Y98R, Y98T, Y98H, Y98C and Y98S, in particular Y98S. Specific examples of the substitutions performed at position 100 of SEQ ID NO: 1 (or a fragment thereof) include V100H, V100D, V100A, V100M, V100N, V100T, V100R, V100S, or V100C, in particular V100T, V100R, V100S or V100C. Include.

In a similar manner, from the point of view of in vivo N-glycosylation at position 25, these sites are D21, V22, A23, D24, G26, L28 and F29 (ie -4, -3, -2, -1 with respect to the N25 position). It can be further optimized by performing modifications such as substitutions at positions selected from the group consisting of) at the +1, +3 or +4 position. Specific examples of the substitutions performed at position 26 of SEQ ID NO: 1 (or a fragment thereof) include G26F, G26N, G26Y, G26Q, G26V, G26A, G26M, G26I, G26K, G26R, G26T, G26H, G26C, and G26S, Preferably G26A, G26M, G26I, G26K, G26R, G26T, G26H, G26C and G26S, more preferably G26A and G26S, in particular G26A. Specific examples of the substitutions performed at position 28 of SEQ ID NO: 1 (or a fragment thereof) are G28H, G28D, G28A, G28M, G28N, G28T, G28R, G28S, or G28S, in particular G28A, G28T, G28R, G28S or Includes G28C.

Obviously, any of the changes mentioned in connection with the optimization of glycosylation at position 97 may be combined with any of the above changes made in connection with optimization of glycosylation at position 25.

Inventive IFNG Variants With Increased AUSsc and / or Increased Serum Half-Life

In another aspect the invention relates to an IFNG polypeptide variant having the amino acid sequence shown in SEQ ID NO: 1, wherein the variant exhibits IFNG activity.

Moreover, the present invention also provides SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16 To a variant of an IFNG polypeptide fragment having an amino acid sequence, wherein said variant exhibits IFNG activity.

Thus, such variants include at least one or more additional modifications relative to SEQ ID NO: 1-16.

To avoid too much disruption of the structure and function of the [S99T] huIFNG polypeptide variant (or fragment thereof), the total number of amino acid residues modified in accordance with the present invention is typically no greater than 15. Usually, IFNG polypeptide variants include 1-10 modifications with respect to the amino acid sequence shown in SEQ ID NO: 1, for example 1-8, 2-8, with respect to the amino acid sequence shown with SEQ ID NO: 1, 1-5, 1-3, or 2-5 variants. Preferably the modification is a substitution. Similar thoughts are understood to be true for variants of IFNG polypeptide variant fragments having the amino acid sequence shown in SEQ ID NO: 1. Thus, when the variant is a variant of any sequence disclosed in SEQ ID NOS: 2-16, such variant generally comprises less than 15 modifications, typically in relation to the relevant amino acid sequence shown in SEQ ID NOS: 2-16. 1-10 modifications and, for example, 1-8, 2-8, 1-5, 1-3, or 2-5 modifications relative to the relevant amino acid sequence shown in SEQ ID NOS: 2-16. Preferably, the change is a substitution.

Thus, typically such IFNG polypeptide variants (ie, variants comprising at least one additional alteration other than S99T substitutions are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). , Amino acid sequences (or fragments thereof) that differ from the amino acid sequence shown in SEQ ID NO: 1 at 15 amino acid residues.

In a preferred embodiment, the IFNG variant (also) comprises at least one introduced and / or removed amino acid residue comprising an attachment group for a nonpolypeptide moiety.

By removing and / or introducing amino acid residues comprising a conjugate to a non-polypeptide moiety, it is possible to specifically conjugate the polypeptide, in order to optimize the conjugation pattern (eg, to optimize the non-polypeptide moiety at the surface of an IFNG polypeptide variant). To ensure dispersion) to make the molecule more sensitive to conjugation to the non-polypeptide selection moiety, thereby obtaining a new conjugate molecule, which has one or more enhanced properties compared to currently available huIFNG- or rhuIFNG-based molecules, Activity. For example, by introducing an attachment group, the IFNG polypeptide variant is enhanced or otherwise altered in the content of the specific amino acid residue to which the relevant nonpolypeptide moiety is bound, thereby achieving more effective, specific and / or extensive conjugation. do. By removing one or more attachment groups, conjugation to non-polypeptide moieties, such as amino acid residues located at or near the functional site of the polypeptide, may be avoided in the portion of the polypeptide where such conjugation is disadvantageous (such site by impaired receptor recognition). Conjugation can lead to inactivation or reduced IFNG activity of the resulting conjugation polypeptide).

It may also be advantageous to remove the attachment located adjacent to other attachments in order to avoid release bonding to such groups. In one embodiment of the invention, one or more IFNG polypeptide amino acid residues are modified and the alteration comprises the introduction and removal of amino acid residues comprising an attachment group for the selection site of the nonpolypeptide. This example is of particular interest because it is possible to design IFNG polypeptides specifically to obtain optimal conjugation to the nonpolypeptide moiety.

In addition to the removal and / or introduction of amino acid residues, polypeptide variants may include other modifications, for example, substitutions that do not involve the introduction and / or removal of amino acid residues comprising attachment groups for non-polypeptide moieties. Examples of such alterations are conservative amino acid substitutions at the N terminus and / or introduction of Cys-Tyr-Cys or Met.

The exact number of attachment groups available for conjugation, and present in dimeric form in IFNG polypeptide variants, depends on the desired effect achieved by conjugation. The effect obtained depends, for example, on the degree of conjugation and nature (e.g., identity of nonpolypeptides, the number of nonpolypeptides for which conjugation is desired or possible to the polypeptide, where they should be conjugated, where conjugation should be avoided, etc.). .

Amino acid residues comprising attachment groups for non-polypeptide moieties, whether introduced or removed, are selected based on the nature of the non-polypeptide selection moiety and in most cases based on the conjugation method used. For example, when the nonpolypeptide moiety is a polymer molecule, eg, a polyethylene glycol- or polyalkylene oxide-derived molecule, amino acid residues that can function as attachment groups include cysteine, lysine, aspartic acid, glutamic acid, and arginine It may be selected from the group consisting of. In particular, cysteine is preferable. If the nonpolypeptide is a sugar moiety, the attachment group is for example an in vivo glycosylation site, preferably an N-glycosylation site.

Whenever an attachment to a nonpolypeptide moiety is introduced into or removed from an IFNG polypeptide having an amino acid sequence shown as SEQ ID NO: 1 (or a fragment thereof), the position of the polypeptide to be modified is typically selected as follows: ;

The position is preferably located on the surface of the IFNG polypeptide, more preferably occupied by an amino acid residue having a side chain exposed to at least 25% solvent, or more preferably an amino acid residue having a side chain exposed to at least 50% solvent. It is preferred to occupy, and can be determined based on a dimer type of IFNG 3D or model, and the structure or model may consist of one or two IFNG receptor molecules. Such locations are listed here in Example 1.

Also of interest is the modification of any of the 23 C-terminal amino acid residues of the parent IFNG, which can be modified by introducing or removing amino acid residues composed of non-polypeptide moiety attachment groups, in particular the surface of the IFNG polypeptide. This is because such residues are shown to be located.

It may also be interesting to modify one or more amino acid residues located in the loop region of an IFNG polypeptide, since most of the amino acid residues in these loop regions are exposed to the surface and are located far enough from the functional groups, thereby providing non-polypeptide moieties, For example, polymer molecules, in particular PEG molecules and / or N-glycosylation sites, can be introduced without compromising the function of the molecule. Such loop regions can be identified by inspection of the three-dimensional structure of huIFNG. The amino acid residues that make up the loop region are N16-K37 ("A-B loop"), F60-S65 ("B-C loop"), N83-S84 ("C-D loop") and Y98-L103 ("D-E loop").

The amino acid residues that make up the IFNG receptor binding site are Q1, D2, Y4, V5, E9, K12, G18, H19, S20, D21, V22, A23, D24, N25, G26, T27, L30, K34, K37, K108, HI111, E112, I114, Q115, A118, E119 (see also Example 2). In general, it is preferred that the attachment group to the nonpolypeptide moiety (additional N glycosylation site and / or cysteine residue) is not introduced into this site of the molecule.

To determine the optimal distribution of the attachment groups, the distance can be calculated for the amino acid residues located on the surface of the IFNG polypeptide based on the 3D structure of the IFNG dimeric polypeptide. More specifically, the distance between the CBs of amino acid residues composed of such attachment groups or another functional group of amino acid residues (NZ for lysine, CG for aspartic acid, CD for glutamic acid, SG for cysteine) and another The distance between CBs of amino acid residues was measured. In the case of glycine, CA is used instead of CB. In some IFNG polypeptides of the invention, a distance of at least 8 microns, in particular 10 microns, is necessary to avoid or reduce heterozygous.

In addition, the amino acid sequence of an IFNG polypeptide may differ from SEQ ID NO: 1 (or fragments thereof), wherein the epitope is substituted by an amino acid consisting of a non-polypeptide portion attachment vessel to destroy or inactivate the epitope. One or more amino acid residues that form part of can be removed. Epitopes of [S99T] huIFNG, huIFNG or rhuIFNG can be identified using methods known in the art, such as epitope mapping (Romagnoli et. Al., Biol Chem, 1999, 380 (5): 553-9, DeLisser HM , Methods Mol Biol, 1999, 96: 11-20, Van de Water et al., Clin Immunol Immunopathol, 1997, 85 (3): 229-35, Saint-Remy JM, Toxicology, 1997, 119 (1): 77 -81, and Land DP and Stephen CW, Curr Opin Immunol, 1993, 5 (2): 268-71). One method is to create a phage display library that expresses, for example, random oligopeptides of 9 amino acid residues. IgGl antibodies were purified from antisera specific for [S99T] huIFNG, huIFNG or rhuIFNG using immunoprecipitation, and active phage was identified using immunoblotting. By sequencing the DNA of the purified active phage, the 3D structure of IFNG can be placed to determine the sequence of the oligopeptide. The moieties identified on the structure may constitute epitopes and may be selected as target moieties for introducing non-polypeptide moiety attachment groups.

In vivo functional half-life and serum half-life depend on the molecular weight of the conjugate and the number of attachment groups needed to provide increased half-life, ie, the molecular weight of the non-polypeptide portion in question. In one embodiment, the IFNG polypeptide variant of the invention has a molecular weight of at least 67 kDa, in particular 70 kDa (Laemmli, UK Nature Vol 227 (1970), p680-85. Determination of molecular weight using SDS-PAGE described by p680-85. ). IFNG has a molecular weight (Mw) in the range of about 35-50 kDa, therefore an additional about 20-40 kDa is required to achieve the desired effect. This can be provided, for example, by providing 2-4 10 kDa PEG molecules or by combining additional in vivo glycosylation sites with additional PEG molecules, or by any of the other methods described herein.

Conjugated IFNG polypeptide variants of the invention comprise 1-10 (additional) non-polypeptide moieties, eg, 1-8, 2-8, 1-5, 1-3, or 2-5 (additional) nonpolypeptides Include the part. Typically, the conjugated variants include 1-3 (additional) nonpolypeptide moieties, such as 1,2, or 3 (additional) nonpolypeptide moieties.

As mentioned above, under physiological conditions, huIFNG is present as a dimeric polypeptide. Polypeptides are normally homodimeric (e.g., can be made up of an association of two IFNG polypeptide molecules prepared as described herein). However, certain IFNG polypeptide variants may be provided in a single chain form, wherein the two IFNG polypeptide monomers are linked via peptide bonds or peptide linkers. The provision of a single chain IFNG polypeptide has the advantage of allowing asymmetric mutation of the polypeptide because the two constituent IFNG polypeptides may be different. For example, one of the monomers may remove the PEGylation site at the receptor binding site and retain the other. Thus, after PEG reaction, one monomer can have a significantly increased molecular weight since one monomer has its own receptor-binding site while the other has a complete PEG site.

Conjugates of the invention wherein the non-polypeptide is a sugar moiety

In a suitable embodiment of the conjugates of the invention, the INFG variant of SEQ ID NO: 1 (or a fragment thereof) comprises at least one removed or introduced in vivo glycosylation site. Preferably, the glycosylation site is an in vivo N-glycosylation site, ie the nonpolypeptide moiety is a sugar moiety, for example an O-linked or N-linked sugar moiety, preferably an N-linked sugar moiety.

In one embodiment of the invention, the variant comprises at least one introduced glycosylation moiety, in particular the introduced in vivo glycosylation moiety. Preferably, the glycosylated moiety introduced is introduced by substitution.

For example, an in vivo glycosylation site can be introduced at the position of SEQ ID NO: 1 (or a fragment thereof) comprising amino acid residues exposed on the surface. Preferably, the amino acid residues exposed on the surface have at least 25% of the side chains exposed on the surface, and preferably have at least 50% of the surface exposed side chains. Details of the measurement of such a position can be found in Example 1.

The N-glycosylation site is introduced so that the N residues at that site are in this position. Similarly, the O-glycosylation reaction site is introduced so that the S or T residues that make up such site are at this position. When the term "at least 25% (or 50%) of the side chain exposed to its surface" is used in connection with the introduction of an in vivo N-glycosylation site, the term refers to the surface of the amino acid side chain in the body where the moiety is substantially attached. It should be understood to refer to accessibility. In many cases, it is necessary to introduce threonine or serine at position + 2 relative to the asparagine residue to which the sugar moiety is substantially attached, and those positions at which the serine or threonine is introduced, ie the surface of the molecule It is acceptable to have at least 25% (or 50%) of these side chains exposed to.

In addition, in order to ensure an effective glycosylation reaction, the S or T residues of the N-residue or O-glycosylation site of the in vivo glycosylation site, in particular the N-glycosylation site, are 118 N-terminus of the IFNG polypeptide. Located within amino acid residues, and more preferably within 93 N-terminal amino acid residues. More suitably, the in vivo glycosylation site is introduced at a position where only one mutation is required to make such a site (e.g., any other amino acid residue already required to make a functional glycosylation site is already present. In the molecule).

For example, the substituents used to induce the introduction of additional N-glycosylation sites at positions occupied by amino acid residues exposed to the surface of the IFNG polypeptide and having at least 25% of the side chains exposed to the surface are as follows; 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 / T, A124N, A124N + T126S, K125N + G127S / T, T126N + K128S / T, G127N + R129S / T, K128N + K130S / T, R129N + R131S / T, K130N, K130N + S132T, R131N + Q133S / T, S132N + M134S / T, Q133N + L135S / T, M134N + F136S / T, L135N + R137S / T, F136N + G138S / T, R137N + R139S / T, G138N + R140S / T, R139N + A141S / T, R140N, R140N + S142T. Such substituents are those shown for [S99T] huIFNG having the amino acids found in SEQ ID NO: 1 (or in relation to their associated fragments having the amino acids found in SEQ ID NOS 2- 16). S / T refers to a serine or threonine residue, preferably a threonine residue substituent.

Substituents that induce the introduction of additional N-glycosylation sites at IFNG polypeptide surface exposed locations having at least 50% of the side chains exposed to the surface are as follows; 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, Q106S / T, P122N + A124S / T, A123N + K125S / T, A124N, A124N + T126S, K125N + G127S / T, T126N + K128S / T, G127N + R129S / T, K128N + K130S / T, R129N + R131S / T, K130N, K130N + S132T, R131N + Q133S / T, S132N + M134S / T, Q133N + L135S / T, M134N + F136S / T, L135N + R137S / T, F136N + G138S / T, R137N + R139S / T, G138N + R140S / T, R139N + A141S / T, R140N, R140N + S142T. Such substituents are pointed in relation to [S99T] huIFNG having the amino acids found in SEQ ID NO: 1 (or in relation to their related fragments having the amino acids found in SEQ ID NOS 2- 16). S / T refers to a serine or threonine residue, preferably a threonine residue substituent.

