WO2008101671A2 - Il-4 fc fusion proteins - Google Patents

Il-4 fc fusion proteins Download PDF

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Publication number
WO2008101671A2
WO2008101671A2 PCT/EP2008/001293 EP2008001293W WO2008101671A2 WO 2008101671 A2 WO2008101671 A2 WO 2008101671A2 EP 2008001293 W EP2008001293 W EP 2008001293W WO 2008101671 A2 WO2008101671 A2 WO 2008101671A2
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Prior art keywords
fusion protein
anyone
domain
polypeptide
carcinoma
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PCT/EP2008/001293
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French (fr)
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WO2008101671A9 (en
WO2008101671A3 (en
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Oliver Hill
Christian Gieffers
Meinolf Thiemann
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Apogenix Gmbh
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Publication of WO2008101671A3 publication Critical patent/WO2008101671A3/en
Publication of WO2008101671A9 publication Critical patent/WO2008101671A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention relates to fusion proteins comprising at least one IL-4 polypeptide domain and a constant immunoglobulin domain.
  • WO 2004/069274 refers to the use of cytokine antagonists which modulate the expression and/or the function of a cytokine in a cell for the downregulation of anti-apoptotic proteins in a cell. In particular, it is referred to the use of cytokine antagonists for the treatment of cancer. Muteins of the cytokines themselves are given as examples of cytokine antagonists, which are able to bind to the respective cell surface receptor, inhibiting the signal cascade triggered by the cytokine itself.
  • US Patents US 6,313,272 and US 6,028,176 describe recombinant agonistic or antagonistic human IL-4 muteins comprising at least one amino acid substitution in the binding surface of either the region of the A- or C- ⁇ -helix of the wild-type IL-4.
  • the IL-4 muteins are indicated as being suitable for the treatment of condition exacerbated by IL-4 production such as asthma, allergy or inflammatory response-related conditions. It is speculated that the IL-4 muteins might be suitable for the treatment of cancers or tumours.
  • US Patent Application US 2005/0059590 describes modified IL-4 mutein receptor antagonists comprising an IL-4 mutein receptor antagonist, and in particular the IL-4 muteins as disclosed in the above-mentioned US Patents US 6,313,272 and US 6,028,176, coupled to polyethylene glycol. Said modified muteins are in particular indicated as useful in the treatment of severe asthma, chronic obstructive pulmonary disease and related lung conditions.
  • US Patent US 5,723,118 describes mutant IL-4 proteins which compete with the wild-type IL-4 for occupation of the IL-4 receptor and act as antagonists or partial agonists of the human interleukin-4. In particular, mutant IL-4 proteins are disclosed wherein one or more of the amino acids occurring at position 121 , 124 or 125 have been replaced. The mutant IL-4 proteins are indicated as being suitable for the treatment and/or prevention of allergic conditions.
  • EP 06 014 080.3 and EP 06 026 609.5 disclose that antagonistic IL-4 muteins are suitable in combination with at least one further chemotherapeutic or pro-apoptotic agent for the treatment of cancer diseases.
  • the antagonistic IL-4 muteins are used for curative cancer therapy.
  • fusion proteins comprising at least one IL-4 polypeptide fused to a constant immunoglobulin domain have favourable pharmaceutical and physiological properties.
  • the present invention relates to a fusion protein comprising (i) at least one first domain comprising an IL-4 polypeptide fused to (ii) at least one heterologous second domain comprising at least a portion of a constant immunoglobulin domain, and (iii) a fusion region between the first and second domain, wherein the fusion region comprises at least one cysteine residue.
  • the first domain is located N-terminally from the second domain.
  • the fusion protein may comprise a signal sequence, e.g. an N-terminal signal peptide, which allows a secretory expression in a host cell, particularly in a eukaryotic host cell.
  • the signal peptide may be a homologous IL-4 signal peptide or a heterologous signal peptide.
  • the IL-4 domain comprises at least one cysteine residue which is capable of forming a disulphide bridge with the at least one cysteine residue in the fusion region.
  • this disulphide bridge the protein structure may be stabilized.
  • Free cysteine residues in the IL-4 polypeptide domain are preferably located at the N-terminus and/or at the C-terminus thereof, more preferably at the N- terminus, e.g. at amino acid position 1-20 of the mature IL-4 polypeptide. More preferably, the free cysteine residue is a naturally occurring cysteine residue, e.g. a cysteine residue at amino acid position 3 of the mature human IL-4 polypeptide.
  • the IL-4 polypeptide is preferably a mammalian IL-4 polypeptide or a mutein thereof, e.g. an agonistic or antagonistic IL-4 mutein.
  • the term "IL-4 mutein” refers to any polypeptide derived from IL-4 polypeptides which has biological activities of IL-4, e.g. increasing the activity of endogenous IL-4 (agonistic muteins) or which inhibits biological activity of IL-4, e.g. inhibiting the activity of endogenous IL-4 (antagonistic mutein).
  • the mutein differs from the native IL-4 polypeptide by substitution, deletion and/or insertion of at least one amino acid.
  • the IL-4 mutein has an identity of at least 85%, 90%, 95%, 97% or 98.5% up to 99.5% with a native IL-4 polypeptide over the entire amino acid sequence.
  • the first domain of the fusion protein comprises human IL-4, an agonistic human IL-4 mutein or an antagonistic human IL-4 mutein, e.g. IL-4 muteins as disclosed in the documents as cited above, which are herein incorporated by reference.
  • human IL-4" refers to a human IL-4 as disclosed in US 5,017,691 , which is herein incorporated by reference, or an allelic variant thereof, e.g. an allelic variant comprising an amino acid substitution at position 29, particularly a substitution of isoleucine by valine.
  • the IL-4 polypeptide is a C-terminally truncated IL-4 polypeptide. More preferably, the last amino acid residue is a cysteine residue (e.g. Cys127 of native human IL-4). In addition to the C-terminal truncation, the IL- 4 polypeptide may comprise the wild-type sequence or further mutations.
  • the present invention refers to a combination of (i) at least one fusion protein as described above and (ii) at least one further medicament.
  • the amino acid sequence of the preferred antagonistic IL-4 muteins differs from the amino acid sequence of the wild-type IL-4 by mutation of one or more amino acids at certain positions of the native protein.
  • mutant as used in the context of the present invention can be understood as substitution, deletion and/or addition of single or multiple amino acids in the target sequence.
  • mutation of the target sequence in particular of the native IL-4 sequence, is a substitution at one or more positions of the native IL-4 polypeptide chain.
  • substitution can occur with different genetically encoded amino acids or by non-genetically encoded amino acids.
  • non-genetically encoded amino acids are homocysteine, hydroxyproline, ornithine, hydroxylysine, citrulline, carnitine, etc.
  • a substitution within the native polypeptide sequence can be a conservative or a non-conservative substitution.
  • the common classification of the amino acid residues on the base of the side-chain characteristics, which determine the amino acid groups for a conservative or a non-conservative substitution, is well known by the person skilled in the art.
  • IL-4 muteins having an antagonistic action with respect to the action of the wild-type IL-4.
  • antagonistic action means that the IL-4 muteins of the invention are capable of modulating the function of the cytokine, in particular are capable of inhibiting the function of endogenous IL-4 cytokine.
  • IL-4 produced in cancer cells promotes the up-regulation of anti-apoptotic proteins which may lead to resistance to cell death and to therapy refractoriness.
  • an antagonistic action of the IL-4 muteins of the invention leads to the inhibition of the internal signal cascade triggered by the endogenous IL-4 which leads to the up-regulation of anti-apoptotic proteins.
  • the IL-4 muteins of the invention may further show a higher affinity for the wild-type IL-4 receptor in comparison to wild-type IL-4.
  • the muteins may compete with the endogenously expressed IL-4 for the binding site on the respective receptor.
  • the present invention comprises the use of IL-4 muteins, wherein mutations of the amino acid sequence of the wild-type IL-4 sequence have been made to the region of the A-, C- and/or D-helices and more preferably to those amino acids comprising the binding surfaces of said helices of the IL-4 protein.
  • the IL-4 mutein of the invention is preferably an IL-4 mutein as described in US 5,723,118 and US 6,130,318, which are herein incorporated by reference in their entirety.
  • a mutation to the region of the D-helix of the wild-type IL-4 protein sequence occurs preferably on at least one of the positions 120, 121 , 122, 123, 124, 125, 126, 127 and/or 128 of the wild-type amino acid sequence. Even more preferably, the mutation occurs on at least one of the positions 121 , 124 and/or 125. Most preferably, the mutation occurs at position 121 and/or 124.
  • a IL-4 mutein of the wild-type is used, wherein the amino acid tyrosine, which occurs naturally at position 124, is replaced by aspartic acid, glycine or glutamic acid, i.e. the amino acid tyrosine, which occurs naturally at position 124, is replaced by aspartic acid, glycine or glutamic acid, i.e. the amino acid tyrosine, which occurs naturally at position 124, is replaced by aspartic acid, glycine or glutamic acid, i.e. the amino acid tyrosine, which occurs naturally at position 124, is replaced by aspartic acid, glycine or glutamic acid, i.e. the amino acid tyrosine, which occurs naturally at position 124, is replaced by aspartic acid, glycine or glutamic acid, i.e. the amino acid tyrosine, which occurs naturally at position 124, is replaced by aspartic acid, glycine or glutamic acid,
  • a IL-4 mutein is preferably used wherein the amino acid arginine, which occurs naturally at position 121, is replaced by aspartic acid or glutamic acid, i.e. the R121 D- and R121 E-IL-4 mutein.