Substituents that require only one amino acid mutation to introduce into the 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, Q106S / T, E119N, A124N, K130N and R140N, especially K12S / T, G18N, G18S / T, K37S / T, E38N, K61S / T, D63N, Q67N, K80S / T, N85S / T, K94N, Q106S / T, A124N , K130N, and R140N (where the side chains are exposed to at least 25% of the surface (in structures without receptor molecules)) or more preferably G18N, E38N, D63N, Q67N, K94N, S99N, A124N, K130N, R140N (( In structures without receptor molecules) with side chains exposed at least 50% of the surface).

Typically, it is not desirable to introduce an N-glycosylation site into the region constituting the receptor binding site (except in exceptional exceptional cases, for example, entitled “Variants with Reduced Receptor Affinity”). Sections). Thus, deformation, Q1N + P3S / T, E9N + L11S / T, G18N, G18N + S20T, H19N + D21S / T, D21N + A23S / T, G26N + L28S / T, K34N + W36S / T, K37N + E39S / T, E119N and E119N + S 121T, should not be performed unless reduced receptor affinity is required.

Particularly preferred variants of the invention include variants of SEQ ID NO: 1 (or fragments thereof having the amino acid sequence of SEQ ID NOS: 2-16), wherein the variants exhibit IFNG activity, and K12S, K12T, G18S, G18T, E38N, E38N + S40T, K61S, K61T, N85S, N85T, K94N, Q106S and Q106T, and more preferably K12T, G18T, E38N + S40T, K61T, N85T, K94N and Q106T Selected from the group consisting of K12T, G18T, E38N + S40T, K61T and N85T, in particular at least one substitution being E38N + S40T.

In another embodiment of the invention, the variant of SEQ ID NO: 1 (or a fragment thereof having the amino acid sequence of SEQ ID NOS: 2-16) comprises at least two introduced glycosylation sites, in particular at least two N-glycosylation sites. At least two modifications, in particular substitutions, lead to the introduction of at least two introduced N-glycosylation sites, K12S, K12T, G18S, G18T, E38N, E38N + S40T, K61S, K61T, N85S, N85T, K94N, Q106S and Q106T Selected from the group consisting of K12T, G18T, E38N + S40T, K61T, N85T, K94N and Q106T, and even more preferably K12T, G18T, E38N + S40T, K61T and N85T. It is selected from the group consisting of. Specific examples of such substitutions resulting in variants comprising two or more additional N-glycosylation sites are K12T + G18T, K12T + E38N + S40T, K12T + K61T, K12T + N85T, G18T + E38N + S40T, G18T + K61T, G18T + N85T, E38N + S40T + K61T, E38N + S40T + N85T and K61T + N85T.

In the list of substituents above, it is preferred to select a substituent located within 118 N-terminal amino acid residues, and in particular, to select a substituent located within 93 N-terminal amino acid residues.

IFNG polypeptide variants of the invention may comprise one additional in vivo glycosylation site per monomer (as compared to SEQ ID NO: 1 and fragments thereof). However, in order to be large enough to increase serum half-life, the polypeptide may have one or more additional in vivo glycosylation sites, in particular 2-7 or 2-5 additional in vivo glycosylation sites, for example two. It is preferred to include three, four or five in vivo glycosylation sites. Such in vivo glycosylation sites are preferably introduced by one or more substitutions in the list above.

It will also be understood that any of the modifications mentioned above may be combined with any of the modifications disclosed in the section entitled “IFNG Variants of the Invention Having Optimized In vivo Glycosylation Sites”, in particular the substitution G26A.

IFNG variants of the invention wherein the nonpolypeptide moiety is a molecule having a cysteine as an attachment group

In another embodiment of the invention, the IFNG variant of SEQ ID NO: 1 (or a fragment thereof) comprises at least one introduced cysteine residue. For example, cysteine residues can be introduced into the position of the IFNG polypeptide of SEQ ID NO: 1 (or a fragment thereof) comprising amino acid residues exposed on the surface. Preferably, the surface exposed amino acid residue has its side chain exposed to at least 25% of its surface, in particular its side chain exposed to at least 50% of its surface. Details in determining such locations can be found herein in Example 1.

For example, substitutions leading to the introduction of cysteine residues at positions occupied by amino acids having side chains exposed to the surface of the IFNG polypeptide and which are exposed to at least 25% of the surface (in structures with receptor molecules) include Q1C, D2C, P3C, K6C, E9C, N10C, K13C, Y14C, N16C, G18C, H19C, D21C, N25C, G26C, G31C, K34C, N35C, K37C, E38C, E39C, S40C, K55C, K58C, N59C, K61C, D62C, D62C, D62C Includes Q64C, S65C, Q67C, K68C, E71C, T72C, K74C, E75C, N78C, V79C, K80C, N83C, S84C, N85C, K86C, K87C, D90C, E93C, K94C, T101C, D102C, L103C, N104C and E119C , Substituents are indicated in relation to [S99T] huIFNG having the amino acids found in SEQ ID NO: 1 (or in relation to their related fragments having the amino acids found in SEQ ID NOS 2- 16).

For example, substitutions leading to the introduction of cysteine residues at positions occupied by amino acids having side chains exposed to the surface of the IFNG polypeptide and which are exposed to at least 50% of the surface (in structures with receptor molecules) include 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, K87C, T101C, D102C, L103C and N104C, substituents are indicated in relation to [S99T] huIFNG having the amino acids found in SEQ ID NO: 1 (or SEQ ID NOS; those having the amino acids found in 2- 16) In relation to the relevant fragments of).

Typically, it is not desirable to introduce cysteine residues (and consequently the cysteine residues attached to non-polypeptide moieties) into the regions making up the receptor binding site (except in exceptional cases, for example, " Section entitled “Variants with Reduced Receptor Affinity”). Thus, modifications, Q1C, E9C, G18C, H19C, D21C, G26C, K34C, K37C and E119C should not be performed unless reduced receptor affinity is required.

More preferably, the constant cysteine residue is introduced by a substitution selected from the group consisting of N10C, N16C, E38C, N59C, N83C, K94C, N104C and A124C.

In another embodiment of the invention, the variant of SEQ ID NO: 1 (or a fragment thereof having the amino acid sequence of SEQ ID NOS: 2-16) comprises at least two introduced cysteine residues. Substitutions leading to the introduction of at least two modifications, in particular at least two cysteine residues, are selected from the group consisting of N10C, N16C, E38C, N59C, N83C, K94C, N104C and A124C. Specific examples of such substitutions resulting in variants comprising at least two introduced cysteine residues are N10C + N16C, N10C + E38C, N10C + N59C, N10C + N83C, N10C + K94C, N10C + N104C, N10C + A124C, N16C + E38C, N16C + N59C, N16C + N83C, N16C + K94C, N16C + N104C, N16C + A124C, E38C + N59C, E38C + N83C, E38C + K94C, E38C + N104C, E38N + A124C, N59C + N83C, N59C + K94 N59C + N104C, N59C + A124C, N83C + K94C, N83C + K94C, N83C + N104C, N83C + A124C, K94C + N104C, K94C + A124C and N104C + A124C.

It will be appreciated that the introduced cysteine residue (s) may preferably be conjugated to the nonpolypeptide moiety and, for example, to PEG or more preferably mPEG. Conjugation between cysteine-containing polypeptide variants and polymer molecules can be accomplished in a suitable manner, for example, as described in the section “Conjugation to Polymer Molecules”, for example the one-step method or step-by-step method mentioned in the section above. Use A preferred method of PEGylating IFNG polypeptide variants is to covalently attach PEG to cysteine residues using cysteine-reactive PEGs. Many highly specific, different groups (eg orthopyridyl-disulfide, maleimide, and vinylsulfone) and different size PEGs (2-20 kDa, for example 5 kDa, 10 kDa, 12 kDa, or 15 kDa) Cysteine reactive PEGs with) are commercially available, for example Shearwater Polymers Inc., Huntsville, AL, USA.

It will also be understood that any of the modifications mentioned above may be combined with any of the modifications disclosed in the section entitled “IFNG Variants of the Invention Having Optimized In vivo Glycosylation Sites”, in particular the substitution G26A.

IFNG variants of the invention wherein the first nonpolypeptide moiety is a sugar moiety and the second nonpolypeptide moiety is a molecule having a cysteine as an attachment group

In another preferred embodiment of the invention, the IFNG variant of SEQ ID NO: 1 (or a fragment thereof) comprises at least one introduced N-glycosylation site and at least one introduced cysteine residue. Such variants can be prepared by selecting the residues described in the two preceding sections describing the appropriate positions to introduce the N-glycosylation sites and cysteine residues, respectively. However, in a preferred embodiment of the invention, the variant comprises a substitution selected from the group consisting of K12T + N16C, K12T + E38C, K12T + N59C, K12T + N83C, K12T + K94C, K12T + N104C, K12T + A124C, G18T + N10C, G18T + E38C, G18T + N59C, G18T + N83C, G18T + K94C, G18T + N104C, G18T + A124C, E38N + S40T + N10C, E38N + S40T + N16C, E38N + S40T + N59C, E38 S40T + N83C, E38N + S40T + K94C, E38N + S40T + N104C, E38N + S40T + A124C, K61T + N10C, K61T + N16C, K61T + E38C, K61T + N83C, K61T + K94C, K61T + N104C, K61T + A N85T + N10C, N85T + N16C, N85T + E38C, N85T + N59C, N85T + K94C, N85T + N104C, N85T + A124C, K94N + N10C, K94N + N16C, K94N + E38C, K94N + N59C, K94N + N83C, K94N + N83C N104C, K94N + A124C, Q106T + N10C, Q106T + N16C, Q106T + E38C, Q106T + N59C, Q106T + N83C, Q106T + K94C and Q106T + A124C, more preferably E38N + S40T + N10C, E38N + S40T + N16C, E38N + S40T + N59C, E38N + S40T + N83C, E38N + S40T + K94C and E38N + S40T + N104C.

It will be appreciated that the introduced cysteine residues may preferably be attached to non-polypeptide moieties, such as PEG, more preferably mPEG. Conjugation between cysteine-containing polypeptide variants and polymer molecules can be accomplished in a suitable manner, for example, as described in the section “Conjugation to Polymer Molecules”, for example the one-step method or step-by-step method mentioned in the section above. Use Suitable polymers are VS-mPEG or OPSS-mPEG.

It will also be understood that any of the modifications mentioned above may be combined with any of the modifications disclosed in the section entitled “IFNG Variants of the Invention Having Optimized In vivo Glycosylation Sites”, in particular the substitution G26A.

IFNG with reduced receptor affinity

One way to increase the serum half-life of IFNG polypeptides is to reduce receptor mediated internalization and thereby reduce receptor mediated clearance.

Receptor mediated internalization depends on the affinity of the IFNG dimer for the IFNG receptor complex, and therefore IFNG variants with reduced affinity for the IFNG receptor complex are expected to be internalized to a lesser extent and thus cleaned up.

The affinity of an IFNG dimer for its receptor complex can be reduced by carrying out one or more modifications, particularly substitutions, at the receptor binding site of the IFNG polypeptide. The amino acid residues that make up the receptor binding site are defined herein in Example 2. One class of substitutions that can be made is conservative amino acid substitutions. In another embodiment, the modifications performed result in the introduction of N-glycosylation sites.

Thus, in a further particularly preferred embodiment of the invention, the IFNG variant fragment of SEQ ID NO: 1 (or a fragment thereof) comprises at least one variant, eg a substitution, at the receptor binding site (as defined herein). More preferably, the IFNG polypeptide comprises at least one modification, preferably substitution, which forms an in vivo N-glycosylation site at said receptor binding site. For example, such substitutions are Q1N + P3S / T, D2N + Y4S / T, Y4N + K6S / T, V5N + E7S / T, E9N + L11S / T, K12N + Y14S / T, G18N, G18N + S20T, H19N + D21S / T, S20N + V22S / T, D21N + A23S / T, V22N + D24S / T, D24N + G26S / T, G26N + L28S / T, L30N + I32S / T, K34N + W36S / T, K37N + E39S / T, K108N + I110S / T, H111N + L113S / T, E112N + I114S / T, I114N + V116S / T, Q115N + M117S / T, A118N + L120S / T, E119N and E119N + S121T, preferably Q1N + P3S / T, D2N + Y4S / T, E9N + L11S / T, K12N + Y14S / T, G18N, G18N + S20T, H19N + D21S / T, S20N + V22S / T, D21N + A23S / T, K34N + W36S / T, K37N + E39S / T, H111N + L113S / T, Q115N + M117S / T, A118N + L120S / T, E119N and E119N + S121T (positions containing amino acid residues having their side chains exposed to at least 25% of the surface) To the N-glycosylation site), more preferably Q1N + P3S / T, D2N + Y4S / T, E9N + L11S / T, G18N, G18N + S20T, H19N + D21S / T, S20N + V22S / T, D21N + A23S / T, K34N + W36S / T, K37N + E39S / T, Q115N + M117S / T, A118N + L120S / T, E119N and E119N + S121T (amino acid residues with their side chains exposed to at least 50% surface) Contains Introduces an N-glycosylation site at the position), even more preferably Q1N + P3T, D2N + Y4T, E9N + L11T, G18N + S20T, H19N + D21T, S20N + V22T, D21N + A23T, K34N + W36T, K37N + E39T, Q115N + M117T, A118N + L120T and E119N + S121T, most preferably G18N + S20T, H19N + D21T, D21N + A23T and E119N + S121T, in particular D21N + A23T.

Such variants are believed to exhibit reduced receptor affinity compared to huIFNG or Acitimmune. Receptor affinity can be specified using methods known in the art. One example of a suitable measure to determine receptor binding affinity is MICHIELS et al. Int. J. Biochem. Cell Biol. 30: 505-516 (1998), a BIAcore measurement. Using the assays identified above, IFNG variants that are considered useful for the purposes described herein are such variants, wherein the binding affinity (Kd) is glycosylated huIFNG. Or 1-95% of Kd of Acitimmune. For example, the Kd-value of an IFNG polypeptide may be 1-75%, or 1-50%, eg 1-25%, for example 1-20%, or 1-15% of Kd of glycosylated huIFNG or Acitimmune. , As low as 1-10%, or 1-5%.

Such IFNG variants, which typically have reduced receptor affinity, will exhibit reduced IFNG activity, for example when tested in the "primary measurements" described herein. For example, IFNG polypeptide variants may comprise 1-95% of the specific activity of huIFNG or Acitimmune, for example 1-75% of the specific activity of huIFNG or Acitimmune, for example 1-50%, for example 1-20%, Or 1-10%.

As mentioned above, such IFNG variants are thought to possess increased serum half-life due to reduced receptor mediated clearance. Therefore, IFNG polypeptide variants according to one aspect of the invention are considered to meet the requirements in terms of increasing the serum half-life described herein above in connection with the definition of increased serum half-life.

Obviously, any of the above-mentioned modifications that result in slowed receptor binding affinity can be described in the other modifications disclosed herein, in particular "IFNG variants of the invention having optimized N-glycosylation sites", "non-polypeptide moieties of the invention IFNG variants "," IFNG variants of the invention wherein the non-polypeptide moiety is a molecule having a cysteine group as the attachment group ", and" the first non-polypeptide moiety is a sugar moiety, and the second non-polypeptide moiety is a molecule having a cysteine group as the attachment group Combinations such as G26A, E39N + S40t, and combinations thereof, mentioned in the section entitled IFNG Variants.

Splicing method

Nonpolypeptide moiety

As further described, the non-polypeptide moiety is preferably selected from the group consisting of polymer molecules, lipophilic compounds, sugar moieties (eg by in vivo N-glycosylation) and organic derivatizing agents. All of these agents can impart certain properties to IFNG polypeptide variants, particularly increased AUCsc, increased serum half-life, and / or reduced immunogenicity. Polypeptide variants are typically conjugated to only one type of nonpolypeptide moiety, but can also be conjugated to two or more different types of nonpolypeptide moieties, for example, polymer molecules and sugar moieties, lipophilic groups and sugar moieties, organic induction. Agents and sugar moieties, affinity groups and polymer molecules. When conjugated to non-polypeptide moieties of two different forms, they are preferably sugar moieties and polymer moieties. Conjugation to two or more non-polypeptide moieties can occur simultaneously or sequentially. The conjugation to certain types of non-polypeptide moieties is described in the sections “conjugation to lipophilic compounds”, “conjugation to polymers”, “conjugation to sugar moieties”, “conjugation to organic derived materials”.

Conjugation to lipophilic compounds

Polypeptide variants and lipophilic compounds may be conjugated to each other directly or via a linker. The lipophilic compound may be composed of natural or synthetic compounds such as saturated or unsaturated fatty acids, fatty acid diketones, terpenes, prostaglandins, vitamins, carotenoids, steroids, such as one or more alkyl, aryl, alkenyl, and other polyunsaturated compounds. The branches may be carbonic acid, alcohols, amines, sulfonic acids and the like. Conjugation between polypeptide variants and lipophilic compounds (conjugation, which may optionally be made via a linker), is described in methods known in the art, for example in Bodanszky in Peptide Synthesis, John Wiley, New York, 1976 and WO 96/12505. It can be carried out by the method.

Bonding to Polymers

The polymer capable of binding to the polypeptide is any suitable polymer, for example a natural or synthetic homo-polymer or hetero-polymer, generally having a molecular weight ranging from 300-100,000 Da, or 1000-50,000 Da, specifically 2000-40,000 Da or 2000-30,000 Da, for example 2000-20,000 Da, 2000-10,000 or 1000-5000 Da. More specifically, polymers, such as PEG, in particular mPEG, typically have a molecular weight of about 2, 5, 10, 15, 20, 30, 40 or 50 kDa, in particular about 5 kDa, about 10 kDa, about 12 kDa, It has a molecular weight of about 15 kDa or about 20 kDa.

As used herein for polymers, the word "about" refers to an approximate average molecular weight and typically reflects the fact that there is some molecular weight distribution for a given polymer preparation.

Homo-polymers include polyols (poly-OH), polyamines (poly-NH ₂ ), polycarbosilic acid (poly-COOH) and the like. The release-polymers consist of one or more different bonding groups such as hydroxyl groups and amine groups.

Suitable polymers are polyalkylene glycols (PAG)-for example polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs-, poly-vinyl alcohol (PVA), poly-carboxylate, poly (vinyl) Pyrrolidone), polyethylene-co-maleic anhydride, polyalkylene oxide (PAO) with polystyrene-co-maleic anhydride, dextran with carboxymethyl-dextran, or immunogenicity It is selected from other biopolymers suitable for decreasing or increasing functional half-life in vivo or increasing serum half-life. Another example of a polymer may be human albumin or other abundant plasma proteins. In general, polyalkylene glycol-derived polymers are biocompatible, non-toxic, non-immunogenic, non-immunogenic, have a wide variety of water soluble properties, and are readily secreted from living organisms.

PEG is a suitable polymer that can be used because there are few cross-linkable reactors when compared to polysaccharides such as dextran. In particular, monofunctional functional PEGs, such as monomethoxypolyethylene glycol (mPEG), are suitable because their binding chemistry is relatively simple (a single reactor can be used for conjugation with attachment groups on polypeptides). Only). As a result, there is no risk of cross-linking, and the resulting polypeptides have considerable homogeneity and are easier to control the reaction of the polymer with the polypeptide.

In order to covalently bind a polymer to a polypeptide variant, the hydroxy end group of the polymer has an active state, i.e. a reactive functional group (e.g., a primary amino group, hydrazide (HZ), thiol, succinate as a reactive functional group) (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl propionate (SPA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC ), N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), tresylate (TRES)). Properly activated polymers may be commercially available, such as those available from Shearwater Polymers, Inc., Huntsville, AL, USA. Alternatively, the polymer can be activated by methods known in the art, for example using the method described in WO 90/13540. Specific examples of activated linear or branched polymers that can be used in the present invention are Sharwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Plymers for Research and Pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Examples of activated PEG polymers include the following; Linear PEGs include NHS-PEG (ie SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG and SCM-PEG), NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, MAL-PEG, and branched PEGs such as PEG2-NHS ( Described in US 5,932,462 and US 5,643,575). In addition, the following publications describe useful polymer or PEGylation reaction chemistries: US5,824,778, US5,476,653, WO97 / 32607, EP229108, EP402378, US4,902,502, US5,281,698, US5,122,614, US5, 219,564, WO92 / 16555, WO94 / 04193, WO94 / 14758, WO94 / 17039, WO94 / 18247, WO94 / 2804, WO95 / 00162, WO95 / 11924, WO95 / 13090, WO95 / 33490, WO96 / 00080, WO97 / 18832, WO98 / 41562, WO98 / 48837, WO99 / 32134, WO99 / 32139, WO99 / 32140, WO96 / 40791, WO98 / 32466, WO95 / 06058, EP439508, WO97 / 03106, WO96 / 21469, WO95 / 13312, EP921131, US5, 736,625, WO98 / 05363, EP809996, US5,629,384, WO96 / 41813, WO96 / 07670, US5,473,034, US5,516,673, EP605963, US5,382,657, EP510356, EP400472, EP183503, EP154316.

Specific examples of activated PEG polymers particularly preferred for binding to cysteine residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG), preferably vinyl sulfone-mPEG] (VS-mPEG); Maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG ). Typically, such PEG or mPEG polymers have a size of about 5 kDa, about 10 kD, about 12 kDa or about 20 kDa.

Conventional methods are used for conjugating a polypeptide to an activated polymer, and the methods described in the following literature are also used (also described as appropriate methods for polymer activation); Harris and Zalipsky, eds., Poly (ethylene glycol ) Chemistry and Biological Applications, AZC, Washington; RF Taylor (1991), "Protein immobilisation. Fundamental 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.). IFNG variants for PEGylation at cysteine residues (see above) are typically treated prior to PEGylation with a reducing agent, such as dithiothreitol (DDT). The reducing agent is subsequently removed by conventional methods, for example by desalting. Conjugation to the cysteine residues of PER typically occurs in the range of 4 ° C. to 25 ° C. for up to 16 hours at an appropriate buffer of about pH 6-9.

Those skilled in the art will appreciate that the activation method and conjugation chemistry may vary depending on the functional group of the polymer (eg, amino, hydroxyl, carboxyl, aldehyde or sulfahydryl) and the attachment group of the IFNG polypeptide. PEGylation reactions can be used to conjugate to all available attachment groups (e.g., attachments exposed on the surface of the polypeptide, etc.) on polypeptide variants, or to conjugate to certain attachment groups such as N-terminal amino groups (US 5,985,265). . Conjugation can also be made in one step or step reactions (as described in WO99 / 55377).

PEGylation reactions are designed to produce molecules that are optimal for the number of PEG molecules attached, the size and shape of the molecules (e.g. they are linear or branched), and where such molecules are attached in the polypeptide. . For example, the molecular weight of the polymer to be used can be selected based on the desired effect to be obtained. For example, if the primary purpose of the conjugation is to obtain a conjugate with a high molecular weight (to reduce the rate of removal from the kidneys), then conjugation should be possible to polymers with slightly higher molecular weights in order to obtain the desired molecular weight. In order to increase the degree of epitope blocking, a large number of low molecular weight polymers (eg, 5,000 Da) may be used to effectively block all or most epitopes of the polypeptide. For example, 2-8, for example, 3-6 polymers can be used.

Where only a single attachment to a protein is to be conjugated (as described in US 5,985,265), it is advantageous for the linear or branched polymer to have a high molecular weight of about 20 kDa.

Normally, polymer conjugation is carried out under conditions aimed at allowing all available polymer attachments to react with the polymer. In general, the molar ratio of activated polymer attachment to the polypeptide can be made 1000-1, in particular 200-1, suitably 100-1, for example 10-1 or 5-1, to obtain an optimal response. However, an equivalent molar ratio may be used.

According to the present invention, it is also conceivable to bind the polymer to the polypeptide via a linker, suitable linkers being known to those skilled in the art. Suitable examples are cyanuric chloride (Abuchowski et al., (1997), J. Biol. Chem., 252, 3578-3581; US 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym) Chem.Ed., 24, 375-378).

In accordance with methods known in the art, it is possible to block residual activated polymers upon conjugation, for example by adding primary amines to the reaction mixture for blocking and the resulting inactivated molecules can be employed using appropriate methods. To remove it.

Combined on our part

It can be bound to the sugar moiety in vivo or in vitro . To obtain the IFNG polypeptide having the activity (already modified to introduce one or more in vivo glycosylation reaction site) of in vivo glycosylation reactions, need to insert the nucleotide sequence encoding the polypeptide part of the glycosylated, eukaryotic expression host do. Expression host cells can be selected from fungi (filamentous fungi or yeasts), insects, animal cells, transgenic plant cells. In addition, the glycosylation reaction can be obtained in the human body by using the nucleotide sequence encoding the polypeptide variant of the present invention in gene therapy. In one embodiment, the host cell is a CHO cell, BHK or HEK cell, for example a mammalian cell such as HEK293 or an insect cell such as SF9 cell or a yeast cell such as Saccharomyces cerevisiae, Pichia pastoris or any other suitable glycosylation host ( May be further explained). Alternatively, the sugar moiety attached to the IFNG polypeptide variant by in vivo glycosylation can be further modified using glycosyltransferases, for example using Neose, Horsham, PA, USA commercially available glycoAdvance ^™ . have. Thus, expression and in vivo glycosylation by CHO cells may be followed by sialination of glycosylated IFNG polypeptide variants.

Covalently binding glycosides to the amino acid residues of IFNG in vitro can be used to modify or increase the number or profile of carbohydrate substituents. Depending on the binding mode employed, a) arginine and histidine; b) free carboxyl groups; c) free sulfahydryl groups such as those in cysteine; d) free hydroxyl groups such as in serine, threonine, tyrosine, hydroxyproline; e) aromatic residues such as those in phenylalanine or tryptophan; f) Sugars can be attached to the amide group of glutamine. Such amino acid residues consist of a group for attachment to a sugar moiety that can be removed or inserted in an IFNG polypeptide of a conjugate of the invention. Suitable methods for in vitro binding are described in WO 87/05330 and Aplin et al., CRC Crit Rev. Biochem., Pp 259-306, 1981. Transglutaminase (TGases) can be used to bind sugar moieties or PEG in vitro to protein and peptide-bound Gln-residues (Sato et al., 1996 Biochemistry 35, 13072-13080; EP 725145).

Binding to Organic Derived Materials

IFNG polypeptides can be covalently modified by reacting the attached group of the polypeptide variant with an organic derived material. Suitable derivatization materials and methods are known in the art. For example, the most commonly used cystenyl moieties are reacted with α-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetaamide to provide carboxymethyl or carboxyamidomethyl. . Bromotriloloacedon, α-bromo-β- (4-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmelimides, 3-nitro-2-pyridyl disulfide, methyl 2- Cysteinyl residues can be induced by reaction with pyridine disulfide, p-chloromercurybenzoate, 2-chloromercury-4-nitrophenol, chloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues can be derived by reaction with diethylpyrocarbonate pH 5.5-7.0 because the material is relatively specific for histidyl side chains. Para-bromophenolacyl bromide is also useful; This reaction is carried out in 0.1M sodium cacodylate (pH 6.0). Lysinyl and amino terminal residues were reacted with succinic or other carboxylic anhydrides. The derivatization reaction using these substances has the effect of reversing the charge of the lysine residue. Other suitable reagents for deriving residues comprising α-amino include imidoesters such as methyl picorinimidate; Pyridoxal phosphate; Pyridoxal; Chloroborohydride; Trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pendanedione; Glyoxylates and transaminase-catalyzed reactions. Arginine residues are modified by reaction with one or several conventional reagents, among which phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, ninhydryl and the like. The derivatization reaction of arginine residues should be carried out under alkaline conditions because the pKa of the guanidine functional group is high. In addition, such reagents may react not only with lysine groups but also with arginine guanidino groups. Carboxyl side groups (aspartyl or glutamyl) can be selectively modified by reaction with carbodiimide (R-N = C = N-R '); Wherein 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 reacted with ammonium ions to convert them into asparaginyl and glutaminyl residues.

Blocking of functional groups

It has been reported that excessive polymer conjugation may cause the polymer to lose activity of the conjugated polypeptide. This problem can be solved by removing the attachment located at the functioning part or blocking the functional group before joining. These latter strategies constitute embodiments of the invention, and the first strategy is further illustrated, for example, by removing lysine residues located in proximity to the functional groups and / or by sites that do not interfere with cysteine residues and / or functional sites Is to introduce glycosylation sites in vivo.

More specifically, according to the second strategy, the conjugation between the polypeptide and the non-polypeptide moiety is such that a helper molecule capable of binding to the functional group of the polypeptide variant is carried out under conditions that can block the functional group of the IFNG polypeptide variant. Suitably, the helper molecule is one that specifically recognizes the function of a polypeptide variant such as a receptor. Alternatively, the helper molecule may be an antibody, in particular a monoclonal antibody that recognizes a polypeptide variant that exhibits IFNG activity. In particular, the helper molecule may be a neutralizing monoclonal antibody.

Before the conjugation, the helper molecule and the polypeptide variant will interact. This shows that the functional sites of the polypeptide are blocked or protected, resulting in their inability to be used for induction by non-polypeptide moieties such as polymers. After eluting from the helper molecule, conjugation can be restored between the non-polypeptide moiety and the polypeptide variant, having at least partially conserved functional groups.

Continuous conjugation to a polymer, lipophilic compound, sugar moiety, organic derived material or any other compound of a polypeptide having a blocked functional moiety can be carried out using conventional methods, e.g. Are described in the section entitled "."

In further embodiments, the helper molecules are first covalently bound to a column packing material, such as Sephadex or agarose beads, or to a solid surface, such as a reaction vessel. The polypeptide was then loaded onto the column material with the helper molecules and conjugation was carried out using methods known in the art, such as those described in the section entitled "Conjugation to ...". Elution can be used in this process to liberate the polypeptide conjugate from the helper molecule. Conjugated polypeptide variants are eluted using conventional techniques under physicochemical conditions, which conditions must be such that substantial degradation of the conjugated polypeptide variants does not occur. A fluidized bed comprising conjugated polypeptide variants is separated from the solid phase, where the helper molecules remain covalently bound. It can also be separated in other ways; For example, a helper molecule can be directed to a second molecule (biotin) that can be recognized by a specific conjugate (streptavidin). Certain binders can be bound to the solid phase, thereby allowing the separation of conjugated polypeptide variants from the helper molecule-second molecular complex, and through the second helper-solid phase column, on subsequent elution, the helper molecule-second molecular complex Will remain, but the conjugated polypeptide variant will not. Conjugated polypeptide variants can be released from the helper molecule in any suitable manner. De-protection reactions can be obtained under conditions that allow helper molecules to dissociate from the functional site. For example, the pH may be adjusted to an acidic or alkaline pH to dissociate the complex between the conjugated antibody and the anti-idiotype antibody.