  • a IL-4 mutein is preferably used wherein the amino acid serine, which occurs naturally at position 125, is replaced by aspartic acid or glutamic acid, i.e. the S125D- and S125E-IL-4 mutein.
  • the IL-4 mutein of the invention is an IL-4 mutein as described in US 6,028,176 and US 6,313,272, which are herein incorporated by reference in their entirety.
  • a mutation to the region of the A-helix of the wild-type IL-4 protein sequence occurs preferably on at least one of the positions 13 and 16.
  • a mutation to the region of the C-helix of the wild-type IL-4 protein sequence occurs preferably on at least one of the positions 81 and 89.
  • a IL-4 mutein of the wild-type is used, wherein the amino acid threonine which occurs naturally at position 13 is replaced by aspartic acid, i.e. the T13D-IL-4 mutein.
  • IL-4 mutein is preferably used wherein the amino acid serine which occurs naturally at position 16 is replaced by one of the amino acids selected from the group alanine, aspartate, isoleucine, leucine, glutane, arginine, threonine, valin, thyrosine (S16A-, S16D-, S16H-, S16I-, S16L-, S16Q-, S16R-, S16T-, S16V- and S16Y-IL-4 mutein).
  • a IL-4 mutein is used, wherein the amino acid arginine which occurs naturally at position 81 is replaced by lysine, i.e. the R81 K-IL-4 mutein. Still further, a IL-4 mutein is preferably used, wherein the amino acid aspargine, which occurs naturally at position 89, is replaced by isoleucine, i.e. the N89I-IL-4 mutein.
  • IL-4 muteins are used which contain a combination of the above-disclosed mutations.
  • a IL-4 mutein is used which contains the mutation R121 D and Y124D on the D-helix and optionally a further substitution on the A- and/or C-helix.
  • the further mutations on either the A- or the C-helix are as defined above.
  • the second domain of the fusion protein comprises at least a portion of a constant immunoglobulin domain, e.g. a constant heavy immunoglobulin domain or a constant light immunoglobulin domain.
  • the second domain comprises at least a portion of a constant heavy immunoglobulin domain.
  • the constant heavy immunoglobulin domain is preferably an Fc fragment comprising the CH2 and CH3 domain and, optionally, at least a part of the hinge region.
  • the immunoglobulin domain may be an IgG, IgM, IgD or IgE immunoglobulin domain or a modified immunoglobulin domain derived therefrom.
  • the second domain comprises at least a portion of a constant IgG immunoglobulin domain.
  • the IgG immunoglobulin domain may be selected from IgGI , lgG2, lgG3 of lgG4 domains or from modified domains such as are described in US 5,925,734.
  • the immunoglobulin domain may exhibit effector functions, particularly effector functions selected from ADCC and/or CDC. In some embodiments, however, modified immunoglobulin domains having modified, e.g. at least partially deleted, effector functions may be used.
  • the constant immunoglobulin domain is preferably a mammalian protein, more preferably a human protein or a mutein of a human protein.
  • a mammalian protein more preferably a human protein or a mutein of a human protein.
  • human proteins or muteins thereof is preferred.
  • the fusion region between the first and the second domain comprises at least one cysteine residue.
  • the fusion region may comprise a region which is heterologous both to the first and the second domain.
  • the fusion region may also be free from heterologous regions and comprise a region which is a portion of the second domain, e.g. the hinge region of constant immunoglobulin domain or a portion thereof. Further, the fusion region may comprise a region of overlap between the first and second domain.
  • the first amino acid of the fusion region is a cysteine residue.
  • a cysteine residue in the fusion region preferably forms a disulphide bridge with a cysteine residue of the IL-4 polypeptide. More particularly, a cysteine residue in the fusion region may form a disulphide bridge with the cysteine residue on position 3 of a mature human IL-4 polypeptide or a mutein thereof.
  • the fusion region comprises at least a portion of an immunoglobulin hinge region, particularly at least a portion of a human immunoglobulin hinge region.
  • suitable hinge regions are hinge regions of human lgG-1 , human lgG-2, human lgG-3, human lgG-3 M15, human lgG-4, mouse lgG-1 and mouse lgG-2a.
  • the length of the fusion region is preferably up to 30 amino acids, more preferably up to 25 amino acids.
  • the fusion region may comprise one or several cysteine residues, e.g. 1 , 2 or 3 cysteine residues.
  • the fusion region comprises at least a portion of a human immunoglobulin lgG-1 hinge region, which e.g. may comprise the sequence CDKTH TCPPC PAPEL LG (SEQ ID NO: 1) or a fragment thereof comprising at least one cysteine residue, e.g. a fragment comprising the sequence CPPCP APELL G (SEQ ID NO: 2) or a fragment thereof comprising at least one cysteine residue.
  • the fusion region comprises at least a portion of a human immunoglobulin hinge region, wherein one cysteine residue has been replaced by a different amino acid residue.
  • the fusion region may comprise at least a portion of a human immunoglobulin lgG-1 hinge region, which e.g. may comprise the sequence (XI)DKTH T(X2)PP(X3) PAPEL LG (SEQ ID NO: 3), wherein (X1), (X2) and (X3) stand for an amino acid residue, wherein at least one of (X1 ), (X2) and (X3) is a cysteine residue and at least one of (X1 ), (XZ) and (X3) is an amino acid residue different from cysteine, e.g. serine, alanine or threonine, particularly serine or a fragment thereof comprising at least cysteine residue.
  • the fragment may e.g.
  • (X2)PP(X3)PAPEL LG SEQ ID NO: 4
  • one of (X2) and (X3) is cysteine and the other of (X2) and (X3) is an amino acid residue different from cysteine, e.g. as described above, or a fragment thereof comprising at least one cysteine residue.
  • (X1) may e.g. be cysteine and one of (X2) or (X3) may be cysteine and the other one an amino acid residue different from cysteine.
  • (X2) may be an amino acid residue different from cysteine, e.g. an amino acid as described above, particularly serine
  • (X3) may be cysteine.
  • (X2) may be cysteine and (X3) may be an amino acid residue different from cysteine as described above, particularly serine.
  • (X1) may be cysteine and both (X2) and (X3) may be amino acid residues different from cysteine, e.g. as described above, particularly serine.
  • immunoglobulin hinge region refers to an amino acid sequence that shares sequence identity with a portion of a naturally- occurring immunoglobulin hinge region sequence. Sequence identity of the hinge region of the present invention with naturally-occurring immunoglobulin hinge region amino sequences can range from at least 50% to about 75%- 80% and typically to at least about 90% or more. Suitable hinge regions are disclosed in US 6,165,476, the content of which is herein incorporated by reference.
  • the fusion region may be homologous or heterologous to the second domain of the fusion protein, i.e. the constant immunoglobulin domain or a portion thereof.
  • the fusion region is a region, which is at least partially homologous to the constant immunoglobulin domain.
  • Most preferred is a human lgG-1 hinge region or a portion thereof and a human lgG-1 constant immunoglobulin domain or a portion thereof.
  • the fusion region comprises a region of overlap between the first IL-4 polypeptide domain and the second constant immunoglobulin domain, e.g. the last amino acid residue(s) of the IL-4 polypeptide correspond(s) to the first amino acid residue(s) of a hinge region.
  • the length of the overlap may be at least one amino acid, e.g. 1 , 2, 3 or more amino acids.
  • Fusion proteins with at least one amino acid overlap between the first domain and the second domain in the fusion region are e.g. disclosed in PCT/EP2004/003239, the content of which is herein incorporated by reference.
  • the IL-4 polypeptide is preferably C-terminally truncated. More preferably, the C-terminal amino acid residue of the IL-4 polypeptide is the last cysteine residue occurring in the respective native IL-4 polypeptide, which is e.g. located at position 127 of the mature human IL-4 polypeptide and wherein the residues Ser 128 and Ser 129 are deleted. This cysteine residue may form an overlap with an immunoglobulin hinge region which preferably also begins with a cysteine residue.
  • a preferred fusion protein of the invention is free from a non-naturally occurring transition between the last amino acid of the IL-4 domain and the first amino acid of a hinge region of a constant immunoglobulin domain.
  • the fusion protein of the invention may also comprise an N-terminal signal sequence which allows secretion from a host cell after recombinant expression.
  • the signal sequence may be a signal sequence which is homologous to the first domain of the fusion protein.
  • the signal sequence may also be a heterologous signal sequence, e.g. the lg ⁇ or the Ig ⁇ signal peptide sequence.
  • the fusion protein is free from an N-terminal sequence, thus representing the mature form of the fusion protein.
  • the fusion protein may be a monomeric protein or a multimeric protein, e.g. a dimeric or tetrameric protein, which may be formed by multimerisation via the constant immunoglobulin domain.
  • the fusion protein is a dimeric protein which is formed by dimerisation via a constant IgG immunoglobulin domain.