Conjugation to Tagged Polypeptide Variants

In another embodiment, the IFNG polypeptide can be expressed as a fusion protein, typically having an amino acid sequence consisting of 1-30, for example 1-20 amino acid residues, or a peptide stretch in tag. For quick and easy purification, the tag is used as a convenient tool for conjugating between tagged IFNG polypeptide variants and non-polypeptide moieties. In particular, a tag is used to conjugate a microtiter plate or other carrier, such as a paramagnetic rod, with the tag, through which the tagged polypeptide is anchored to the surface. Conjugation to tagged IFNG polypeptide variants, eg, microtiter plates, is that the tagged polypeptide variants can be immobilized directly to microtiter plates directly from the culture (no further purification is required), so that conjugation is required. Thus, the total number of process steps (from expression to splicing) can be reduced. The tag can also function as a space molecule that improves access to the immobilized polypeptide to be conjugated. Conjugation using tagged polypeptide variants may be for a non-polypeptide moiety described herein, eg, a polymer such as PEG.

Identification of the specific tag that can be used is not critical if the tag is expressed with polypeptide variants and can be immobilized on a suitable surface or carrier material. Many suitable tags are available commercially (Unizyme Laboratories, Denmark). For example, a tag can consist of the following sequences;

His-His-His-His-His-His (SEQ ID NO: 35)

Met-Lys-His-His-His-His-His-His (SEQ ID NO: 36)

Met-Lys-His-His-Ala-His-His-Gln-His-His (SEQ ID NO: 37)

Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln (SEQ ID NO: 38)

(All products of Unizyme Laboratories, Denmark)

or

EQKLI SEEDL (SEQ ID NO: 39) (C-terminal tag as described in Mol. Cell. Biol. 5: 3610-16, 1985)

DYKDDDDK (SEQ ID NO: 40) (C- or N-terminal tag)

YPYDVPDYA (SEQ ID NO: 41)

Antibodies to this tag available from ADI, Aves Lab, Research Diagnostics.

Commercially available enzymes can be used to continuously cleave tags in polypeptides.

Methods of Preparing IFNG Polypeptide Variants of the Invention

IFNG polypeptide variants can be made by any suitable method known in the art. Such methods include constructing a nucleotide sequence that encodes a polypeptide and expressing the sequence in an appropriately altered or transfected host. However, polypeptide variants of the present invention can be produced (somewhat inefficient) by chemical synthetic or recombinant DNA techniques and combinations thereof.

Nucleotide sequences of the present invention encoding IFNG polypeptide variants (monomer or single chain form) isolate or synthesize nucleotide sequences encoding parent IFNG, such as huIFNG having the amino acid sequence of SEQ ID NO 17 or fragments thereof, The synthesized nucleotide sequence is modified to introduce (insert or substitute) or remove (delete or substitute) relevant amino acid residues.

According to methods known in the art, nucleotide sequences can be modified via site-directed mutation formation (Mark et al., "Site-specific Mutagonesis of the Human Fibroblast Interferon Gene", Proc. Natl. Acad. Sci. USA, 81, pp. 5662-66 (1984); and US 4,588,585).

Alternatively, the nucleotide sequence can be made by chemical synthesis methods, for example using an oligonucleotide synthesizer, wherein the oligonucleotide is designed based on the amino acid sequence of the desired polypeptide and suitably the host from which the recombinant polypeptide is made. Selected cords of cells are selected and constructed. For example, several small oligonucleotides encoding portions of the desired polypeptide are synthesized and assembled using PCR, ligation or ligation chain reaction (LCR). Individual oligonucleotides generally contain 5 'or 3' overhangs for complementary assembly.

Once assembled (synthetic, site-directed mutation formation, or another method), the nucleotide sequence encoding the polypeptide variant is inserted into a recombinant vector and acts on regulatory sequences necessary for expressing IFNG in the desired transformed host cell. Connect as much as possible.

Of course, it should be recognized that not all vectors and expression control sequences do the same to express the nucleotide sequences encoding the IFNG polypeptide variants described herein. Not all hosts function equally against the same vector system. However, one of ordinary skill in the art would be able to select such vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must be replicated in the host or inserted into the chromosome. The number of copies of the vector, the ability to control the number of copies, the expression of any other protein encoded by the vector, for example the expression of antibiotic markers, etc. are also factors to be considered. In selecting an expression control sequence, various factors should be considered. This includes the relative strength, controllability of sequences, suitability with nucleotide sequences encoding polypeptides, in particular secondary structures. The host should be selected in consideration of its compatibility with the selected vector, the toxicity of the products encoded by the nucleotide sequences, their secretory characteristics, the ability to fold correctly into the polypeptide, and the degree of purification of the products encoded by the nucleotide sequences.

Recombinant vectors can also be autologous replication vectors, which can exist as extrachromosomes, such as plasmids, whose replication is separate from chromosomal replication. Alternatively, when the vector is introduced into a host cell, it may be integrated into the host cell genome and replicated into the integrated chromosome.

The vector is preferably an expression vector, wherein the nucleotide sequence encoding the IFNG polypeptide for nucleotide sequence transcription is operably linked to additional fragments as needed. Such vectors generally become plasmids or viral DNA. Suitable expression vectors for expression in the host cells referred to herein are those that are commercially available. Useful expression vectors for eukaryotic hosts include SV40, bovine papilloma virus, adenovirus, vectors consisting of expression control sequences of cytomegalovirus, and the like. Specific vectors include pCDNA3.1 (+) ＼Hyg (Invitrogen, Carlsbad, CA, USA) and pCI-neo (Stratagene, LA Jola, CA, USA). Useful expression vectors suitable for bacterial hosts include known bacterial plasmids such as plasmids from E. Coli , i.e. a wide range of host plasmids, such as pBR322, pET3a, pET12a (Novagen Inc., WI, USA) and RP4, phage DNAs ( Various phage lambda derivatives) such as NM989 and other DNA phages such as M13 and filamentary single stranded DNA phages. Useful vectors for yeast cells include 2μplasmid and derivatives thereof, POT1 vector (US 4,931,373), pJSO37 vector (Okkels, Ann.New York Acad. Sci. 782, 202-207, 1996), pPICZ A, B, C (Invitrogen) Etc. are included. Useful vectors for insect cells include pVL941, pBG31l (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. 685-98 (1986) , pBluebac 4.5, pMelbac (Invitrogen), and the like.

Other vectors that can be used in the present invention also include vectors that allow nucleotide sequences encoding IFNG polypeptides to be replicated. Vectors that enable such amplification are also known in the art. For example, amplification vectors using DHFR amplification (Kaufman, US Pat. No. 4,470,461, Kaufman and Sharp, "Construction OF A Modular Dihydrafolate Reductaso cDNA Gene: Analysis Of Signals Utilized For Efficient Expression", Mol. Cell. Biol., 2, pp. 1304-19 (1982)) and amplification vectors (US 5,122,464 and EP 388,841) using glutamine synthetase ("GS") amplification.

Recombinant vectors further include DNA sequences that allow the vector to replicate in the host cell in question. An example of such a sequence, where the host cell is a mammalian cell, may be an SV40 replication origin. If the host cell is a yeast cell, the appropriate sequences capable of replicating the vector are the yeast plasmid 2μ replication gene REP 1-3 and the origin of replication.

Vectors are selectivity markers, which are genes, for example, genes in which the product of a particular gene can compensate for defects in a host cell, that is, a gene encoding dihydrofolate reductase (DHFR) or a Schizosaccaromyces pombe TPI gene (described by PR Russell). , Gene 40, 1985, pp. 125-130) or drugs such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin, methotrexate. In the case of filamentous fungi, selectivity markers include amdS , pyrG, arcB , niaD , sC and the like.

A "regulatory sequence" is defined herein to include all components necessary for or assist in the expression of an IFNG polypeptide. Each regulatory sequence can be a sequence unique to a nucleic acid sequence encoding a polypeptide or a sequence introduced exogenously. Such regulatory sequences include, but are not limited to, leader, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal peptide sequence and transcription terminator. At a minimum, regulatory sequences include promoters.

Various ranges of expression control sequences can be used in the present invention. Useful expression control sequences include not only expression control sequences associated with the structural genes of the above-described expression vectors, but also any sequences known to regulate expression of prokaryotic or eukaryotic cells, viral genes thereof, or various complexes thereof.

Examples of appropriate regulatory sequences that direct transcription in mammalian cells include the early and late promoters of SV40 and adenovirus, namely the two major late promoters of adenovirus, the MT-1 (metallothionine gene) promoter, and human cytomegalo. Viral super electric gene promoter (CMV), human elongation factor 1α (EF-1α) promoter, Drosophila Drosophila minimal heat shock protein 70 promoter, Raus Salcoma virus (RSV) promoter, human ubiquitin C (UbC) promoter, human Growth hormone terminators, SV40 or adenovirus Elb partial polyadenylation reaction signals and Kozak consensus sequences (Kozak, MJ Mol. Biol. 1987 Aug 20; 196 (4): 947-50).

To improve expression in mammalian cells, synthetic introns can be inserted in the 5 'untranslated portion of the nucleotide sequence encoding the IFNG polypeptide. Synthetic introns include, for example, synthetic introns obtained from plasmid pCI-Neo (Promega Corporation, WI, USA).

Suitable regulatory sequences used to direct transcription in insect cells include polyhedrin promoter, P10 promoter, Autographa californica polyhedrosis virus based protein promoter, baculovirus hyperelectric gene 1 promoter, baculovirus 39K delayed-initial gene promoter, SV40 Polyadenylation reaction sequences and the like.

Suitable regulatory sequences available in yeast host cells include, for example, the promoters of the yeast α-mating system, the promoters of the yeast triose phosphate isomerase (TP1) promoter, the yeast glocotic gene or the alcohol dehydrogenase gene, ADH2-4c promoter and inducible GAL promoter.

Suitable regulatory sequences available for filamentous fungal host cells include, for example, the ADH3 promoter and terminator, Aspergillus oryzae TAKA amylase triose phosphate isomerase or alkaline protease, A. niger α-amylase, A. niger or A. nidulans Glycoamylase, A. nidulans acetamidases, Rhizomucor miehei aspartic proteinases or lipases, TPI1 terminators, promoters derived from genes encoding ADH3 terminators, and the like.

Suitable regulatory sequences available for bacterial host cells include, for example, promoters of the lac system, trp system, TAC or TRC system and major promoter portions of phage lambda.

The nucleotide sequence of the present invention (prepared by site-directed mutation formation, synthesis and other methods, etc.) may or may not include the nucleotide sequence encoding the signal peptide. When the polypeptide is secreted in the cell in which it is expressed, a signal peptide is present. If signal peptides are present, such signal peptides must be recognizable by the cell selected for polypeptide expression. The signal peptide may be homologous to the polypeptide (normally associated with huIFNG) or heterologous (obtained from a source other than huIFNG), or homologous or heterologous to the host cell, i.e., the signal peptide is normally expressed in the host cell. It may be a signal peptide (if homologous) or a peptide (if heterozygous) that is not normally expressed in the host cell. Thus, the signal peptide may be a prokaryotic peptide derived from a bacterium such as E. coli or a eukaryotic cellular peptide derived from a mammal or insect or yeast cell.

The presence or absence of signal peptides may vary depending on whether the expression host cell used to produce the polypeptide, the protein to be expressed (either intracellular or intercellular protein), or whether secretion is desired. If used on filamentous fungi, Asperhillus sp . Signal peptides are typically derived from a gene encoding amylase or glucoamylase or a gene encoding Rhizomucor miehei lipase or protease or a gene encoding Humicola lanuginosa lipase. The signal peptide is preferably derived from genes encoding A. oryzae TAKA amylase, A. niger neutral α-amylase, A. niger acid-stable amylase, A. niger glucoamylase. For use in insect cells, it is convenient to derive from the insect gene (WO90 / 05783), e.g. lepidopteran Manduca sexta adipocaine hormone precursor (US 5,023,328), bee melittin (Invitrogen), activator UDP glue Signal peptides were identified in rosyltransferase (egt) (Murphy et. Al. Protein Expression and Purification 4,349-357 (1993) or human pancreatic lipase (hpl) (Methods in Enzymology 284, pp. 262-272, 1997). Can be induced.

Suitable signal peptides available for mammalian cells are huIFNG or murine Ig kappa light chain signal peptide (Coloma, M (1992) J. Imm. Methods 152: 89-104). Suitable for yeast cells, α-factor signal peptide of S. cereviciae (US 4,870,008), signal peptide of mouse saliva amylase (O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), modified carboxy Peptidase Signal Peptides (LA Valls et al., Cell 48, 1987, pp. 887-897), East BARl Signal Peptides (WO 87/02670), East Aspartic Protease 3 (YAP3) Signal Peptides (M. Egel-Mitani) et al., Yeast, 6, 1990, pp. 127-137) found the appropriate signal peptide.

Any suitable host used to produce the IFNG polypeptide includes bacteria, fungi (including yeast), plants, insects, mammals or other suitable animal cells or cell lines, as well as transgenic animals or plants. Bacterial host cells include, for example, Gram positive bacteria such as Bacillus strains (ie, B. brevls, B. subtilis ) Pseudomonas or Streptomyces ; Gram-negative bacteria such as E. Coli . Protoplast transformation (Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221; Electroporation (Shigekawa and Dower, 1988, Biotechniques 6: 742-751); Conjugation (Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-5278) and the like are methods used to introduce vectors into bacterial cells.

Filamentous fungal cells include, for example, Aspergillus strains ( A. oryzae, A. niger, A. nidulane ), Fusarium or Trichoderma . Fungal cells can be transformed by a series of processes such as protoplast formation, protoplast transformation, and cell wall regeneration by known methods. Suitable transformation procedures for Aspergillus host cells are described in EP 238 023 and US 5,679,543. Methods for transforming Fusarium species are described in Malardier et al., 1989, Gene 78: 147-156 and WO 96/0787. Becker and Guarente, In Abelson, JN and Simon, MI, edlitors, 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; Yeast is transformed using the process described in and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Suitable yeast host cells include Saccharomyces species ( S. cerevisiae, Schizosaccharomyces ), Kluyvsromyces, Plchia ( P. pastoris, P. methanolica ), Hansenula ( H. Polymorpha ) or Yarrowia species. Methods for transforming yeast cells with heterologous DNA to produce heterologous polypeptides from yeast are described in Clontech Laboratories, Inc., Palo Alto, CA USA (protocol of the Yeastmaker ^™ Yeast Tranformation System Kit) and Reeves et. al., FEMS Microbiology Letters 99 (1992) 193-198, Manivasakam and Schies, Nucleic Acids Research, 1993, Vol. 21, No. 18, pp. 4414-4415 and Geneva et al., FEMS Microbiology Letters 121 (1994) 159-164.

Suitable gonadotrophic host cells include, for example, Lepidoptora cell lines such as Spodoptera frugiperda (Sf9 or Sf21), Trichoplusioa ni cells (High Five) (US 5,077,214) and the like. Transformation of insect cells and production of heterologous polypeptides can be carried out as described in Invitrogen.

Examples of suitable mammalian host cells include the Chinese Hamst Ovari (CHO) cell line (CHO.K1; ATCC CCL-61), the green monkey cell line (COS) (COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-). 1651)); Mouse cells (NS / O), Baby Hamster Kindney (BHK) cell line (ATCC CRL-1632 or ATCC CCL-10), human cells (HEK 293 (ATCC CRL-1573)) and plant cells in tissue cultures. . Another suitable cell line is known in the art and can be distributed from public depositories such as the American Type Culture Collection, Rockville, Maryland. In addition, mammalian cells, such as CHO cells, are modified to express cyclyltransferases such as 1,6-cyaryltransferase (US 5,047,335) to improve glycosylation of IFNG polypeptides.

Calcium phosphate-mediated transfection, electroporation, DEAE-dextran mediated transfection, liposome-mediated transfection, viral vectors, methods described by Life Technologies Ltd, Paisley, UK using Lipofectamin 2000, and the like. It is a method used to introduce exogenous DNA into mammalian host cells. Such methods are already known in the art, and Ausber et. al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. Cultivation of mammalian cells is established by methods such as Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc, Totowa, New Jorsey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture, Cambridge It was carried out according to the method described in University Press 1997.

In order to produce glycosylated polypeptides, it is preferred to use eukaryotic host cells, for example the cells mentioned above.

In the production method of the present invention, the cells were cultured in a nutrient medium suitable for producing the polypeptide using methods known in the art. For example, using flask shake culture, small or large scale culture (including continuous fermentation, batch, feed-batch, solid state fermentation, etc.), laboratory scale in an appropriate medium under conditions under which the polypeptide can be expressed and / or separated. Alternatively, cells can be cultured on an industrial scale. Culture is carried out in a suitable nutrient medium consisting of carbon, nitrogen source, inorganic salts using methods known in the art. Appropriate media can be obtained from the supplier or prepared according to known compositions (see American Typer Culture Collection Catalog). Once the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

The resulting polypeptide can be recovered using any method known in the art. For example, the polypeptide can be recovered from the nutrient medium using conventional procedures, including but not limited to centrifugation, filtration, extraction, spray drying, evaporation, precipitation, and the like.

Polypeptides can be purified using a variety of methods known in the art, for example, chromatography (ion exchange chromatography, affinity chromatography. Hydrophobic chromatography, isoelectric focus chromatography), electrical processes (preliminary) Isoelectric point focus), use of differential solubility (eg, ammonium sulphate precipitation), SDS-PAGE or extraction (Protein Purification, JC Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) It can be purified using. Particular methods for purifying polypeptides exhibiting IFNG activity are described in EP 110044 and also in Japanese patent application 186995/84 (unexamined).

The biological activity of IFNG polypeptide variants can be examined by any suitable method known in the art. Such tests include antibody neutralization of antiviral activity, protein kinase induction, oligoadenylate 2,5-A synthetase or phosphodiesterase activity (EP 41313 B1), and the like. Such tests include immunomodulation tests (US 4,753,795), growth inhibition tests, and measuring the extent of binding to cells expressing interferon receptors. Specific tests are described in the Materials and Methods section of this specification.

Pharmacological Compositions and Their Use

The invention also relates to inflammatory diseases such as interstitial lung diseases, for example idiopathic pulmonary fibrosis and granulomatosis diseases; Cancer, especially uterine cancer; Infections such as idiopathic atypical mycobacterial infections, bone diseases (bone metabolic diseases such as malignant osteoporosis), autoimmune diseases such as rheumatoid arthritis and multiresistent pulmonary tuberculosis; Yeast meningitis; A method for treating and preventing cystic fibrosis and hepatic fibrosis, in particular secondary fibrosis for hepatitis C, and the like, wherein the method comprises a mammal, in particular a human being in need of an effective amount of a polypeptide variant of the invention, or a composition of the invention. Important benefits include effective treatment while reducing the frequency of invasive administration and reducing the risk of immune response to the therapeutically active ingredient.

The molecules of the invention are preferably administered in a composition such as a pharmaceutically acceptable carrier or excipient. "Pharmaceutically acceptable" means a carrier or excipient that does not cause side effects in a patient receiving the composition. Such pharmaceutically acceptable carriers and excipients are known in the art (Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, s. Frokjaer and K. Hovgaard, Eds., Taylor% Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]).

The molecules of the present invention can be used in the form of prototypes and salts. Suitable salts include, but are not limited to, alkali or alkaline earth metals such as sodium, potassium, calcium, magnesium and zinc salts. Such salts or complexes exist in crystalline or amorphous structures.

Variants of the invention can be administered in similar dosages used in the treatment with known commercially available IFNGs such as Actimmune or as mentioned in EP 795332. The exact dosage to be administered depends on the environment. Normally, the dosage should be able to prevent or alleviate the symptoms of the condition being treated. The effective amount of the variant or composition of the present invention depends on the disease, dosage, schedule of administration, and also depends on whether the polypeptide variant is administered alone or in combination with other therapeutic agents, and the serum half-life of the composition and the patient's overall health. Different.

The invention also relates to the pharmaceutical use of the IFNG polypeptide variant of the invention or the pharmaceutical composition according to the invention.

The invention also relates to the use of a) an IFNG variant according to the invention or b) a pharmaceutical composition of the invention for the manufacture of a medicament, a pharmaceutical composition, or a part of a kit, the disease name being for example an inflammatory disease, for example For example, interstitial lung diseases, such as idiopathic pulmonary fibrosis and granulomatosis diseases; Cancer, especially uterine cancer; Infections such as idiopathic atypical mycobacterial infections, bone diseases (bone metabolic diseases such as malignant osteoporosis), autoimmune diseases such as rheumatoid arthritis and multiresistent pulmonary tuberculosis; Yeast meningitis; Secondary fibrosis for cystic fibrosis and hepatic fibrosis, especially hepatitis C. Most preferred is interstitial lung disease, especially idiopathic pulmonary fibrosis.

Improved means of transporting molecules or preparations that optionally further comprise glucocorticoids are also described.

Suitable polypeptide components per dose are 1-4 μg / kg, more preferably 2-3 μg / kg polypeptide per patient weight. More suitable dosages are 100-350 μg / kg glucocorticoids, more preferably 100-150 μg / kg glucocorticoids.

The invention also provides a kit suitable for treating interstitial lung disease, the kit comprising: a) a first pharmaceutical composition consisting of the active ingredient b) a second pharmaceutical composition comprising the above-mentioned or at least one glucocorticoid It consists of. Wherein each component is optionally provided with a pharmaceutically acceptable carrier or excipient.

The conjugates of the present invention are formulated into pharmaceutical compositions using known methods. Suitable preparation methods are described in Remington's Pharmaceutical Sciences (E.W.Martin) and US 5,183,746.

Pharmaceutical compositions can be formulated in a variety of forms, for example, liquid, gel, freeze-dried, powder, compressed solid or any other suitable zero. Appropriate form depends on the disease to be treated, but will be apparent to those skilled in the art.

The pharmaceutical composition may be administered by oral cavity, subcutaneous, intravenous, brain, nasal, transdermal, peritoneal, muscle, lung, vaginal, rectal, ocular or the like or using any other suitable method such as PowderJect or ProLease technology. . The composition may be administered continuously via infusion, but pill infusion is also possible using methods (pumps or implants) known in the art. In some cases, the composition may be provided directly as a solution or spray. Appropriate dosage forms will vary depending on the particular disease being treated, but are known in the art.

The pharmaceutical composition of the present invention can be administered in combination with other therapeutic agents. Such materials may be included in part of the same pharmaceutical composition or administered separately from the polypeptides or conjugates of the invention. In addition, polypeptide variants of the invention, or pharmaceutical compositions, can be used as an adjuvant in other therapies. In particular, complexes with glucocorticoids as described in EP 795332 can also be considered.

In another aspect, the invention relates to a method of treating a mammal having a circulating antibody against huIFNG or rhuIFNG, the method comprising administering an IFNG variant that has bioactivity of IFNG and does not react with the antibody. . The compound is preferably the variant described herein, and the mammal is preferably a human. Mammals to be treated can suffer from any of the diseases listed above, wherein IFNG is a useful treatment. The invention also relates to a method of making a medicament for use in treating a mammal having a circulating antibody against huIFNG or rhuIFNG, wherein an IFNG variant that has bioactivity of IFNG and does not react with the antibody is administrable or Otherwise it is formulated with an appropriate formulation. The term "cyclic antibody" is intended to refer to an autoantibody formed in a mammal that is responsive to treatment with any commercially available IFNG preparation.

Also contemplated is the use of nucleotide sequences encoding IFNG polypeptide variants of the invention in the field of gene therapy. In particular, it is of interest to use nucleotide sequences encoding the IFNG polypeptide variants described in the above section entitled " IFNG variants of the invention wherein the non-polypeptide moiety is a sugar moiety. &Quot; Glycosylation of polypeptide variants is thus accomplished during gene therapy after the nucleotide sequence is expressed in the human body.

Gene therapy contemplated will include treatment of diseases in which the polypeptide is expected to provide effective treatment.

Local delivery of IFNG variants using gene therapy can provide therapeutic agents to the target region, while avoiding potential toxicity issues related to non-specific administration.

Both in vivo and in vitro gene therapy are contemplated.

Several methods of delivering potential therapeutic agents to limited cell populations are known. For further reference, see Mulligan, The Basic Science Of Gene Therapy, "Science, 260, pp. 926-31 (1993).

Direct gene transfer. For example, Wolff et al., "Direct Gene transfer Into Mouse Muscle In vivo", Science 247, pp. 1465-68 (1990)

Liposome-mediated DNA delivery, for example, 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)

Retrovirus-mediated DNA delivery, for example, Kay 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 delivery, eg, DNA viruses, may include adenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses (preferably herpes simplex virus based vectors), and parvoviruses (preferably Preferably, "fatal" or non-autonomous parvovirus based vectors, more preferably adeno-associated viral based vectors, most preferably AAV-2 based vectors) Ali et al., "The Use Of DNA Viruses as Vectors for Gene" Therapy ", Gene Therapy, 1, pp. 367-84 (1994); US 4,797,368, and US 5,139,941. See

Parenteral administration

Pharmaceutical compositions can be, for example, solutions for the purpose of extra-intestinal administration. In most cases, the pharmaceutical solution phase composition may be readily available in liquid form, and such extratracheal compositions may be provided in frozen or lyophilized form. In the case described above, the composition is thawed before use. The latter case is used when one wishes to enhance the stability of the active compound included in the composition under various storage conditions, and those skilled in the art will appreciate that the lyophilized preparation is generally more stable than the liquid phase. Such lyophilized preparations are reconstituted by adding one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological salt solution.

In the case of extra-intestinal administration, in the preparation of a lyophilized composition or aqueous solution for storage, one or more pharmaceutically acceptable carriers, excipients or stabilizers (collectively, all those used in the art) And excipients include buffers, stabilizers, preservatives, isotonic solutions, non-ionic surfactants, antioxidants or other additives.

It is a buffer that helps to adjust the pH within a range close to physiological conditions. It generally has a concentration range of 2 mM to 50 mM. Suitable buffers for use in the present invention include organic and inorganic acids and salts thereof, for example citrate buffers (e.g. monosodium citrate, disodium citrate mixtures, citric acid-citrate trisodium mixtures, citric acid-citric acid monosodium mixtures). ), Succinic acid buffer (e.g., succinic acid-sodium citrate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartarate buffer (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartarate mixture, tin Acid salt-sodium hydroxide mixtures, etc., fumaric acid buffer solutions (e.g., fumaric acid-fumaric acid monosodium fumarate mixtures, fumaric acid-fumaric acid disodium mixtures, fumaric acid monosodium fumaric acid disodium mixtures, etc.), gluconate buffers (gluconic acid-glyconate mixtures) , Gluconic Acid-Sodium Hydroxide Mixture, Gluconic Acid-Glucoside Yate mixtures, etc.), oxalate buffers (e.g., oxalic acid-oxalate salt mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures) , Lactic acid-potassium lactate mixtures, and the like, and acetate buffers (eg, acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Phosphate buffers, histidine buffers, and trimethylamine salts such as Tris are also possible.

Preservatives are added to retard the growth of microorganisms, typically 0.2% to 1% (w / v). Suitable preservatives for use in the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octedecyldimethylbenzyl ammonium chloride, benzalkonium halide (e.g. benzalkonium chloride, benzalko bromide, benzyl iodide). Cornium), hexamethonium chloride, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclonuxanol, and 3-pentanol.

Isotonic solutions are added for isotonicity of the liquid composition, including polyhydric sugar alcohols, suitably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, gyretol, sorbitol, mannitol, and the like. . The polyhydric alcohol is included in the range of 0.1% to 25wt%, generally 1% to 5% in consideration of the relative amount of the other components.

Stabilizers refer to a wide range of excipients that function primarily as swelling agents, as additives to dissolve therapeutic agents, to prevent degeneration or to prevent adhesion to container walls. Alcohols (mentioned above); Amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, omitin, L-leucine, 2-phenylalanine, glutamic acid and threonine; Organic sugars or sugar alcohols such as lactose, trehalose, stakiose, manditol, sorbitol, ziretol, myoinositol, galactitol, glycerol; Sugar alocol analogs such as cyclitol such as inositol; Polyethylene glycol; Amino acid polymers; Reducing substances including sulfur such as urea, glutathione, tiotic acid, thioglycolate sodium, thioglycerol, α-monothioglycerol and thiosulfate sodium; Low molecular weight polypeptides (eg, less than 10 amino acid residues); Proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Monosaccharides such as gyrose, mannose, fructose, glucose; Disaccharides such as lactose, maltose and sucrose; Trisaccharides such as raffinose; Polysaccharides such as dextran. Generally, stabilizers are present in the range of 0.1 to 10,000 parts by weight based on the weight of the active protein.

Non-ionic surfactants or surfactants (also known as wetting agents) assist in dissolving the therapeutic agent and agitate, thereby causing the composition to be exposed to shear surface stress without causing modification of the polypeptide. It can be added to protect. Suitable non-ionic surfactants include polyoleate (20, 80, etc.), polyoxamers (184, 188), Pluronic ^® polyols, polyoxyethylene sorbitan monoethers (Tween ^® -20, Tween ^® -80, etc.) There is this.

Other additional excipients include swelling agents or fillers (eg, starches), chelating agents (EDTA), antioxidants (ascorbic acid, methionine, vitamin E), cosolvents, and the like.

The active ingredient may also contain microcapsules (eg hydroxymethylcellulose, gelatin, poly- (methylmethacrylate) microcapsules) made using coacervation technology or an interfacial polymerization reaction; Colloidal drug delivery systems (liposomes, albumin vesicles, microemulsions, nanoparticles, nanocapsules) and macroemulsions can also be captured. See Remingtons' Pharmaceutical Science for such a technique.

Extra- intestinal compositions used for in vivo administration must be sterile. It can be sterilized through filtration through sterile filtration membranes.

In a preferred embodiment of the invention, the pharmaceutical composition comprises i) an IFNG variant of the invention, ii) a buffer, a salt of an especially organic acid capable of maintaining a pH of 5.0 to 6.5, iii) a stabilizer, in particular an organic sugar or a sugar alcohol, iv) Nonionic surfactants, and v) sterilized water. More specifically, the buffer is selected from the acetate, succinate, and citrate groups, the stabilizer is mannitol, or sorbitol, and the nonionic surfactant is Tween-20 or Tween-80. Preferably the pharmaceutical composition does not contain any preservatives.

Delayed release preparation

Suitable examples of delayed release preparations include semipermeable matrices of solid hydrophobic polymers containing conjugates, matrices of the appropriate type such as films or microcapsules. Examples of delayed release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide, L-glutamic acid and ethyl-L-glutamate copolymers, Non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Prolease ^® technology or Lupron Depot ^® (injectable microspheres consisting of lactic acid-glycolic acid copolymer and roprolide acetate), poly-D-(- ) -3-hydroxybutyl acid, and the like. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for long periods of time up to 100 days or more, but certain hydrogels release proteins for shorter periods. Encapsulated polypeptides remain in the body for a long time, which are denatured or aggregated upon exposure to moisture at 37 ° C., resulting in loss of biological activity and altered immunogenicity. Depending on the mechanism involved, reasonable strategies should be devised for stabilization. For example, if a cohesion mechanism is found in which intermolecular SS bonds are formed through thio-disulfide interactions, the sulfahydryl moiety is modified, lyophilized with an acidic solution, water additives are adjusted with appropriate additives, Certain polymeric matrix compositions can be developed to achieve stabilization.