  • the fusion protein may be a glycosylated or non-glycosylated protein. Further, the fusion protein may comprise covalent modifications, e.g. with acyl, lipid and/or polymeric groups. A preferred modification comprises a coupling to one or more poly(oxyalkylene) groups, e.g. polyethylene glycol (PEG) groups. PEGylated IL-4 polypeptides and methods for their production are e.g. described in US 2005/059590, the content of which is herein incorporated by reference.
  • a further aspect of the present invention relates to a nucleic acid molecule encoding a fusion protein as described above.
  • the nucleic acid molecule may be a DNA molecule, e.g. a double-stranded or single-stranded DNA molecule, or an RNA molecule.
  • the nucleic acid molecule may encode the fusion protein or a precursor thereof, e.g. a pro- or pre-proform of the fusion protein which may comprise a signal sequence or other heterologous amino acid portions for secretion or purification which are preferably located at the N- and/or C-terminus of the fusion protein.
  • the heterologous amino acid portions may be linked to the first and/or second domain via a protease cleavage site, e.g. a Factor X 3 , thrombin or IgA protease cleavage site.
  • the nucleic acid molecule may be operatively linked to an expression control sequence, e.g. an expression control sequence which allows expression of the nucleic acid molecule in a desired host cell.
  • the nucleic acid molecule may be located on a vector, e.g. a plasmid, a bacteriophage, a viral vector, a chromosal integration vector, etc. Examples of suitable expression control sequences and vectors are described for example by Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, and Ausubel et al. (1989), Current Protocols in Molecular Biology, John Wiley & Sons.
  • Suitable host cells include, but are not limited to, prokaryotic cells such as bacteria, e.g. E.coli, eukaryotic host cells such as yeast cells, insect cells, plant cells or animal cells, preferably mammalian cells and, more preferably, human cells.
  • prokaryotic cells such as bacteria, e.g. E.coli
  • eukaryotic host cells such as yeast cells, insect cells, plant cells or animal cells, preferably mammalian cells and, more preferably, human cells.
  • the invention relates to a non-human organism transformed or transfected with a nucleic acid molecule as described above.
  • Such transgenic organisms may be generated by known methods of genetic transfer including homologous recombination.
  • a further aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising as an active agent at least one fusion protein or nucleic acid molecule, vector, host cell or non-human host organism as described above.
  • the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, diluent and/or adjuvant.
  • the pharmaceutical composition may comprise a further pharmaceutical active ingredient, which may be formulated with the fusion protein as a single composition or as a kit comprising two separate compositions.
  • the first domain of the fusion protein is an IL-4 polypeptide or an agonistic mutein
  • it may be useful for treating conditions wherein the presence of IL-4 is desired e.g. autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus etc. More particularly, the autoimmune diseases are characterized by a lack of the T helper cell population Th2, the production of which is increased by IL-4 and IL-4 agonists.
  • the first domain of the fusion protein is an IL-4 antagonist, it maybe useful in treating disorders in which a suppression or inhibition of IL-4 production is desired, e.g. inflammatory response related conditions such as asthma and allergy.
  • antagonist IL-4 mutein fusion proteins are used for the treatment of cancer, particularly in combination with an antitumor agent such as a chemotherapeutic agent, an anti-tumor antibody or a pro-apoptotic agent.
  • the chemotherapeutic agents which are used in combination with the antagonistic IL-4 mutein fusion protein of the present invention preferably are antineoplastic compounds.
  • Such compounds included in the present invention comprise, but are not restricted to, (a) antimetabolites, such as cytarabine, fludarabine, ⁇ -fluoro ⁇ '-deoxyuridine, gemcitabine, hydroxyurea or methotrexate; (b) DNA-fragmenting agents, such as bleomycin; (c) DNA- crosslinking agents, such as chlorambucil, platinum compounds, e.g.
  • cisplatin carboplatin, satraplatin or oxaliplatin, cyclophosphamide or nitrogen mustard
  • intercalating agents such as adriamycin (doxorubicin) or mitoxantrone
  • protein synthesis inhibitors such as L-asparaginase, cycloheximide, puromycin or diphteria toxin
  • topoisomerase I inhibitors such as camptothecin or topotecan
  • topoisomerase Il inhibitors such as etoposide (VP- 16) or teniposide
  • microtubule-directed agents such as colcemide, colchicine, taxanes, e.g.
  • kinase inhibitors such as flavopiridol, staurosporine or derivatives thereof, e.g. STI571 (CPG 57148B) or UCN-01 (7-hydroxystaurosporine);
  • miscellaneous agents such as thioplatin, PS-341 , phenylbutyrate, ET-18- OCH3, or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof;
  • hormones such as glucocorticoids or fenretinide
  • hormone antagonists such as tamoxifen, finasteride or LHRH antagonists.
  • the chemotherapeutic agent is selected from the group consisting of platinum compounds, e.g. cisplatin, doxorubicin and taxanes, e.g. paclitaxel.
  • anti-tumor antibody used in combination with antagonistic IL-4 mutein fusion proteins are e.g. antibodies directed against anti-tumour antigens such as receptor-tyrosine kinases, e.g. EGFR or HER2.
  • the pro-apoptotic agents used in combination with antagonistic IL-4 mutein fusion proteins are preferably TRAIL or CD95 ligand or fusion proteins of TRAIL or CD95 ligand with a constant immunoglobulin domain.
  • the antagonistic IL-4 mutein fusion protein in combination with the chemotherapeutic agent, the antibody or the pro-apoptotic agent may be administered locally or systematically.
  • the agents are administered parenterally, e.g. by injection or infusion, in particular intravenously, intramuscularly, transmucosally, subcutaneously or intraperitoneally.
  • the IL-4 mutein is formulated as a pharmaceutical composition in a physiologically acceptable carrier, optionally together with physiologically acceptable excipients.
  • the daily dose may vary depending on the mode of administration and/or the severity of the disease and is preferably in the range of 0.01 mg/kg to 100 mg/kg body weight.
  • the combination therapy is carried out for a time period sufficient to obtain the desired beneficial effect, e.g. induction of a tumour response to treatment.
  • administration of the fusion protein may be terminated after a predetermined time period and chemo- and/or radiation therapy may be continued alone.
  • the administration of (i) at least one antagonistic IL-4 mutein fusion protein and (ii) at least one chemotherapeutic agent, antibody or pro-apoptotic agent may be simultaneous, separate or sequential, respectively.
  • the administration of agent (i) and agent (ii) is started simultaneously.
  • the combination therapy can be started stepwise.
  • the start of administration of the antagonistic IL-4 mutein agent (i) is ⁇ 1 week before the administration of the chemotherapeutic agent, antibody or pro-apoptotic agent (ii).
  • the administration of the chemotherapeutic agent, antibody or pro-apoptotic agent (ii) may in turn start ⁇ 1 week before the administration of the IL-4 mutein agent (i).
  • the appropriate administration scheme of agent (i) and (ii) will be set up by a person skilled in the art, i.e. by a physician.
  • the use of a combined therapy of the above agents (i) and (ii) which can further be in combination surgery and/or radiation therapy is also considered within the scope of this invention.
  • the medicament combination is for simultaneous, separate or sequential combination therapy with surgery and/or radiation therapy.
  • the combination therapy is particularly useful for the treatment of cancer types classified as cytokine-expressing tumours and in particular cancer associated with increased IL-4 expression.
  • Said cancer types may be at least partially resistant to apoptosis due to the expression of anti-apoptotic proteins.
  • a method for the identification and diagnosis of cancer types and cells which express anti-apoptotic cytokines and which can be classified as cytokine-expressing tumours is disclosed in the European Patent Application EP 06 012 754.
  • the teaching of said Application EP 06 012 754 is herein incorporated by reference in its entirety.
  • cancer types comprise neuroblastoma, intestine carcinoma such as rectum carcinoma, colon carcinoma, familiary adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer, esophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma, renal carcinoma, kidney parenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma, testis carcinoma, breast carcinoma, bladder carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkin lymphoma, and peripheral
  • the combination therapy according to the present invention can be used for the prevention and/or treatment of non-lymphoid and non-myeloid cancers, most preferably solid cancers, even more preferably epithelial cancers.
  • epithelial cancer types include all forms of thyroid carcinomas (medullary thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma), breast carcinoma, lung carcinoma, prostate carcinoma, bladder carcinoma, gastric carcinoma, pancreas carcinoma, kidney carcinoma, liver carcinoma and colon carcinoma.
  • thyroid carcinomas medullary thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma
  • breast carcinoma lung carcinoma, prostate carcinoma, bladder carcinoma, gastric carcinoma, pancreas carcinoma, kidney carcinoma, liver carcinoma and colon carcinoma.
  • the combination therapy according to the present invention is particularly useful for the prevention and/or treatment of minimal residual cancer disease (MRD).
  • MRD minimal residual cancer disease
  • residual cancer cells often remain in the patient's body. These cancer cells can give rise to secondary cancers after the primary cancer has been removed. Therefore, one major task of successful cancer therapy must be the eradication of such residual cancer cells and in particular the eradication of cancer stem cells, e.g. colon cancer stem cells.
  • the combination therapy of the present invention is therefore particularly suitable to inhibit, reduce and/or eliminate residual cancer cells, in particular residual cancer stem cells, after an apparently complete regression or surgical excision of the primary tumour.
  • the hinge region of human lgG-1 starts at amino acid 127 and extends to amino acid 143. Thus, there is an overlap of one amino acid between the C- terminus of the IL-4 domain and the hinge region, i.e. the cysteine residue at position 127.
  • C-terminally of the lgG-1 hinge region a human lgG-1 immunoglobulin constant region extends from amino acid residue 144 to amino acid residue 354. Disulphide bridges between cysteine residues are indicated. Disulphide bridges between cysteine residues C 2 (position 24) and C 4 (position 65), or C 3 (position 46) and C 5 (position 99) may be formed within the IL-4 domain.
  • the disulphide bridge between cysteine residues Ci (position 3) and C 6 (position 127) may be formed of one cysteine residue within the IL-4 polypeptide and one cysteine residue which forms the overlap between the IL-4 domain and the fusion region, i.e. the hinge region.
  • FIG. 2a A further embodiment of the invention is shown in Figures 2a (IL4DM- C2Fc/SEQ ID NO: 7) and 2b (IL4DMI29-C2Fc/SEQ ID NO: 8).
  • a part of the hinge region namely the amino acid sequence CDKTHT has been deleted.
  • the truncated hinge region begins with a cysteine.
  • Disulphide bridges may be formed between cysteine residues Ci and C 6 , C 2 and C 4 and C 3 and C 5 , respectively.
  • the amino acid residue at position 29 is valine.
  • the amino acid residue at position 29 is isoleucine.
  • FIG. 3 IL-4DM- C1SerFc/SEQ ID NO: 9
  • This fusion protein corresponds to the fusion protein as shown in Fig. 1a (SEQ ID NO: 3), except that the cysteine residue at position 133 has been replaced by serine and point mutations in the Fc portion have been replaced.
  • IL4DM-Fc Fusion proteins For the expression purpose, synthetic genes encoding the respective proteins were designed and cloned into the pCDNA4-HisMax vector (INVITROGEN), using the unique Hind-lll/Not-l sites of the plasmid. All IL4DM-Fc variants were expressed with an N-teminal secretory signal peptide (METDTLLLWVLLLWVPAGNG) (SEQ ID NO: 10) and a C-terminal Streptag-ll affinity- tag (SAWSHPQFEK) (SEQ ID NO: 11 ).
  • Hek 293T cells grown in DMEM + GlutaMAX (GibCo) supplemented with 10% FBS, 100 units/ml Penicillin and 100 ⁇ g/ml Streptomycin were transiently transfected with the pCDNA4-based expression plasmids encoding the IL4DM-C1-Fc, IL4DM-C2-Fc and IL4DM-C1Ser-Fc proteins with their expression cassettes under control of the CMV-promotor.
  • Cell culture supernatants containing secreted recombinant IL4DM-C1-Fc, IL4DM- C2-Fc and IL4DM-C1Ser-Fc proteins were harvested three days post transfection and clarified by centrifugation at 300 g followed by filtration through a 0.22 ⁇ m sterile filter.
  • affinity purification 1 ml Streptactin Sepharose (IBA GmbH, G ⁇ ttingen, Germany) was packed to a column and equilibrated with 15 ml buffer W (100 mM Tris-HCI, 150 mM NaCI pH 8.0). The cell culture supernatant was applied to the column with a flow rate of 4 ml/min.
  • FIG 4 an SDS-PAGE analysis of affinity purified IL4DM-C1-Fc, IL4DM- C2-Fc and IL4DM-C1Ser-Fc from transfected HEK293T cells is shown. From each protein, 100 ng were diluted into SDS-PAGE sample buffer and separated on Nu-PAGE 4-12%. Separated proteins were subsequently visualized by silver stain. To analyse the apparent MW of the purified protein, standard proteins of known MW were used.
  • the human erythroleukemic cell line TF1 (ATCC: CRL-2003) shows factor dependent proliferation. Exposition of TF1 cells to IL4 or GM-CSF results in a proliferative stimulus that can be analysed by the non radioactive proliferation Assays "CellTiter 96 AQ” (Promega, Madison, USA).
  • TF1 proliferation assay to analyse if the recombinant ⁇ expressed and purified proteins IL4DM-C1-Fc, IL4DM-C2-Fc and IL4DM-C1Ser-Fc are able to compete with wt-IL4 (wildtype lnterleukin-4, human) for binding to the respective IL4-receptor.
  • TF1 cells were plated to 96 wells plates in the presence of 40ng human wt-IL4 and various amounts of the respective IL4DM-FC constructs were added. After 72 hours of cultivation the proliferation rate of the TF1 cells was measured according to the instructions of the manufacturer by "CellTiter 96 AQ".

Abstract

The invention relates to fusion proteins comprising at least one IL-4 polypeptide domain and a constant immunoglobulin domain.

Description

IL-4 Fc fusion proteins
Description
The invention relates to fusion proteins comprising at least one IL-4 polypeptide domain and a constant immunoglobulin domain.
WO 2004/069274 refers to the use of cytokine antagonists which modulate the expression and/or the function of a cytokine in a cell for the downregulation of anti-apoptotic proteins in a cell. In particular, it is referred to the use of cytokine antagonists for the treatment of cancer. Muteins of the cytokines themselves are given as examples of cytokine antagonists, which are able to bind to the respective cell surface receptor, inhibiting the signal cascade triggered by the cytokine itself.
US Patents US 6,313,272 and US 6,028,176 describe recombinant agonistic or antagonistic human IL-4 muteins comprising at least one amino acid substitution in the binding surface of either the region of the A- or C-α-helix of the wild-type IL-4. The IL-4 muteins are indicated as being suitable for the treatment of condition exacerbated by IL-4 production such as asthma, allergy or inflammatory response-related conditions. It is speculated that the IL-4 muteins might be suitable for the treatment of cancers or tumours.
US Patent Application US 2005/0059590 describes modified IL-4 mutein receptor antagonists comprising an IL-4 mutein receptor antagonist, and in particular the IL-4 muteins as disclosed in the above-mentioned US Patents US 6,313,272 and US 6,028,176, coupled to polyethylene glycol. Said modified muteins are in particular indicated as useful in the treatment of severe asthma, chronic obstructive pulmonary disease and related lung conditions. US Patent US 5,723,118 describes mutant IL-4 proteins which compete with the wild-type IL-4 for occupation of the IL-4 receptor and act as antagonists or partial agonists of the human interleukin-4. In particular, mutant IL-4 proteins are disclosed wherein one or more of the amino acids occurring at position 121 , 124 or 125 have been replaced. The mutant IL-4 proteins are indicated as being suitable for the treatment and/or prevention of allergic conditions.
US Patent US 6,130,318 describes novel IL-4 antagonist or partial agonist mutant proteins and their use as medicaments, in particular in association with overshooting, falsely regulated immuno reactions and autoimmune diseases. Further, it is speculated that the IL-4 mutant proteins can be employed in the palliative therapy of tumour diseases.
EP 06 014 080.3 and EP 06 026 609.5 disclose that antagonistic IL-4 muteins are suitable in combination with at least one further chemotherapeutic or pro-apoptotic agent for the treatment of cancer diseases. Preferably, the antagonistic IL-4 muteins are used for curative cancer therapy.
According to the present invention it is contemplated that fusion proteins comprising at least one IL-4 polypeptide fused to a constant immunoglobulin domain have favourable pharmaceutical and physiological properties.
Thus, the present invention relates to a fusion protein comprising (i) at least one first domain comprising an IL-4 polypeptide fused to (ii) at least one heterologous second domain comprising at least a portion of a constant immunoglobulin domain, and (iii) a fusion region between the first and second domain, wherein the fusion region comprises at least one cysteine residue.
In the fusion protein of the invention the first domain is located N-terminally from the second domain. Additionally, the fusion protein may comprise a signal sequence, e.g. an N-terminal signal peptide, which allows a secretory expression in a host cell, particularly in a eukaryotic host cell. The signal peptide may be a homologous IL-4 signal peptide or a heterologous signal peptide.
Preferably, the IL-4 domain comprises at least one cysteine residue which is capable of forming a disulphide bridge with the at least one cysteine residue in the fusion region. By means of this disulphide bridge the protein structure may be stabilized.
Free cysteine residues in the IL-4 polypeptide domain are preferably located at the N-terminus and/or at the C-terminus thereof, more preferably at the N- terminus, e.g. at amino acid position 1-20 of the mature IL-4 polypeptide. More preferably, the free cysteine residue is a naturally occurring cysteine residue, e.g. a cysteine residue at amino acid position 3 of the mature human IL-4 polypeptide.
The IL-4 polypeptide is preferably a mammalian IL-4 polypeptide or a mutein thereof, e.g. an agonistic or antagonistic IL-4 mutein. The term "IL-4 mutein" refers to any polypeptide derived from IL-4 polypeptides which has biological activities of IL-4, e.g. increasing the activity of endogenous IL-4 (agonistic muteins) or which inhibits biological activity of IL-4, e.g. inhibiting the activity of endogenous IL-4 (antagonistic mutein). Preferably, the mutein differs from the native IL-4 polypeptide by substitution, deletion and/or insertion of at least one amino acid. Further, it is preferred that the IL-4 mutein has an identity of at least 85%, 90%, 95%, 97% or 98.5% up to 99.5% with a native IL-4 polypeptide over the entire amino acid sequence.