Oral administration

For oral administration, the pharmaceutical composition must be in solid or liquid form, for example in the form of capsules, tablets, suspensions, emulsions or solutions. Pharmaceutical compositions are suitably formulated in unit dosage forms containing a given amount of active ingredient. Appropriate daily dosages available to humans or other mammals can vary widely depending on the condition of the patient and other factors, but will be determined by those skilled in the art using conventional methods.

Solid dosage forms for oral administration include capsules, tablets, suppositories, powders, granules, and the like. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose, starch and the like. Such dosage forms may include additional materials such as lubricants such as magnesium stearate. In the case of capsules, tablets and pills, the dosage form also includes a buffer. Tablets and pills may be coated with enteric skin.

The binder may be mixed with an adjuvant, for example, lactose, sucrose, starch powder, alkanoic acid cellulose ester, stearic acid, talc, magnesium stearate, magnesium oxide, phosphoric acid and sodium sulfate and calcium salts, acacia Gelatin, sodium alginate, polyvinyl-pyrrolidone or polyvinyl alcohol may be mixed and, for conventional administration, made into tablets or encapsulated. Alternatively, it may be dissolved in salt, water, polyethylene glycol, propylene glycol, ethanol, oil (corn oil, peanut oil, cottonseed oil, sesame oil), tracatan gum or various buffers. Carriers or diluents include time delay materials such as glyceryl monostearate, glyceryl distearate, which may be used alone or in admixture with waxes or other known materials.

The pharmaceutical composition undergoes conventional pharmaceutical operations, such as stabilization reactions, and may include conventional adjuvant such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers and the like.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, pensions liquids, including inert diluents commonly used in the art, ie, water. Such compositions may also include adjuvant such as wetting agents, sweetening agents, flavoring and fragrances and the like.

Topical administration

Formulations for topical administration include liquid or semi-liquid preparations (eg, applied medications, lotions, ointments, creams, plasters) that can penetrate the skin or are suitable for administration to the eyes, ears, or nose.

Transport by lung

Compositions suitable for using jet or ultrasonic nebulizers include, for example, polypeptide variants dissolved in water at a concentration of about 0.01-25 mg / ml per ml of solution, about 0.1-10 mg / ml. The compositions may include buffers, simple sugars (eg, sugars to stabilize proteins and control osmotic pressure), and / or 0.1 to 10 mg / ml human serum albumin. Buffers that may be used include acetate sodium, citrate and glycine. Suitably the buffer also has a molarity suitable for adjusting the pH of the solution in the range of 3 to 9 with the composition. Generally buffers in the molar range of 1 mM to 50 mM are suitable for this purpose. Sugars that may be used for stabilization include maltose, lactose, mannitol, sorbitol, trehalose, gyrose and the like, in amounts ranging from 1% to 10% by weight of the composition.

The nebulizer composition may also contain a surfactant to form aerosols to reduce or prevent surface aggregation of the protein upon atomization of the solution. Typically various surfactants may be used, for example polyoxyethylene fatty acid esters, alcohols and polyoxyethylene sorbitan fatty acid esters and the like. The amount generally ranges from 0.001% to 4% of the weight of the composition. Particularly suitable surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleates.

Methods and specific compositions for making suitable liquid particle dispersions of the present invention are described in WO94 / 20069, US5,915,378, US5,960,792, US5,957,124, US5,934,272, US 5,915,378, US 5,855,564, US 5,826,570, US 5,522,385.

Compositions that can be used in a given dose of inhalation device generally consist of finely divided powders. Such powders may be made by lyophilizing and then pulverizing the liquid conjugate composition, which may include stabilizers such as human serum albumin (HSA). Generally more than 0.5% (w / w) HSA is added. In addition, one or more sugars or sugar alcohols are added to the preparation as needed. Sugars or sugar alcohols include lactose, maltose, mannitol, sorbitol, sorbitol, trehalose, xylitol, xylose and the like. The amount added to the composition is in the range of 0.01 to 200% (w / w), suitably about 1 to 50% of the conjugate present. This preparation is lyophilized and ground to the desired particle size.

Appropriately sized particles are then suspended in a propellant with the aid of a surfactant. Propellants can be any conventional materials used for this purpose, for example chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons or trichlorofluoromethane, dichlorodifluoromethane, dichlorotetra Hydrocarbons including fluoroethanol, 1,1,1,2-tetrafloorethane or a combination thereof. Suitable surfactants include sorbitan treoleate, soy lecithin and the like. Oleic acid may also be useful as a surfactant. This mixture is then added to the conveying device. An inhaler that provides a suitable commercially available dose amount available for the purposes of the present invention is Ventolin supplied by Glaxo Inc., Research Triangle Park, N.C.

Formulations for powder inhalers include finely ground dry powder comprising a conjugate, such that an amount of swelling agent such as lactose, sorbitol, sucrose or mannitol can disperse the powder from the device, for example 50% of the weight. To 90%. The powder particles have an average diameter of about 10 μm, suitably 0.5 to 5 μm, most suitably 1.5 to 3.5 μm, and may have motility in the lungs corresponding to particles having a density of 1 g / cm 2. Powder inhalers that can be used in accordance with the techniques described herein include Spinhaler from Fisons Corp., Bedford, Mass.

Such powders for devices can be made and transported by the methods described in US 5,997,848, US 5,993,783, US 5,985,248, US 5,976,574, US 5,922,354, US 5,785,049, US 5,654,007.

Mechanical devices designed to deliver therapeutic products to the lungs include, but are not limited to, nebulizers, prescribed dose inhalers, powder inhalers, and the like, all of which are well known to those skilled in the art. Commercially available devices suitable for practicing the present invention include Mallinckrodt, Inc., St. Ultravent Nebulizers supplied by Louis, Missouri .; Acorn II Nebulizers supplied by Marquest Medical Procucts, Englewood, Colorado; Ventolin metered dose inhaler supplied by Glaxo Inc., Research Triangle Park, North Carolina; Spinhaler powder inhaler supplied by Fision Corp., Bedfor, Massachusetts; "Standing cloud" supplied by Inhale Therapeutic Systems, Inc., San Carlos, California; AIR inhalers supplied by Alkermes, Cambridge, Massachusetts; Systems to deliver drugs to AERx lungs from Aradigm Corporation, Hayward, California.

The invention is further described in the following examples. The embodiments are not to be understood in any way as limiting the generality of the specification and claims of the invention.

Materials and methods

material

CHO-K1 cells (available from American Type Culture Collection (ATCC # CCL-61))

HeLa cells (available from American Type Culture Collection (ATCC # CCL-2)).

ISRE-Luc was obtained from FROMSTRATAGENE, La Jolla USA.

PCDNA 3.1 / Higro was obtained from Invitrogen, Carlsbad USA.

Restriction enzymes and polymerases were obtained from New England Biolabs INC., Beverly, USA.

DMEM medium: Dulbecco's Modified Eagle Media (DMEM), 10% calf serum and Hygromycin B were obtained from Life Technologies A / S, Copenhagen, Denmark.

LucLite substrates were obtained from Packard Bioscience, Groningen, The Netherlands.

TopCount luminometer was obtained from Packard Bioscience, Groningen, The Netherlands.

Biotinylated polyclonal anti-human IFNG antibody, BAF285, was used from R & D Systems Inc., MINNEAPOLIS, USA.

Horse Radish peroxidase-conjugated streptavidin, P0397, was obtained from DAKO, Copenhagen, Denmark.

TMB blot reagents were obtained from KEM-EN-TEC, Copenhagen, Denmark.

Way

Overview of Interferon Test

As already disclosed, IFNG interacts with IFNG receptors on HeLa cells to activate the receptors. As a result, transcription is activated in a promoter comprising a reactive element (ISRE) stimulated with interferon. Thus, screening for agonists of interferon receptors is possible using the ISRE-coupled luciferase reporter gene (ISRE-luc) located in HeLa cells.

1st inspection

HeLa cells are co-transformed with ISRE-Luc and pCDNA 3.1 / hygro, and lesions (cell clones) can be selected in DMEM medium containing hygromycin B to obtain transformed cell clones. Cellular clones can be screened using luciferase activity in the presence and absence of IFNG. Cell clones with the highest ratio of stimulated luciferase activity relative to unstimulated luciferase activity were used for further testing.

To screen for polypeptide variants, 15,000 cells per well were inoculated on 96 well culture plates and incubated overnight in DMEM medium. The next day, the standard as well as the polypeptide variant was added to the cells at various concentrations. Actimmune was used as a "known standard" and the vial of Actimmune was diluted to 300 IU / ml in DMEM, 5% FBS and stored at -80 ° C until use.

The plates were incubated for 6 hours at 37 ° C. under 5% CO ₂ atmosphere. Successively LucLite substrate (Packard Bioscience, Groningen The Netherlands) was added to each well. The plate was sealed and luminescence was measured by SPC method (single photon count method) on a TopCount Luminometer (Packard).

Individual plates have wells containing IFNG as a stimulated criterion and other wells containing normal medium as a non-stimulated criterion. The ratio between the stimulated luciferase activity and the unstimulated luciferase activity is used as an internal reference for IFNG activity and interlaboratory variation. For each IFNG sample, unit amounts are measured against Actimmune standards and given as AU.

Determination of Increased Glycosylation

To measure various degrees of glycosylation of IFNG variant monomers, SDS-PAGE gels are run under standard conditions and transferred to nitrocellulose membranes. Western blots are performed according to the standard mode, biotinylated polyclonal anti-human IFNG antibody as the first antibody (BAF285 from R & D System), and horse redish peroxidase-conjugated streptavidin as the secondary antibody. (Horse Radish Peroxidase-conjugated streptavidin) (PO397 from DAKO), followed by staining with the following TMB blot reagent (KEM-EN-TEC, Copenhagen, Denmark). The distribution of IFNG variant monomers with varying degrees of glycosylation is made by visual inspection of the stained membrane.

Measurement of AUCsc

AUCsc is measured by subcutaneous administration of one 200 μl bolus of the same amount (activity criteria) of the inventive IFNG polypeptide variant in mice.

For these experiments, Sprag-Dawley rats weighing between 220 and 260 grams are used. IFNG polypeptide variants are formulated at sodium succinate (720 mg / l), mannitol (40 g / l), polysorbet 20 (100 mg / l) pH 6.0.

Prior to subcutaneous administration, one blood sample is taken from the tail-vein to determine what background IFNG activity is detected. After dosing, blood samples were removed from the tail vein after 10, 20, 40, 60, 120, 240, 480, 720, 1440, 1620, 1920 and 2880 minutes (sometimes 3600 minutes). Is collected. Serum is prepared by leaving the blood sample at room temperature for 20 minutes, followed by centrifugation at 5000 g, 20 minutes at room temperature. The serum is then separated and stored at -80 ° C until the IFNG activity is measured using the "primary measurement" above. It should be noted that when units are measured in serum samples from PK studies in rats, Actimmune standards are diluted in DMEM, 5% FBS and 5% rat serum.

Unit amounts (AU / ml) in serum over time (min) are then plotted, and AUCsc is calculated using GraphicsPad Prism 3.01.

In order to measure the increase in AUCsc of the IFNG polypeptide variant of the invention relative to its glycosylated huIFNG and / or Actimmune, aborted experiments are performed on the glycosylated huIFNG and / or Actimmune.

Serum half-life

Serum half-life is measured by subcutaneous administration of one 200 μl bolus of the same amount (activity criteria) of the inventive IFNG polypeptide variant in mice.

For these experiments, Sprag-Dawley rats weighing between 220 and 260 grams are used. IFNG polypeptide variants are formulated at sodium succinate (720 mg / l), mannitol (40 g / l), polysorbet 20 (100 mg / l) pH 6.0.

Prior to subcutaneous administration, one blood sample is taken from the tail-vein to determine what background IFNG activity is detected. After dosing, blood samples were removed from the tail vein after 10, 20, 40, 60, 120, 240, 480, 720, 1440, 1620, 1920 and 2880 minutes (sometimes 3600 minutes). Is collected. Serum is prepared by leaving the blood sample at room temperature for 20 minutes, followed by centrifugation at 5000 g, 20 minutes at room temperature. The serum is then separated and stored at -80 ° C until the IFNG activity is measured using the "primary measurement" above. It should be noted that when units are measured in serum samples from PK studies in rats, Actimmune standards are diluted in DMEM, 5% FBS and 5% rat serum.

Unit quantities (AU / ml) in serum over time (min) are then plotted, and serum half-life is calculated using WinNonLin Pro 3.0.

In order to measure the increase in the serum half-life of the inventive IFNG polypeptide variant relative to its glycosylated huIFNG and / or Actimmune, aborted experiments are performed on the glycosylated huIFNG and / or Actimmune.

Identification of surface exposed amino acid residues

rescue

Ealick et.al. Science 252: 698-702 (1991) reported on the experimental 3D structure of huIFNG identified by X-ray crystallography, for the C-alpha trace of IFNG homodimer. Walter et. al. Nature 376: 230-235 (1995) also reported on the IFNG homodimer structure having two molecules of the IFNG receptor in soluble form. Such structural coordinates are never made available. Thiel at al., Structure 8: 927-937 (2000) describe an IFNG homodimer structure complexed with two molecules of IFNG soluble receptors with a third receptor molecule that does not interact with the IFNG homodimer in the structure. It has been reported about.

Accessible Surface Area (ASA)

Using the computer program Access (B. Lee and FMRichards, J. Mol. Biol. 55: 379-400 (1971)) version 2 (Copyright (c) 1983 Yale University), the accessible surface area of individual atoms in the structure ( ASA) was calculated. This method generally uses a 1.4 micron probe and defines an accessible surface area as the area formed by the probe center. Before performing this calculation, all water molecules, hydrogen atoms, and other atoms not directly related to the protein are removed from the coordinates.

Fragment ASA of Side Chain

The fragment ASA of the side chain can be calculated by dividing the ASA sum of the atoms in the side chain by the value representing the ASA of the side chain atom of each residue type in the extended ALA-x-ALA trivalent peptide (Hubbard, Campbell & Thornton (1991) J. Mol. Biol .: 220,507-530). For example, a CA atom is considered not part of the side chain of the glycine residue or part of the remaining residues. The following table uses 100% ASA criteria for the side chains;

Ala 69.23Å ²

Arg 200.35Å ²

Asn 106.25Å ²

Asp 102.06Å ²

Cys 96.69Å ²

Gln 140.58Å ²

Glu 134.61Å

Gly 32.28Å

His 147.00Å

Ile 137.91Å

Leu 140.76Å

Lys 162.50Å

Met 156.08Å

Phe 163.90Å

Pro 119.65Å

Ser 78.16Å

Thr 101.67Å

Trp 210.89Å

Tyr 176.61Å

Val 114.14Å

Residues not detected in the structure are defined as 100% exposed because they reside in flexible parts.

Measure distance between atoms

InsightⅡ v. Calculate the distance between atoms using 98.0, MSI INC.'S molecular graphics software.

Determining Receptor Binding Sites

The receptor-binding site was defined as consisting of all residues with accessible surfaces that charge upon receptor binding. This is determined by at least two ASA calculations, one on the isolated ligand in the ligand / receptor complex and the other on the complete ligand / receptor complex.