In a preferred embodiment of the invention, the first domain of the fusion protein comprises human IL-4, an agonistic human IL-4 mutein or an antagonistic human IL-4 mutein, e.g. IL-4 muteins as disclosed in the documents as cited above, which are herein incorporated by reference. The term "human IL-4" refers to a human IL-4 as disclosed in US 5,017,691 , which is herein incorporated by reference, or an allelic variant thereof, e.g. an allelic variant comprising an amino acid substitution at position 29, particularly a substitution of isoleucine by valine.
Preferably, the IL-4 polypeptide is a C-terminally truncated IL-4 polypeptide. More preferably, the last amino acid residue is a cysteine residue (e.g. Cys127 of native human IL-4). In addition to the C-terminal truncation, the IL- 4 polypeptide may comprise the wild-type sequence or further mutations.
Further, the present invention refers to a combination of (i) at least one fusion protein as described above and (ii) at least one further medicament.
The amino acid sequence of the preferred antagonistic IL-4 muteins differs from the amino acid sequence of the wild-type IL-4 by mutation of one or more amino acids at certain positions of the native protein.
The term "mutation" as used in the context of the present invention can be understood as substitution, deletion and/or addition of single or multiple amino acids in the target sequence. Preferably, the mutation of the target sequence, in particular of the native IL-4 sequence, is a substitution at one or more positions of the native IL-4 polypeptide chain.
The substitution can occur with different genetically encoded amino acids or by non-genetically encoded amino acids. Examples for non-genetically encoded amino acids are homocysteine, hydroxyproline, ornithine, hydroxylysine, citrulline, carnitine, etc.
Moreover, in the context of this invention, a substitution within the native polypeptide sequence can be a conservative or a non-conservative substitution. The common classification of the amino acid residues on the base of the side-chain characteristics, which determine the amino acid groups for a conservative or a non-conservative substitution, is well known by the person skilled in the art.
Preferably, mutations of the native IL-4 amino acid sequence are being contemplated which result in IL-4 muteins having an antagonistic action with respect to the action of the wild-type IL-4. The term "antagonistic action" as used herein means that the IL-4 muteins of the invention are capable of modulating the function of the cytokine, in particular are capable of inhibiting the function of endogenous IL-4 cytokine. IL-4 produced in cancer cells promotes the up-regulation of anti-apoptotic proteins which may lead to resistance to cell death and to therapy refractoriness. Hence, an antagonistic action of the IL-4 muteins of the invention leads to the inhibition of the internal signal cascade triggered by the endogenous IL-4 which leads to the up-regulation of anti-apoptotic proteins.
Moreover, the IL-4 muteins of the invention may further show a higher affinity for the wild-type IL-4 receptor in comparison to wild-type IL-4. In particular, the muteins may compete with the endogenously expressed IL-4 for the binding site on the respective receptor.
Preferably, the present invention comprises the use of IL-4 muteins, wherein mutations of the amino acid sequence of the wild-type IL-4 sequence have been made to the region of the A-, C- and/or D-helices and more preferably to those amino acids comprising the binding surfaces of said helices of the IL-4 protein.
According to one preferred embodiment, the IL-4 mutein of the invention is preferably an IL-4 mutein as described in US 5,723,118 and US 6,130,318, which are herein incorporated by reference in their entirety.
Thus, a mutation to the region of the D-helix of the wild-type IL-4 protein sequence occurs preferably on at least one of the positions 120, 121 , 122, 123, 124, 125, 126, 127 and/or 128 of the wild-type amino acid sequence. Even more preferably, the mutation occurs on at least one of the positions 121 , 124 and/or 125. Most preferably, the mutation occurs at position 121 and/or 124.
In a very preferred embodiment of the present invention, a IL-4 mutein of the wild-type is used, wherein the amino acid tyrosine, which occurs naturally at position 124, is replaced by aspartic acid, glycine or glutamic acid, i.e. the
Y124D-, the Y124G- and the Y124E-IL-4 mutein. Further, a IL-4 mutein is preferably used wherein the amino acid arginine, which occurs naturally at position 121, is replaced by aspartic acid or glutamic acid, i.e. the R121 D- and R121 E-IL-4 mutein. Further, a IL-4 mutein is preferably used wherein the amino acid serine, which occurs naturally at position 125, is replaced by aspartic acid or glutamic acid, i.e. the S125D- and S125E-IL-4 mutein.
According to a further preferred embodiment, the IL-4 mutein of the invention is an IL-4 mutein as described in US 6,028,176 and US 6,313,272, which are herein incorporated by reference in their entirety.
Thus, a mutation to the region of the A-helix of the wild-type IL-4 protein sequence occurs preferably on at least one of the positions 13 and 16. A mutation to the region of the C-helix of the wild-type IL-4 protein sequence occurs preferably on at least one of the positions 81 and 89.
In a very preferred embodiment of the present invention, a IL-4 mutein of the wild-type is used, wherein the amino acid threonine which occurs naturally at position 13 is replaced by aspartic acid, i.e. the T13D-IL-4 mutein. Further, a IL-4 mutein is preferably used wherein the amino acid serine which occurs naturally at position 16 is replaced by one of the amino acids selected from the group alanine, aspartate, isoleucine, leucine, glutane, arginine, threonine, valin, thyrosine (S16A-, S16D-, S16H-, S16I-, S16L-, S16Q-, S16R-, S16T-, S16V- and S16Y-IL-4 mutein).
In a still further preferred embodiment, a IL-4 mutein is used, wherein the amino acid arginine which occurs naturally at position 81 is replaced by lysine, i.e. the R81 K-IL-4 mutein. Still further, a IL-4 mutein is preferably used, wherein the amino acid aspargine, which occurs naturally at position 89, is replaced by isoleucine, i.e. the N89I-IL-4 mutein.
Moreover, according to the present invention, IL-4 muteins are used which contain a combination of the above-disclosed mutations. According to a preferred embodiment of the invention, a IL-4 mutein is used which contains the mutation R121 D and Y124D on the D-helix and optionally a further substitution on the A- and/or C-helix. Preferably, the further mutations on either the A- or the C-helix are as defined above.
The second domain of the fusion protein comprises at least a portion of a constant immunoglobulin domain, e.g. a constant heavy immunoglobulin domain or a constant light immunoglobulin domain. Preferably, the second domain comprises at least a portion of a constant heavy immunoglobulin domain. The constant heavy immunoglobulin domain is preferably an Fc fragment comprising the CH2 and CH3 domain and, optionally, at least a part of the hinge region. The immunoglobulin domain may be an IgG, IgM, IgD or IgE immunoglobulin domain or a modified immunoglobulin domain derived therefrom.
Preferably, the second domain comprises at least a portion of a constant IgG immunoglobulin domain. The IgG immunoglobulin domain may be selected from IgGI , lgG2, lgG3 of lgG4 domains or from modified domains such as are described in US 5,925,734. The immunoglobulin domain may exhibit effector functions, particularly effector functions selected from ADCC and/or CDC. In some embodiments, however, modified immunoglobulin domains having modified, e.g. at least partially deleted, effector functions may be used.
The constant immunoglobulin domain is preferably a mammalian protein, more preferably a human protein or a mutein of a human protein. For therapeutic purposes, in particular the use of human proteins or muteins thereof is preferred.
The fusion region between the first and the second domain comprises at least one cysteine residue. The fusion region may comprise a region which is heterologous both to the first and the second domain. The fusion region may also be free from heterologous regions and comprise a region which is a portion of the second domain, e.g. the hinge region of constant immunoglobulin domain or a portion thereof. Further, the fusion region may comprise a region of overlap between the first and second domain.
In an especially preferred embodiment, the first amino acid of the fusion region is a cysteine residue. A cysteine residue in the fusion region preferably forms a disulphide bridge with a cysteine residue of the IL-4 polypeptide. More particularly, a cysteine residue in the fusion region may form a disulphide bridge with the cysteine residue on position 3 of a mature human IL-4 polypeptide or a mutein thereof.
Preferably, the fusion region comprises at least a portion of an immunoglobulin hinge region, particularly at least a portion of a human immunoglobulin hinge region. Examples of suitable hinge regions are hinge regions of human lgG-1 , human lgG-2, human lgG-3, human lgG-3 M15, human lgG-4, mouse lgG-1 and mouse lgG-2a.
The length of the fusion region is preferably up to 30 amino acids, more preferably up to 25 amino acids. The fusion region may comprise one or several cysteine residues, e.g. 1 , 2 or 3 cysteine residues.
More preferably, the fusion region comprises at least a portion of a human immunoglobulin lgG-1 hinge region, which e.g. may comprise the sequence CDKTH TCPPC PAPEL LG (SEQ ID NO: 1) or a fragment thereof comprising at least one cysteine residue, e.g. a fragment comprising the sequence CPPCP APELL G (SEQ ID NO: 2) or a fragment thereof comprising at least one cysteine residue. In an especially preferred embodiment, the fusion region comprises at least a portion of a human immunoglobulin hinge region, wherein one cysteine residue has been replaced by a different amino acid residue. For example, the fusion region may comprise at least a portion of a human immunoglobulin lgG-1 hinge region, which e.g. may comprise the sequence (XI)DKTH T(X2)PP(X3) PAPEL LG (SEQ ID NO: 3), wherein (X1), (X2) and (X3) stand for an amino acid residue, wherein at least one of (X1 ), (X2) and (X3) is a cysteine residue and at least one of (X1 ), (XZ) and (X3) is an amino acid residue different from cysteine, e.g. serine, alanine or threonine, particularly serine or a fragment thereof comprising at least cysteine residue. The fragment may e.g. comprise the sequence (X2)PP(X3)PAPEL LG (SEQ ID NO: 4), wherein one of (X2) and (X3) is cysteine and the other of (X2) and (X3) is an amino acid residue different from cysteine, e.g. as described above, or a fragment thereof comprising at least one cysteine residue.
In this embodiment, (X1) may e.g. be cysteine and one of (X2) or (X3) may be cysteine and the other one an amino acid residue different from cysteine. For example, (X2) may be an amino acid residue different from cysteine, e.g. an amino acid as described above, particularly serine, and (X3) may be cysteine. Alternatively, (X2) may be cysteine and (X3) may be an amino acid residue different from cysteine as described above, particularly serine. In a still further embodiment, (X1) may be cysteine and both (X2) and (X3) may be amino acid residues different from cysteine, e.g. as described above, particularly serine.
As used herein, the term "immunoglobulin hinge region" refers to an amino acid sequence that shares sequence identity with a portion of a naturally- occurring immunoglobulin hinge region sequence. Sequence identity of the hinge region of the present invention with naturally-occurring immunoglobulin hinge region amino sequences can range from at least 50% to about 75%- 80% and typically to at least about 90% or more. Suitable hinge regions are disclosed in US 6,165,476, the content of which is herein incorporated by reference.
The fusion region may be homologous or heterologous to the second domain of the fusion protein, i.e. the constant immunoglobulin domain or a portion thereof. Preferably, the fusion region is a region, which is at least partially homologous to the constant immunoglobulin domain. Most preferred is a human lgG-1 hinge region or a portion thereof and a human lgG-1 constant immunoglobulin domain or a portion thereof.
Further, it is preferred that the fusion region comprises a region of overlap between the first IL-4 polypeptide domain and the second constant immunoglobulin domain, e.g. the last amino acid residue(s) of the IL-4 polypeptide correspond(s) to the first amino acid residue(s) of a hinge region. The length of the overlap may be at least one amino acid, e.g. 1 , 2, 3 or more amino acids. Fusion proteins with at least one amino acid overlap between the first domain and the second domain in the fusion region are e.g. disclosed in PCT/EP2004/003239, the content of which is herein incorporated by reference.
In a preferred embodiment, the IL-4 polypeptide is preferably C-terminally truncated. More preferably, the C-terminal amino acid residue of the IL-4 polypeptide is the last cysteine residue occurring in the respective native IL-4 polypeptide, which is e.g. located at position 127 of the mature human IL-4 polypeptide and wherein the residues Ser 128 and Ser 129 are deleted. This cysteine residue may form an overlap with an immunoglobulin hinge region which preferably also begins with a cysteine residue. Thus, a preferred fusion protein of the invention is free from a non-naturally occurring transition between the last amino acid of the IL-4 domain and the first amino acid of a hinge region of a constant immunoglobulin domain.
The fusion protein of the invention may also comprise an N-terminal signal sequence which allows secretion from a host cell after recombinant expression. The signal sequence may be a signal sequence which is homologous to the first domain of the fusion protein. Alternatively, the signal sequence may also be a heterologous signal sequence, e.g. the lgκ or the Igλ signal peptide sequence. In a different embodiment, the fusion protein is free from an N-terminal sequence, thus representing the mature form of the fusion protein.
The fusion protein may be a monomeric protein or a multimeric protein, e.g. a dimeric or tetrameric protein, which may be formed by multimerisation via the constant immunoglobulin domain. Preferably, the fusion protein is a dimeric protein which is formed by dimerisation via a constant IgG immunoglobulin domain.
The fusion protein may be a glycosylated or non-glycosylated protein. Further, the fusion protein may comprise covalent modifications, e.g. with acyl, lipid and/or polymeric groups. A preferred modification comprises a coupling to one or more poly(oxyalkylene) groups, e.g. polyethylene glycol (PEG) groups. PEGylated IL-4 polypeptides and methods for their production are e.g. described in US 2005/059590, the content of which is herein incorporated by reference.
A further aspect of the present invention relates to a nucleic acid molecule encoding a fusion protein as described above. The nucleic acid molecule may be a DNA molecule, e.g. a double-stranded or single-stranded DNA molecule, or an RNA molecule. The nucleic acid molecule may encode the fusion protein or a precursor thereof, e.g. a pro- or pre-proform of the fusion protein which may comprise a signal sequence or other heterologous amino acid portions for secretion or purification which are preferably located at the N- and/or C-terminus of the fusion protein. The heterologous amino acid portions may be linked to the first and/or second domain via a protease cleavage site, e.g. a Factor X3, thrombin or IgA protease cleavage site.
The nucleic acid molecule may be operatively linked to an expression control sequence, e.g. an expression control sequence which allows expression of the nucleic acid molecule in a desired host cell. The nucleic acid molecule may be located on a vector, e.g. a plasmid, a bacteriophage, a viral vector, a chromosal integration vector, etc. Examples of suitable expression control sequences and vectors are described for example by Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, and Ausubel et al. (1989), Current Protocols in Molecular Biology, John Wiley & Sons.
Various expression vector/host cell systems may be used to express the nucleic acid sequences encoding the fusion proteins of the present invention. Suitable host cells include, but are not limited to, prokaryotic cells such as bacteria, e.g. E.coli, eukaryotic host cells such as yeast cells, insect cells, plant cells or animal cells, preferably mammalian cells and, more preferably, human cells.
Further, the invention relates to a non-human organism transformed or transfected with a nucleic acid molecule as described above. Such transgenic organisms may be generated by known methods of genetic transfer including homologous recombination.
A further aspect of the present invention relates to a pharmaceutical composition comprising as an active agent at least one fusion protein or nucleic acid molecule, vector, host cell or non-human host organism as described above. Additionally, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, diluent and/or adjuvant.
In a particularly preferred embodiment, the pharmaceutical composition may comprise a further pharmaceutical active ingredient, which may be formulated with the fusion protein as a single composition or as a kit comprising two separate compositions.
If the first domain of the fusion protein is an IL-4 polypeptide or an agonistic mutein, it may be useful for treating conditions wherein the presence of IL-4 is desired, e.g. autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus etc. More particularly, the autoimmune diseases are characterized by a lack of the T helper cell population Th2, the production of which is increased by IL-4 and IL-4 agonists.
If the first domain of the fusion protein is an IL-4 antagonist, it maybe useful in treating disorders in which a suppression or inhibition of IL-4 production is desired, e.g. inflammatory response related conditions such as asthma and allergy. Most preferably, however, antagonist IL-4 mutein fusion proteins are used for the treatment of cancer, particularly in combination with an antitumor agent such as a chemotherapeutic agent, an anti-tumor antibody or a pro-apoptotic agent.
The chemotherapeutic agents which are used in combination with the antagonistic IL-4 mutein fusion protein of the present invention preferably are antineoplastic compounds. Such compounds included in the present invention comprise, but are not restricted to, (a) antimetabolites, such as cytarabine, fludarabine, δ-fluoro^'-deoxyuridine, gemcitabine, hydroxyurea or methotrexate; (b) DNA-fragmenting agents, such as bleomycin; (c) DNA- crosslinking agents, such as chlorambucil, platinum compounds, e.g. cisplatin, carboplatin, satraplatin or oxaliplatin, cyclophosphamide or nitrogen mustard; (d) intercalating agents such as adriamycin (doxorubicin) or mitoxantrone; (e) protein synthesis inhibitors, such as L-asparaginase, cycloheximide, puromycin or diphteria toxin; (f) topoisomerase I inhibitors, such as camptothecin or topotecan; (g) topoisomerase Il inhibitors, such as etoposide (VP- 16) or teniposide; (h) microtubule-directed agents, such as colcemide, colchicine, taxanes, e.g. paclitaxel, vinblastine or vincristine; (i) kinase inhibitors such as flavopiridol, staurosporine or derivatives thereof, e.g. STI571 (CPG 57148B) or UCN-01 (7-hydroxystaurosporine); G) miscellaneous agents such as thioplatin, PS-341 , phenylbutyrate, ET-18- OCH3, or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; (k) hormones such as glucocorticoids or fenretinide; (I) hormone antagonists, such as tamoxifen, finasteride or LHRH antagonists.
In an especially preferred embodiment of the present invention, the chemotherapeutic agent is selected from the group consisting of platinum compounds, e.g. cisplatin, doxorubicin and taxanes, e.g. paclitaxel.
The anti-tumor antibody used in combination with antagonistic IL-4 mutein fusion proteins are e.g. antibodies directed against anti-tumour antigens such as receptor-tyrosine kinases, e.g. EGFR or HER2.