Example 1-Measurement of Surface Exposed Amino Acid Residues

The X-ray structures used are two of the soluble IFNG receptors with the third molecule of the IFNG receptor in a structure that does not interact with the IFNG homodimer reported in Thiel at al., Structure 8: 927-937 (2000). It is the structure of an IFNG homodimer complexed with a molecule. This structure consists of an IFNG homodimer, with the two molecules labeled A and B. For construction purposes, an additional methionine is located before the IFNG labeled MO, the sequence being C-terminally truncated to 10 residues (Q133 is the final residue of the truncated molecule). All calculations in the examples subtract MO from the structure. The two IFNG monomer structures have a very weak electron density after residue 120, but only modeled before residue T126. Thus, in this example residues S121-T126 are removed from the structure prior to calculation. Two receptor fragments labeled C and D were allowed to interact directly with the IFNG homodimer and the third receptor molecule labeled E was excluded from this calculation because it was not in contact with the IFNG homodimer.

Surface exposure

Except for M0 and S121-T126 in the two molecules A and B, fractional ASA calculations on homodimers of the molecules yield residues having at least 25% of these side chains exposed to the surface in at least one monomer. Is the same as; 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, E119.

The following residues are those having at least 50% of the side chains exposed to the surface in at least one monomer; 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.

Performing fractional ASA calculations on homodimers of the molecules, including receptor molecules C and D, and excluding the M0 and S121-T126 in two molecules A and B, those exposed to the surface in at least one monomer Residues having at least 25% of the side chains are as follows; 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. The next residue is a residue having 50% of the amino acid side chains exposed to the surface in at least one monomer; 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, D102, L103, N104.

All these positions are the target of the modifications according to the invention.

Comparing the two lists, K12, S20, A23, D24, T27, H111, Q115, and A118 were removed from the 25% side chain ASA list upon receptor binding, and Q1, D2, E9, G18, H19, S20, A23, D24, T27, Q115, A118, E119 are removed.

Residues not determined structurally include residues S121, P122, A123, A124, K125, T126, G127, K128, R129, K130, R131, S132, Q133, M134, L135, F136, R137, G138, R139, R140, A141, Treated as completely surface exposed, such as S142, Q143. These residues also constitute separate targets for introducing the attachment groups in accordance with the present invention (or appear to belong to a group at a surface exposed amino acid residue, such as having at least 25% or at least 50% of the exposed side chains).

Example 2- Receptor Binding Site Measurement

ASA calculations as described above resulted in a decrease in ASA at the next residue in the IFNG molecule at at least one monomer in the complex as compared to that calculated at the isolated dimer; 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.

Example 3

Design of an Expression Cassette to Express IFNG with Optimized Codon Usage

To express at high levels in CHO cells, the DNA sequence (GenBank accession No. X13274) containing the full-length cDNA encoding the complete huIFNG was modified without native signal peptide. Codons in the huIFNG nucleotide sequence were modified to bias codon usage against codons frequently used in homosapiens. As a result, in order to introduce a restriction enzyme recognition site for the DNA restriction enzyme, certain nucleotides in the sequence were substituted with another. Primers were designed to allow genes to be synthesized.

Primers were assembled into synthesized genes using one-step PCR and PCR cycling parameters using the Platinum Pfx kit (Life Technologies). Amplify the assembled gene by PCR using the same conditions, the gene has the sequence in SEQ ID NO 42. The synthesized gene is cloned into pcDNA3.1 / hugof (InVitrogen) between the BamHI and XbaI sites to obtain pIGY-22.

pIGY-22 was transfected into CHO K1 cells by the use of Lipofectamin2000 (Life Technologies) as a transfection agent. After 24 hours the culture medium was incubated and measured for IFNG activity and concentration by Elisa. Using the primary measurement described here, 1.4 × 10 ⁷ AU / ml activity was obtained.

Example 4- Sited Modifications

Generation of Glycosylation Variants

In order to induce IFNG alterations, PCR-generated changes were designed in such a way that they can be introduced into the expression plasmid (pIGY-22) by traditional two-step PCR.

Two vector primers were used with specific modified primers: ADJ013: 5'-GATGGCTGGCAACTAGAAG-3 '(semi-significant down vector primer) (SEQ ID NO: 43) and ADJ014: 5'-TGTACGGTGGGAGGTCTAT-3' (SEQ ID NO : 44) (meaning up vector primer). S99T variants were generated by traditional two-step PCR, using ADJ013 and ADJ014 as vector primers, using ADJ093 (5'-GTTCAGGTCTGTCACGGTGTAATTGGTCAGCTT-3 ') (SEQ ID NO: 45) and ADJ094 (5'-AAGCTGACCAATTACACCGTGACAGACCTGAAC-3') (SEQ ID NO: 46) was used as a modification primer and pIGY-22 as a template. 447 bp PCR product was subcloned with pcDNA3.1 / Hygro (Invitron) using BAMHI and XbaI up to plasmid pIGY-48.

pIGY-48 was transfected into CHO K1 cells by the use of Lipofectamin2000 (Life Technologies) as a transfection agent. After 24 hours the culture medium was measured for IFNG activity and concentration. Using the primary measurements described herein, 5.1 × 10 ⁶ AU / ml activity was obtained.

E38N + S40T + S99T variants were generated via traditional two-stage PCR, with ADJ013 and ADJ014 as vector primers, ADJ091 5'-CATGATCTTCCGATCGGTCTC GTTCTTCCAATT-3 ') (SEQ ID NO: 47) and ADJ092 (5'-AATTGGAAGAACGAGACC GATCGGAAGATCATG-3 ') (SEQ ID NO: 48) is used as a modification primer and pIGY-48 as a template. The 447 bp PCR product was subcloned with pcDNA3.1 / Hygro (Invitron) using BAMHI and XbaI up to plasmid pIGY-54.

pIGY-54 was transfected into CHO K1 cells by the use of Lipofectamin2000 (Life Technologies) as a transfection agent. After 24 hours the culture medium was measured for IFNG activity and concentration. Using the primary measurements described herein, 1.3 × 10 ⁷ AU / ml activity was obtained.

Using similar standard techniques, a number of full-length IFNG glycosylation variants have been prepared. These variants were compiled in Table 1 below.

Generation of C-terminal Fragment Variants

C-terminal truncated INFG variants containing stop codons just downstream of the codons for Leu135 are generated by one-step PCR using pIGY-22, pIGY-48 and pIGY-541 as templates, and pcDNA3.1 / Hygro Tron) followed by subclones of PCR products using BAMHI and XbaI. Primers used for construction of these variants are ADJ014 (see above, upstream) and: 5'-GAGTCTAGATTACAGCATCTGGCTTCTCTT-3 '(SEQ ID NO: 49) (downstream). The resulting plasmids are named pIGY-72 (wild type IFNG, truncated after Leul35), pIGY-73 (tripped after S99T variant LEUL35) and pIGY-74 (truncated after E38N + S40T + S99T LEU35).

Generation of Cysteine-Containing IFNG Variants

Cysteine residue containing IFNG variants were generated according to the Stratagene's QUIKCHANGE ^™ XL site designated modified kit, manufacturer's specification. Seven IFNGs contain one introduced cysteine and were generated using pIGY-48 as a template: N10C + S99T, N16C + S99T, E38C + S99T, N59C + S99T, N83C + S99T, K94C + S99T and S99T + N104C. Similarly, IFNG variants containing six one introduced cysteine were generated using pIGY-54 as a template: N10C + E38N + S40T + S99T, N16C + E38N + S40T + S99T, E38N + S40T + N59C + S99T, E38N + S40T + N83C + S99T, E38N + S40T + K94C + S99T and E38N + S40T + S99T + N104C.

Example 5- PEGylation of Cysteine-Containing Variants

All buffers were de-oxygenated before use. Protein concentration was determined by measuring A280.

PEGylation Using OPSS Coupling Chemistry

Reduction of 7.2 ml 1.3 mg / ml IFNG variant N16C + S99T (full length), 4% mannitol, 0.01% Tween 20, pH 6.0 in 5 mM sodium succinate by incubating for 30 minutes at room temperature with 300 μl 0.5 M DTT It became. IFNG variants were desalted by performing a 2.5 ml three aliquot run on NAP 25 gel filtration column (Pharmacia) in Buffer A (50 mM sodium phosphate, 1 mM EDTA, pH 8.1). Each aliquot was eluted at 3.5 ml.

mPEG-OPSS (10 kDa) was dissolved in buffer to a concentration of 2 mg / ml, and reduced to eastern blood and administered to desalted IFNG, and incubated at room temperature with gentle shaking for 60 minutes.

11 ml of reaction mixture was concentrated to 1-6 ml using Vivaspin20 column (Vivascience), and the remaining mPEG was removed by gel filtration using Sepjacryl S-100 column (Pharmacia) equilibrated in Buffer A.

PEGylated IFNG variants were diafiltered using a Vivaspin 6 column (Vivascience) at 5 mM sodium succinate, 4% mannitol, pH 6.0, and Tween 20 was added to 0.01%. Purified PEGylated IFNG variants had an inactivation of 1.3 × 10 ⁶ AU / mg as measured in the first assay described herein (15% of inactivation corresponding to non-PEGylated IFNG variants).

PEGylation Using MAL Coupling Chemistry

Reduction by incubating 1.6 ml of 1.5 mg / ml IFNG variant N59C + S99T (full length), 4% mannitol, 0.01% Tween 20, pH 6.0 in 5 mM sodium succinate with 64 μl 0.5 M DTT for 30 minutes at room temperature. It became. IFNG variants were desalted on a NAP 25 gel filtration column (Pharmacia) in Buffer A (50 mM sodium phosphate, 1 mM EDTA, pH 8.1). IFNG variants were eluted at 3.5 ml.

mPEG-MAL (5 kDa) was dissolved in buffer to a concentration of 0.5 mg / ml, then reduced to eastern blood and administered to desalted IFNG, and incubated at room temperature with gentle shaking for 120 minutes.

Ammonium sulfate was added to a concentration of 0.9 M, and PEGylated IFNG variants were applied to a 1 ml Resource ^™ phenyl column (Pharmacia) equilibrated with buffer B (20 mM sodium phosphate, 0.9 M ammonium sulfate, pH 6.6). The column was washed with 5 column volumes of buffer B prior to elution of PEGylated IFNG variants immobilized in 0 to 50% Buffer C (20 mM sodium phosphate, pH 6.6) in a straight gradient over 30 column volumes. PEGylated IFNG variants were eluted near 0.6 M ammonium sulfate.

Fractions containing PEGylated IFNG variants were collected, diafiltered using a Vivaspin 6 column (Vivascience) at 5 mM sodium succinate, 4% mannitol, pH 6.0, and Tween 20 was added to 0.01%. . Purified PEGylated IFNG variants had an inactivation of 2.4 × 10 ⁶ AU / mg as measured in the primary measurements described herein (15% of inactivation corresponding to non-PEGylated IFNG variants).

Example 6 Expression of IFNG and IFNG Variants in Mammalian Cells

For immediate expression of IFNG, cells were cultured with 1:10 calf serum (BioWhittacker cat # 02-701F) and 1: 100 penicillin and streptomycin (BioWhittaker cat # 17-602E) (Dulbecco's MEM / Nut.-Mix F). Incubated in −12 (Ham) L-glutamine, 15 mM Hepes, Pyridoxine-HCl (Life Technologies Cat # 31330-038) up to 95% confluency. IFNG encoding plasmids were infected with cells using Lipofectamine2000 (Life Technologies) according to the manufacturer's instructions. After 24 infection, media was collected and measured for IFNG activity. In addition, to quantify the relative number of glycosylation sites used, western blots were performed using cultured culture medium.

CHO K1 cells were infected with an IFNG encoding plasmid, and the cells were cultured in medium containing 0.36 mg / ml hygromycin to generate stable clones expressing IFNG. Stable infected cells were isolated and subcloned at limited dilution. Clones producing high levels of IFNG were identified by ELISA.

Example 7. Mass Production

Stable cell lines expressing IFNG or variants include Dulbecco's MEM / Nut.-Mix F-12 (Ham) L-glutamine, 15 mM Hepes, Pyridoxine-HCl (Life Technologies Cat # 31330-038), 1:10 Calf Serum (BioWhittacker). cat # 02-701F) and 1: 100 penicillin and streptomycin (BioWhittaker cat # 17-602E) were incubated from 1700 cm 2 roller bottle (Corning, # 431200) to confluency. The medium was then 300 ml UltraCHO with L-glutamine (BioWhittacker cat # 02-724Q), 1: 500 EX-CYTE VLE (Serological Proteins Inc. # 81-129), and 1: 100 penicillin and streptomycin (BioWhittacker). modified with the addition of cat # 17-602E). After 48 h growth, the medium was transferred to Dulbecco's MEM / Nut.-Mix F-12 (Ham) L-glutamine, pyridoxine-HCl (Life Technologies Cat # 21041-025), 1: 100 penicillin and streptomycin (BioWhittaker Cat # 17). -602E), 1: 500 EX-CYTE VLE (Serological Proteins Inc. # 81-129), and 1: 100 penicillin and streptomycin (BioWhittacker cat # 17-602E) addition. Then every 24 hours. The culture medium was incubated and replaced with 300 ml of fresh serum-free medium with the same additives. Recruited medium was filtered through 0.22 μm to remove cells.

Example 8-Tablet

The filtrate was microfiltered (0.22 μm) prior to ultrafiltration using a Millipore TFF system to 1/15 volume. In the same system, the concentrate was diafiltered using 10 mM Tris, pH 7.6. Ammonium sulfate was administered to a concentration of 1.7 M, and after stirring, the precipitate was removed by centrifugation at 8000 rpm for 25 minutes in a Sorvall centrifuge using a GS3 rotor.

The supernatant was applied to a 25 ml column of Phenyl High Performance (Pharmacia) previously equilibrated with 10 mM Tris, 1.7 M ammonium sulfate, pH7.6. After application, the column was washed with 3 column volumes of 10 mM Tris, 1.7 M ammonium sulfate, pH7.6, and the immobilized IFNG variants eluted over 10 column volumes with a linear gradient up to 100% 10 mM Tris, pH 7.6. . Pass-through and eluted IFNG variants were fractionated. Fractions rich in IFNG variants were collected and buffered by diafiltration to 10 mM Tris, pH 9.0, using a Vivaflow 200 system (VivaScience) with a cutoff molecular weight of 10,000 Da.

The IFNG variant was then applied to an 18 ml Q-sepharose Fast Flow column previously equilibrated with 10 mM Tris, pH 9.0. After application, the column was washed with 3 column volumes of 10 mM Tris, pH 9.0, and the immobilized IFNG variants eluted over 15 column volumes with a gradient to 0-100% 10 mM Tris, 0.5 M NaCl, pH 9.0. Pass-through and eluted IFNG variants were fractionated. Fractions rich in IFNG variants were collected and buffered by diafiltration to 10 mM sodium phosphate, pH 7.0, using a Vivaflow 200 system (VivaScience) with a cutoff molecular weight of 10,000 Da.

Next, an IFNG variant was applied with 8 ml of CHT ceramic hydroxyapatite column (Biorad) previously equilibrated with 10 mM sodium phosphate, pH 7.0. After application, the column was washed with 5 column volumes of 10 mM sodium phosphate, pH 7.0, and the immobilized IFNG variants eluted over 30 column volumes in a gradient to 0-60% 500 mM sodium phosphate, pH 7.0. Pass-through and eluted IFNG variants were fractionated. Fractions rich in IFNG variants were collected, converted to 5 mM sodium succinate, 4% mannitol, pH 6.0 using a VivaSpin 20 column (VivaScience), and Tween 20 was added to a 0.01% concentration. IFNG variants were sterile filtered and stored at -80 ° C.

Optionally, IFNG variants can be purified according to the following purification schemes.