The pro-apoptotic agents used in combination with antagonistic IL-4 mutein fusion proteins are preferably TRAIL or CD95 ligand or fusion proteins of TRAIL or CD95 ligand with a constant immunoglobulin domain.
The antagonistic IL-4 mutein fusion protein in combination with the chemotherapeutic agent, the antibody or the pro-apoptotic agent may be administered locally or systematically. Preferably, the agents are administered parenterally, e.g. by injection or infusion, in particular intravenously, intramuscularly, transmucosally, subcutaneously or intraperitoneally. For this purpose, the IL-4 mutein is formulated as a pharmaceutical composition in a physiologically acceptable carrier, optionally together with physiologically acceptable excipients. The daily dose may vary depending on the mode of administration and/or the severity of the disease and is preferably in the range of 0.01 mg/kg to 100 mg/kg body weight. The combination therapy is carried out for a time period sufficient to obtain the desired beneficial effect, e.g. induction of a tumour response to treatment. Alternatively, administration of the fusion protein may be terminated after a predetermined time period and chemo- and/or radiation therapy may be continued alone.
According to a preferred embodiment of the present invention, the administration of (i) at least one antagonistic IL-4 mutein fusion protein and (ii) at least one chemotherapeutic agent, antibody or pro-apoptotic agent may be simultaneous, separate or sequential, respectively. For example, the administration of agent (i) and agent (ii) is started simultaneously. Alternatively, the combination therapy can be started stepwise. According to one preferred embodiment of the present invention, the start of administration of the antagonistic IL-4 mutein agent (i) is ≤ 1 week before the administration of the chemotherapeutic agent, antibody or pro-apoptotic agent (ii). The administration of the chemotherapeutic agent, antibody or pro-apoptotic agent (ii) may in turn start ≥ 1 week before the administration of the IL-4 mutein agent (i). The appropriate administration scheme of agent (i) and (ii) will be set up by a person skilled in the art, i.e. by a physician.
Moreover, the use of a combined therapy of the above agents (i) and (ii) which can further be in combination surgery and/or radiation therapy is also considered within the scope of this invention. In particular, the medicament combination is for simultaneous, separate or sequential combination therapy with surgery and/or radiation therapy.
The combination therapy is particularly useful for the treatment of cancer types classified as cytokine-expressing tumours and in particular cancer associated with increased IL-4 expression. Said cancer types may be at least partially resistant to apoptosis due to the expression of anti-apoptotic proteins. A method for the identification and diagnosis of cancer types and cells which express anti-apoptotic cytokines and which can be classified as cytokine-expressing tumours is disclosed in the European Patent Application EP 06 012 754. The teaching of said Application EP 06 012 754 is herein incorporated by reference in its entirety.
Examples of such cancer types comprise neuroblastoma, intestine carcinoma such as rectum carcinoma, colon carcinoma, familiary adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer, esophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma, renal carcinoma, kidney parenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma, testis carcinoma, breast carcinoma, bladder carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acute myeolid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, hepatocellular carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lung carcinoma, non- small cell lung carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmocytoma.
In a particularly preferred embodiment, the combination therapy according to the present invention can be used for the prevention and/or treatment of non-lymphoid and non-myeloid cancers, most preferably solid cancers, even more preferably epithelial cancers.
Especially preferred examples of epithelial cancer types include all forms of thyroid carcinomas (medullary thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma), breast carcinoma, lung carcinoma, prostate carcinoma, bladder carcinoma, gastric carcinoma, pancreas carcinoma, kidney carcinoma, liver carcinoma and colon carcinoma.
In a further particularly preferred embodiment, the combination therapy according to the present invention is particularly useful for the prevention and/or treatment of minimal residual cancer disease (MRD). In fact, after cancer therapy, residual cancer cells often remain in the patient's body. These cancer cells can give rise to secondary cancers after the primary cancer has been removed. Therefore, one major task of successful cancer therapy must be the eradication of such residual cancer cells and in particular the eradication of cancer stem cells, e.g. colon cancer stem cells. In this context, the combination therapy of the present invention is therefore particularly suitable to inhibit, reduce and/or eliminate residual cancer cells, in particular residual cancer stem cells, after an apparently complete regression or surgical excision of the primary tumour.
In Figure 1a (IL4DM-C1 Fc/SEQ ID NO: 5) and 1b (IL4DMI29-C1 Fc/SEQ ID NO: 6), the amino acid sequences of preferred fusion proteins of the invention are shown. Amino acids 1-127 correspond to a human IL-4 mutein R121D/Y124D, which has been C-terminally truncated (deletions of Ser 128 and Ser 129). In Figure 1a, the amino acid residue at position 29 is valine. In Figure 1b, the amino acid residue at position 29 is isoleucine. The amino acid residue Cys127 (C6) is the last amino acid of the IL-4 polypeptide. The hinge region of human lgG-1 starts at amino acid 127 and extends to amino acid 143. Thus, there is an overlap of one amino acid between the C- terminus of the IL-4 domain and the hinge region, i.e. the cysteine residue at position 127. C-terminally of the lgG-1 hinge region a human lgG-1 immunoglobulin constant region extends from amino acid residue 144 to amino acid residue 354. Disulphide bridges between cysteine residues are indicated. Disulphide bridges between cysteine residues C2 (position 24) and C4 (position 65), or C3 (position 46) and C5 (position 99) may be formed within the IL-4 domain. The disulphide bridge between cysteine residues Ci (position 3) and C6 (position 127) may be formed of one cysteine residue within the IL-4 polypeptide and one cysteine residue which forms the overlap between the IL-4 domain and the fusion region, i.e. the hinge region.
A further embodiment of the invention is shown in Figures 2a (IL4DM- C2Fc/SEQ ID NO: 7) and 2b (IL4DMI29-C2Fc/SEQ ID NO: 8). In this fusion protein, a part of the hinge region, namely the amino acid sequence CDKTHT has been deleted. The truncated hinge region (starting at amino acid position 127) begins with a cysteine. Thus, there is still one amino acid overlap between the human IL-4 domain and the hinge region. Disulphide bridges may be formed between cysteine residues Ci and C6, C2 and C4 and C3 and C5, respectively. In Figure 2a, the amino acid residue at position 29 is valine. In Figure 2b, the amino acid residue at position 29 is isoleucine.
Still a further embodiment of the invention is shown in Figure 3 (IL-4DM- C1SerFc/SEQ ID NO: 9). This fusion protein corresponds to the fusion protein as shown in Fig. 1a (SEQ ID NO: 3), except that the cysteine residue at position 133 has been replaced by serine and point mutations in the Fc portion have been replaced.
Further, the present invention is explained in more detail in the following examples.
Example 1
Cloning, expression and purification of IL4DM-Fc Fusion proteins For the expression purpose, synthetic genes encoding the respective proteins were designed and cloned into the pCDNA4-HisMax vector (INVITROGEN), using the unique Hind-lll/Not-l sites of the plasmid. All IL4DM-Fc variants were expressed with an N-teminal secretory signal peptide (METDTLLLWVLLLWVPAGNG) (SEQ ID NO: 10) and a C-terminal Streptag-ll affinity- tag (SAWSHPQFEK) (SEQ ID NO: 11 ).
Hek 293T cells grown in DMEM + GlutaMAX (GibCo) supplemented with 10% FBS, 100 units/ml Penicillin and 100 μg/ml Streptomycin were transiently transfected with the pCDNA4-based expression plasmids encoding the IL4DM-C1-Fc, IL4DM-C2-Fc and IL4DM-C1Ser-Fc proteins with their expression cassettes under control of the CMV-promotor. Cell culture supernatants containing secreted recombinant IL4DM-C1-Fc, IL4DM- C2-Fc and IL4DM-C1Ser-Fc proteins were harvested three days post transfection and clarified by centrifugation at 300 g followed by filtration through a 0.22 μm sterile filter. For affinity purification 1 ml Streptactin Sepharose (IBA GmbH, Gόttingen, Germany) was packed to a column and equilibrated with 15 ml buffer W (100 mM Tris-HCI, 150 mM NaCI pH 8.0). The cell culture supernatant was applied to the column with a flow rate of 4 ml/min. Subsequently, the column was washed with buffer W and bound IL4DM-C1-Fc, IL4DM-C2-Fc or IL4DM-C1Ser-Fc was eluted stepwise by addition of 7 x 1 ml buffer E (100 mM Tris-HCI , 150 mM NaCI, 2.5 mM Desthiobiotin, pH 8.0). For each construct, fractions E2-E5 were pooled, concentrated by ultrafiltration, analysed by SDS-PAGE and used for subsequent bioassays.
In Figure 4 an SDS-PAGE analysis of affinity purified IL4DM-C1-Fc, IL4DM- C2-Fc and IL4DM-C1Ser-Fc from transfected HEK293T cells is shown. From each protein, 100 ng were diluted into SDS-PAGE sample buffer and separated on Nu-PAGE 4-12%. Separated proteins were subsequently visualized by silver stain. To analyse the apparent MW of the purified protein, standard proteins of known MW were used.
Example 2
Competition of IL4 dependent proliferation of TF1 cells
The human erythroleukemic cell line TF1 (ATCC: CRL-2003) shows factor dependent proliferation. Exposition of TF1 cells to IL4 or GM-CSF results in a proliferative stimulus that can be analysed by the non radioactive proliferation Assays "CellTiter 96 AQ" (Promega, Madison, USA).
We used a TF1 proliferation assay to analyse if the recombinant^ expressed and purified proteins IL4DM-C1-Fc, IL4DM-C2-Fc and IL4DM-C1Ser-Fc are able to compete with wt-IL4 (wildtype lnterleukin-4, human) for binding to the respective IL4-receptor. Briefly, TF1 cells were plated to 96 wells plates in the presence of 40ng human wt-IL4 and various amounts of the respective IL4DM-FC constructs were added. After 72 hours of cultivation the proliferation rate of the TF1 cells was measured according to the instructions of the manufacturer by "CellTiter 96 AQ". The analysis of the data revealed that the constructs IL4DM-C1-Fc and IL4DM-C1Ser-Fc showed a clear dose dependent inhibition of TF1 cell proliferation (Figure 5a, b). However, the construct IL4DM-C2Fc showed only a minimal inhibitory effect on the proliferation of TF1 cell at higher concentrations (Figure 5c). These results demonstrate that the constructs IL4DM-C1-Fc and IL4DM-C1Ser-Fc are capable to inhibit IL4 signalling.

Claims

Claims
1. A fusion protein comprising (i) at least one first domain comprising an IL-4 polypeptide fused to
(ii) at least one heterologous second domain comprising at least a portion of a constant immunoglobulin domain, and (iii) a fusion region between the first and second domain, wherein the fusion region between the first and second domain comprises at least one cysteine residue.
2. The fusion protein of claim 1 , wherein the first domain comprises an IL-4 polypeptide, an agonistic IL-4 mutein polypeptide or an antagonistic IL-4 mutein polypeptide.
3. The fusion protein of anyone of claim 1 or 2, wherein the IL-4 polypeptide is human.
4. The fusion protein of anyone of claims 1-3, wherein the IL-4 polypeptide or mutein polypeptide is C-terminally truncated.
5. The fusion protein of claim 4, wherein the last amino acid residue of the IL-4 polypeptide or mutein polypeptide is a cysteine residue.
6. The fusion protein of anyone of claims 1-5, wherein the IL-4 mutein polypeptide comprises a mutation to the wild-type IL-4 in the region located on A-, C- or/and D-helix of the wild-type human IL-4 protein.
7. The fusion protein of claims 1-6, wherein the IL-4 mutein polypeptide comprises a mutation in the region of the D-helix, selected from at least one mutation, at position 120, 121 , 122, 123, 124, 125, 126, 127 and/or 128 of the wild-type human IL-4 protein sequence.
8. The fusion protein of claim 7, wherein the mutation occurs at position 121 , 124 and/or 125, preferably at position 121 and/or 124.
9. The fusion protein of claim 8, wherein in the IL-4 mutein polypeptide the amino acid tyrosine naturally occurring at position 124 is replaced by the amino acid aspartic acid, glycine or glutamic acid (Y124D-IL-4, Y124G- IL-4 and Y124E-IL-4 mutein).
10. The fusion protein of claim 8, wherein in the IL-4 mutein polypeptide the amino acid arginine naturally occurring at position 121 is replaced by the amino acid aspartic acid or glutamic acid (R121 D-IL-4 and R121 E-IL-4 mutein).
11. The fusion protein of claim 8, wherein in the IL-4 mutein polypeptide the amino acid serine naturally occurring at position 125 is replaced by the amino acid aspartic acid or glutamic acid (S125D-IL-4 and S125E-IL-4 mutein).
12. The fusion protein of anyone of claims 1-11 , wherein the IL-4 mutein polypeptide comprises a mutation in the region of the A-helix selected from at least one mutation at position 13 and/or 16 of the wild-type human IL-4 protein sequence.
13. The fusion protein of anyone of claims 1-12, wherein the IL-4 mutein polypeptide comprises a mutation in the region of the C-helix of the wild- type human IL-4 protein sequence from at least one mutation at position 81 and/or 89.
14. The fusion protein of anyone of claims 1-13, wherein the IL-4 mutein polypeptide comprises a mutation R121 D and Y124D in the D-helix of the wild-type IL-4.
15. The fusion protein of anyone of claims 1-14, wherein the second domain comprises at least a portion of a constant heavy immunoglobulin domain.
16. The fusion protein of anyone of claims 1-15, wherein the second domain is an Fc fragment of a constant heavy immunoglobulin domain comprising the CH2 and CH3 domain.
17. The fusion protein of anyone of claims 1-16, wherein the second domain comprises at least a portion of a constant IgG immunoglobulin domain.
18. The fusion protein of anyone of claims 1-17, wherein the second domain comprises at least a portion of a constant IgGI , lgG2, lgG3 or lgG4 immunoglobulin domain or a variant thereof.
19. The fusion protein of anyone of claims 1-18, wherein the immunoglobulin domain exhibits effector functions, particularly effector functions selected from ADCC and/or CDC.
20. The fusion protein of anyone of claims 1-19, wherein the second domain is derived from a human immunoglobulin.
21. The fusion protein of anyone of claims 1-20, wherein the fusion region comprises at least a portion of an immunoglobulin hinge region.
22. The fusion protein of anyone of claims 1-21 , wherein the fusion region comprises 1 , 2, or 3 cysteine residues.
23. The fusion protein of anyone of claims 1-22, wherein there is at least one amino acid overlap between the first IL-4 polypeptide domain and the second constant immunoglobulin domain.
24. The fusion protein of anyone of claims 1-23, wherein the overlap comprises a cysteine residue.
25. The fusion protein of anyone of claims 1-24, wherein the fusion region comprises the sequence:
(a) CDKTH TCPPC PAPEL LG (SEQ ID NO: 1) or a fragment thereof comprising at least one cysteine residue, or (b) (X1 )DKTH T(X2)PP(X3) PAPEL LG (SEQ ID NO: 3), wherein (X1), (X2) and (X3) each independently denote an amino acid residue, wherein at least one of (X1), (X2) and (X3) is a cysteine residue and at least one of (X1 ), (X2) and (X3) is an amino acid residue different from cysteine, or a fragment thereof comprising at least one cysteine residue.
26. The fusion protein of anyone of claims 1-25, wherein at least one cysteine residue in the fusion region forms a disulphide bridge with a cysteine residue of the IL-4 polypeptide.
27. The fusion protein of claim 26, wherein the cysteine residue of the IL-4 polypeptide is the cysteine residue at position 3 (human IL-4).
28. The fusion protein of anyone of claims 1-27, which is a dimer.
29. A nucleic acid molecule encoding a fusion protein of anyone of claims 1- 27.
30. A vector comprising a nucleic acid molecule encoding a fusion protein of anyone of claims 1-27.
31. A host cell or non-human host organism transformed or transfected with the nucleic acid of claim 28 or the vector of claim 29.
32. A method of producing a fusion protein of anyone of claims 1-27, wherein a host cell nor non-human host organism of claim 31 is cultivated under conditions wherein the fusion protein is produced and the fusion protein is recovered.
33. A pharmaceutical composition comprising the fusion protein of anyone of claims 1-27, the nucleic acid of claim 28, the vector of claim 29 or the host cell or non-human host organism of claim 30.
34. The composition of claim 32 or 33, which comprises a pharmaceutically acceptable carrier, diluent and/or adjuvant.
35. The composition of claim 32, 33 or 34, which comprises a further pharmaceutically active ingredient.
36. The composition of claim 34, wherein the further pharmaceutically active ingredient is an anti-tumour agent, particularly a chemotherapeutic agent, an anti-tumour antibody and/or a pro-apoptotic agent.
37. The composition of claim 36, wherein the chemotherapeutic agent is selected from antimetabolites, DNA-fragmenting agents, DNA- crosslinking agents, intercalating agents, protein synthesis inhibitors, topoisomerase I and Il inhibitors, microtubule-directed agents, kinase inhibitors, hormones and hormone antagonists.
38. The composition of claim 36 or 37, wherein the chemotherapeutic agent is selected from taxanes, platinum compounds and doxorubicin.
39. Use of a composition of anyone of claims 33-38 for the manufacture of a medicament for the prevention and/or treatment of cancer.
40. The use of claim 39 for the prevention and/or treatment of cancer types which have been classified as cytokine-expressing cancer types.
41. The use of anyone of claims 39 or 40, wherein the cancer disease is a solid tumour, in particular an epithelial cancer.
42. The use of anyone of claims 39-41 , wherein the cancer is selected from the group consisting of thyroid carcinoma, breast carcinoma, lung carcinoma, prostate carcinoma, bladder carcinoma, gastric carcinoma, pancreas carcinoma, kidney carcinoma, liver carcinoma and colon carcinoma.
43. The use of anyone of claims 39-42, wherein the cancer disease is a thyroid carcinoma, such as a medullary thyroid carcinoma, a papillary thyroid carcinoma, a follicular thyroid carcinoma or a anaplastic thyroid carcinoma.
44. The use of anyone of claims 39-43 for the prevention and/or treatment of minimal residual cancer disease.
45. The use of anyone of claims 39-44 in combination with radiation therapy and/or a further anti-tumour agent.
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