The filtrate was microfiltered (0.22 μm) prior to ultrafiltration using a Millipore TFF system to 1/15 volume. In the same system, the concentrate is diafiltered using 10 mM Tris, pH 7.6, then the pH is adjusted to 9.0 and the precipitate is removed by microfiltration.

Samples were applied to Q-sepharose Fast Flow columns with IFNG variants pre-equilibrated with 10 mM Tris, pH 9.0. After application, the column was washed with 3 column volumes of 10 mM Tris, pH 9.0, and the immobilized IFNG variants eluted over 15 column volumes with a gradient to 0-100% 10 mM Tris, 0.5 M NaCl, pH 9.0. Pass-through and eluted IFNG variants were fractionated. Fractions rich in IFNG variants were pooled and the pH adjusted to 7.6. Ammonium sulphate was added to 1.5 M, and the stirred precipitate was removed by centrifugation.

IFNG variants were applied to Phenyl High Performance (Pharmacia) columns previously equilibrated with 10 mM Tris, 1.5 M ammonium sulfate, pH7.6. After application, the column was washed with 3 column volumes of 10 mM Tris, 1.5 M ammonium sulfate, pH7.6, and the immobilized IFNG variants eluted over 10 column volumes with a linear gradient up to 100% 10 mM Tris, pH 7.6. . Pass-through and eluted IFNG variants were fractionated. Fractions rich in IFNG variants were collected and ammonium sulfate adjusted to 1.7 M.

Next, the IFNG variant was applied on a butylsepharose column previously equilibrated with 10 mM sodium phosphate, 1.7 M ammonium sulfate, pH 7.6. After application, the column was washed with 10 mM sodium phosphate, 1.7 M ammonium sulfate, pH 7.6, and the immobilized IFNG variants were eluted stepwise using 10 mM sodium phosphate, pH 6.5. Pass-through and eluted IFNG variants were fractionated.

Fractions rich in IFNG variants were collected and applied to a hydroxy-apatite column previously equilibrated with 10 mM sodium phosphate, pH 6.6. After application, the column was washed with 5 column volumes of 10 mM sodium phosphate, pH 6.5, and the immobilized IFNG variants eluted over 30 column volumes in a gradient to 0-100% 500 mM sodium phosphate, pH 6.5. Pass-through and eluted IFNG variants were fractionated.

Fractions containing IFNG variants were pooled and the buffer was converted to a buffer containing 5 mM sodium succinate, 4% mannitol, pH 6.0. Tween 20 was added to a concentration of 0.01%. IFNG variants were sterile filtered and stored at -80 ° C.

Example 9- Variants and Activity of PEGylated Variants

Using this "primary measurement", the following activity data (after instantaneous expression) were obtained.

TABLE 1 Activity of full-length and truncated rhuIFNG polypeptide variants after momentary infection

Using this "primary measurement", the following detailed activity data (after instantaneous expression) were obtained.

TABLE 2 Activity of full-length rhuIFNG polypeptide variants after purification

The activities of multiple PEGylated variants went through the same PEGylation procedure, but were measured by comparing the activity of the PEGylated product by a sample without the PEGylation addition in the reaction medium. The results are described in Table 3 below.

Table 3 Activity of PEGylated variants of full length rhuIFNG polypeptides

1) ODSS coupling chemistry was used. MAL coupling schemes were applied to all other PEGylated variants.

It should be noted that the activity data shown in Table 1 reflects the combination of expression level and inactivity in CHO K1 cells. Therefore, it can be concluded that all variants exhibit comparable expression levels / inactivity with respect to rhuIFNG variants.

As shown in Table 2, all cysteine variants show reduced inactivation compared to rhuIFNG, whereas variants containing E38N, S40T and / or S99T modifications show inactivation comparable to rhuIFNG. When the cysteine variants were variants PEGylated at 5 or 10 kDa, a 2-3 fold reduction in inactivity was observed (Table 3). Without being limited to any specific theory, it should be considered that the decrease in inactivation may be due to reduced receptor binding caused by steric hindrance of the conjugated PEG group.

Example 10-Measurement of Use of N-Glycosylation Sites

To quantify the relative number of glycosylation sites used, western blots were performed using culture medium (see FIG. 1), for wild type rhuIFNG (full length), about 50% of both glycosylation sites (2N) Was used, about 40% used the glycosylation site (1 N), and about 10% was not glycosylated (0 N).

These results are summarized in Hooker et al., 1998, J. Interferon and [CYTOKINE] Res. 18,287-295 and Sarenva et al., 1995, Biochem J., 308, 9-14.

As shown in FIG. 1, the S99T variant (full length) uses the two glycosylation sites significantly more efficiently than the corresponding wild type. For S99T, about 90% used both glycosylation sites (2N), about 7% used glycosylation sites (1 N), and about 3% was not glycosylated (0 N).

It is also shown that the glycosylation site introduced at position 38 from FIG. 1 is significantly better used for variant E38N + S40T (full length) compared to non-optimal variant E38N (full length).

These data clearly indicate whether these sites are naturally occurring or introduced, and that better utilization of glycosylation sites can be achieved by introducing threonine residues than serine residues at the +2 position with respect to asparagine residues. Shows.

Example 11-Pharmacokinetic Studies

AUC for subcutaneous administration (AUCsc) was measured for a number of IFNG variants described herein in mice. The results are described in Table 4 and in FIGS. 2 and 3.

Table 4 Pharmacokinetic Data on Subcutaneous Administration in Rats

1) Battlefield

2) 5 kDa mPEG attached to the full length, introduced cysteine residue.

3) 10 kDa mPEG attached to full length, introduced cysteine residues

Referring to Figures 2, 3 and Table 4, it is clear that the variants (PEGylated variants) have significantly higher AUC compared to rhuIFNG, especially when compared to commercially available Actimmune when subcutaneously administered. Referring to FIG. 3, it should be noted that the dose administered of the two PEGylated variants was reduced 2.5 times compared to the dose administered of the [E38N + S40T + S99T] variant.

Clearly, this opens up the possibility of administering small doses, thereby allowing fewer side effects, and / or less frequent administration of the active element, thereby obtaining improved patient comfort.

Claims (62)

An interferon gamma (IFNG) polypeptide variant having the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof exhibiting IFNG activity and exhibiting IFNG activity. The variant of claim 1, wherein said variant has the amino acid sequence shown in SEQ ID NO: 1. The variant of claim 1, wherein said variant is a fragment of an amino acid sequence as shown in SEQ ID NO: 1 truncated C-terminally with 1-15 amino acid residues. The method of claim 2, wherein the fragment is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID N0: 8 , SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 Variants selected from the group. 5. The variant according to claim 1, wherein said variant is glycosylated. The variant of claim 1, wherein the variant comprises at least one further modification and exhibits IFNG activity. The variant of claim 6, wherein the variant comprises a 1-10 modification. The method of claim 6, wherein the variant is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID N0: 8 , SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 Variants comprising 1-10 modifications with respect to amino acid sequences selected from the group. The variant according to claim 7 or 8, wherein said modification is substitution. The variant of claim 6, wherein the variant comprises at least one introduced and / or at least one removed amino acid residue comprising an attachment group to a non-polypeptide moiety. The variant of claim 10, wherein said variant comprises at least one introduced glycosylation site. The variant of claim 11, wherein said glycosylation site is an N-glycosylation site. The variant of claim 12, wherein said N-glycosylation site is introduced at a position comprising an amino acid residue having its side chain exposed to at least 25% of the surface (as defined in Example 1). The variant of claim 13, wherein said N-glycosylation site is introduced at a position comprising an amino acid residue having its side chain exposed to at least 50% of the surface (as defined in Example 1). The variant according to claim 13 or 14, wherein said N-glycosylation site is introduced by substitution. The variant of claim 15 wherein said substitution is selected from the group consisting of K12S, K12T, G18S, G18T, E38N, E38N + S40T, K61S, K61T, N85S, N85T, K94N, Q106S and Q106T. The variant of claim 16 wherein said substitution is selected from the group consisting of K12T, G18T, E38N + S40T, K61T, N85T, K94N and Q106T. The variant of claim 17, wherein said substitution is E38N + S40T. The variant of claim 10, wherein said variant comprises at least one introduced cysteine residue. The variant of claim 19 wherein said cysteine residue is introduced at a position comprising an amino acid residue having its side chain exposed to at least 25% of the surface (as defined in Example 1). The variant of claim 20, wherein said cysteine residue is introduced at a position comprising an amino acid residue having its side chain exposed to at least 50% of the surface (as defined in Example 1). 22. The variant according to claim 20 or 21, wherein said cysteine residue is introduced by substitution. The variant of claim 22, wherein said substitution is selected from the group consisting of N16C, E38C, N59C, N83C, K94C, N104C and A124C. The variant of claim 19-23, wherein said cysteine residue is covalently attached to a non-polypeptide moiety. The variant of claim 24, wherein said non-polypeptide moiety is a polymer moiety. The variant of claim 25, wherein said polymer molecule is a linear or branched polyethylene glycol. The variant of claim 10, wherein the variant comprises at least one introduced N-glycosylation site and at least one introduced cysteine residue. The method of claim 27, wherein the N-glycosylation site is introduced at a position defined in any one of claims 13-18, and the cysteine residue is introduced at a position defined in any one of claims 20-23. Variants. The variant of claim 28 wherein said variant is K12T + N16C, K12T + E38C, K12T + N59C, K12T + N83C, K12T + K94C, K12T + N104C, K12T + A124C, G18T + N10C, G18T + E38C, G18T + N59C, G18T + N83C, G18T + K94C, G18T + N104C, G18T + A124C, G18N + S20T + N10C, G18N + S20T + N16C, G18N + S20T + E38C, G18N + S20T + N59C, G18N + S20T + N83C, G18N + S20T , G18N + S20TN104C, G18N + S20T + A124C, E38N + S40T + N10C, E38N + S40T + N16C, E38N + S40T + N59C, E38N + S40T + N83C, E38N + S40T + K94C, E38N + S40T + N104C, E38C40 + A124C, K61T + N10C, K61T + N16C, K61T + E38C, K61T + N83C, K61T + K94C, K61T + N104C, K61T + A124C, N85T + N10C, N85T + N16C, N85T + E38C, N85T + N59C, N85T + N59C94 , N85T + N104C, N85T + A124C, K94N + N10C, K94N + N16C, K94N + E38C, K94N + N59C, K94N + N83C, K94N + N104C, K94N + A124C, Q106T + N10C, Q106T + N16C, Q106T + E38C A variant comprising a substitution selected from the group consisting of + N59C, Q106T + N83C, Q106T + K94C and Q106T + A124C. The method of claim 29, wherein the variant is E38N + S40T + N10C, E38N + S40T + N16C, E38N + S40T + N59C, E38N + S40T + N83C, E38N + S40T + K94C, E38N + S40T + N104C, and E38N + S40T + A variant comprising a substitution selected from the group consisting of A124C. 31. The variant of any one of claims 27-30, wherein the cysteine residue is covalently attached to the non-polypeptide moiety. 32. The variant of claim 31, wherein the non-polypeptide variant is defined in any one of claims 25-26. The variant of claim 6-32, wherein the variant comprises a substitution selected from the group consisting of G26F, G26N, G26Y, G26Q, G26V, G26A, G26M, G26K, G26R, G26T, G26H, G26C and G26S. Variants. The variant of claim 33, wherein the variant comprises a substitution selected from the group consisting of G26A, G26M, G26K, G26R, G26T, G26H, G26C a and G26S. The variant of claim 34, wherein the variant comprises a G26A or G26S substitution. 36. The variant of claim 35, wherein said variant comprises a substitution G26A. The variant of claim 10, wherein the variant comprises at least one removed N-glycosylation site and at least one introduced cysteine residue. The method of claim 37, wherein the variant comprises at least one removed N-glycosylation site, at least one introduced N-glycosylation site, and at least one introduced cysteine residue, wherein the introduced N-glyco The variant site is introduced at a position different from the position occupied by the removed N-glycosylation site. 39. The variant of claim 37 or 38, wherein said cysteine residue is covalently attached to a non-polypeptide moiety. The variant of claim 39, wherein the non-polypeptide moiety is defined in any one of claims 25-26. A nucleotide sequence that encodes a polypeptide variant as defined in claims 1-40. An expression vector comprising the nucleotide sequence defined in claim 41. A glycosylated host cell comprising the expression vector according to claim 42 or the nucleotide sequence defined in claim 41. The host cell according to claim 43, which is a CHO cell or a BHK cell. An IFNG polypeptide variant population comprising an IFNG polypeptide variant as defined in any one of claims 1-40, or a composition comprising an IFNG polypeptide variant population. A pharmaceutical composition comprising a polypeptide variant as defined in any one of claims 1-40 and a pharmacologically acceptable diluent, carrier or excipient. A polypeptide variant as defined in any one of claims 1-40 for a medicament or a pharmaceutical composition as defined in claim 46. Use of a medicament for the treatment of an interstitial pulmonary disease of a polypeptide variant as defined in any one of claims 1-40 or a pharmaceutical composition as defined in claim 46. 49. The use of claim 48, wherein the interstitial lung disease is idiopathic pulmonary fibrosis. Treating or preventing an interstitial lung disease comprising administering to a mammal in need thereof an effective amount of a polypeptide variant as defined in claim 1 or a pharmaceutical composition as defined in claim 46. How to. 51. The method of claim 50, wherein said interstitial lung disease is idiopathic pulmonary fibrosis. A method of increasing in vivo glycosylation of a parent IFNG polypeptide comprising at least one in vivo N-glycosylation site having an NXS amino acid sequence, wherein X is an amino acid residue excluding proline, and the method yields an IFNG variant. And replacing the serine residue at the NXS amino acid residue with a threonine residue. The method of claim 52, wherein said parental IFNG polypeptide has the amino acid sequence shown in SEQ ID NO: 17. The method of claim 52, wherein said parent IFNG polypeptide has the amino acid sequence shown in SEQ ID NO: 17 wherein the parental IFNG polypeptide is C-terminally truncated to 1-15 amino acid residues. 55. The fragment of claim 54, wherein the fragment is SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 With SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33 Method selected from the group consisting of: (a) culturing a glycosylated host cell comprising a nucleotide sequence encoding an IFNG polypeptide variant as defined in any one of claims 1-40 under conditions favorable for expression of the polypeptide variant; (b) optionally reacting said polypeptide portion with an in vitro non-polypeptide moiety under favorable conditions for conjugation to occur; And (c) recovering the polypeptide variants A method for producing an IFNG polypeptide variant as defined in any one of claims 1-40. The method of claim 56, wherein (a) culturing a glycosylated host cell comprising a nucleotide sequence encoding an IFNG polypeptide variant as defined in any one of claims 1-40 under conditions favorable for expression of the polypeptide variant; (b) reacting said polypeptide portion with an in vitro non-polypeptide moiety under favorable conditions for conjugation to occur; And (c) recovering the polypeptide variants How to include. 58. The method of claim 57, wherein said variant is defined in any of claims 19-40. The method of claim 56, wherein (a) culturing a glycosylated host cell comprising a nucleotide sequence encoding an IFNG polypeptide variant as defined in any one of claims 1-40 under conditions favorable for expression of the polypeptide variant; And (b) recovering the polypeptide variants How to include. 62. The method of claim 61, wherein the polypeptide variant is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID N0: 13, SEQ ID NO: 14, SEQ ID N0: 15 and SEQ ID NO: 16 A method having an amino acid sequence selected from the group consisting of. 60. The method of claim 59, wherein said variant comprises at least one further modification. 61. The method of claim 60, wherein the variant is defined in any one of claims 11-18 or 33-36.