MXPA06009522A - Il-4/il-13 sepecific polypetides and therapeutic uses thereof - Google Patents

Abstract

Polypeptides and multimeric polypeptides capable of binding unterleukin-4 (IL-4) and interleukin-13 (IL-13) which are useful therapeutically in methods of treating IL-4 and IL-13-related conditions or diseases. The invention features a nucleic acid molecule encoding an interleukin 4 (IL-4) and IL-13-binding fusion polypeptide (R1)x-(R2)y-F, wherein R1 is a modified IL-4 receptor alpha (IL-4R-alpha) component capable of specifically inhibiting IL-4 activity, R2 is an IL-13 receptor alpha 1 or 2 (IL-13R-alpha1 or IL-13R-alpha2) capable of specifically inhibiting IL-13 activity, F is a fusion component, an d x and y are each independently a positive integer>or=1.

Description

SPECIFIC POLYPEPTIDES OF IL-4 / IL-13 AND THERAPEUTIC USES THEREOF FIELD OF THE INVENTION The invention encompasses IL-4 / IL-13 specific polypeptides, as well as therapeutic uses of said polypeptides to inhibit the activity of IL-4 and / or IL-13.

BACKGROUND OF THE INVENTION In US 6,472,179 Stahl et al. describe cytokine fusion protein fusion polypeptides that can bind to a cytokine to form a non-functional complex consisting of two receptor components and a multimerization component. The interleukin-4 alpha receptor (IL-4R0C), and the alpha receptor component of IL-13 (IL-13R0C), are described, for example, in US 5,856,296, and 5,840,869, and EP 876482.

BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the invention features a nucleic acid molecule that codes for a fusion polypeptide binding to interleukin 4 (IL-4) and IL-13 (Rl) x- Ref: 174587 (R2) yF, in which Rl is a component of the IL-4 alpha receptor (IL-4Ra) modified that can specifically inhibit the activity of IL-4 with an IC50 of at least 10"10 molar when present as a component in the fusion polypeptide, R2 is an alpha 1 or 2 receptor of IL-13 (IL-13Ral or IL-13Ra2) that can specifically inhibit the activity of IL-13 with an IC50 of at least 10"10 molar when present as a component in the fusion polypeptide, F is a fusion component, and "x" and "and" are each independently a positive integer > 1. The components of the fusion polypeptide can be arranged in a different order, for example, F- (R1) x (R2) y, (R1) X-F- (R2) y, or (R2) y-F (R1) X. More specifically, Rl is obtained from a precursor IL-4Ra comprising amino acids 1-231, 24-231, 28-231, or 24-221 of SEQ ID NO: 2 (encoded by the nucleic acid sequence of SEQ ID NO: 1), or an allelic variant thereof, and is modified with one to ten modifications defined in modification group I, and R2 is a component of the IL-13 alpha receptor (IL-13Ral or IL -13Ra2) comprising 1-343 or 27-343 of SEQ ID NO: 3, or a fragment thereof, optionally modified with one or more of the modifications defined in modification group II, or comprises amino acids 1-343 or 23-343 of SEQ ID NO: 4, optionally modified with one or more of the modifications defined in modification group III.

Optionally, (R1) x- (R2) y-F also comprises a signal sequence (SS). In one embodiment, the Rl component of the fusion polypeptide is modified to exhibit increased or reduced IL-4 inhibitory activity and / or increased or reduced IL-13 inhibitory activity relative to the unmodified component, preferably modifications to Rl increase the inhibition of both IL-4 and IL-13.

Rl The wild-type IL-4Ra protein normally present in nature is an 800 amino acid protein having the extracellular domain shown in SEQ ID NO: 2. The known allelic variants of SEQ ID NO: 2 include, but are not limited to, , Phe, Val, or Leu at position 75 (Ile75Phe / Val / Leu) and / or Vall31Leu. In one embodiment, Rl comprises amino acids 24-231 of SEQ ID NO: 2, or an allelic variant thereof, optionally also modified with one or more modifications defined in modification group I. In another embodiment, R 1 is the amino acids 1-231 of SEQ ID NO: 2, or an allelic variant thereof, with at least one of the modifications selected from those listed in modification group I. These modifications provide novel polypeptides with specifically desired properties , such as, for example, improved solubility, reduced immunogenicity, improved PK, improved production characteristics and / or improved ability to block the activity of IL-4 and / or IL-13.

Modification Group I The amino acid at position 67, 68, 71, 152, 164, 171, 172, 175, 198, and / or 207 of SEQ ID NO: 2 is (are) replaced with a different amino acid. In the preferred embodiments, the amino acid or amino acid substitution (s) is (are) as follows: Leu at position 67 is replaced with Tyr (Leu67Tyr): Leu68Asn, which can remove a hydrophobic patch and may be desirable in situations specific to improve the solubility and / or ability to block IL-4 and / or IL-13; Aspl7lTyr / Phe; Phel72Ser, which can neutralize an acidic electric potential and reduce the size of a hydrophobic patch, could therefore be desirable for improved solubility and / or folding of the fusion polypeptide; Tyrl52Phe, which changes an amino acid at the ligand binding site, and therefore it may be desirable to improve the inhibitory activity for IL-13 and / or IL-4; Argl98Ser, which removes a positively charged patch and therefore could be desirable to improve the purification properties; and Cys207Ser, which reduces the formation of aberrant disulfide bridges and therefore may be desirable to reduce covalent aggregation and / or incorrect disulfide bridge formation. Modifications that result in the addition of a glycosylation site include Ala7lAsn and Trpl64Ser. In some embodiments, the addition of one or more glycosylation sites is desirable to reduce the immunogenicity, or increase the solubility or stability in vivo relative to the same protein without additional site or glycosylation sites. In the preferred embodiments, Rl comprises 1-231 of SEQ ID NO: 2 with Cys207Ser, further modified by changes in one or more of positions 67, 68, 152, 171 and 172. In preferred embodiments, Rl comprises modifications in (i) ) 67, 68 and 207; (ii) 67, 68, 152 and 207; (iii) 152 and 207; (iv) 67, 171, 172 and 207; (v) 68, 171, 172, and 207; (vi) 67, 68, 171 and 207; (vii) 67, 68, 172 and 207; (viii) 152, 171, 172 and 207; (ix) 67, 68, 171, 172 and 207; (x) 67, 68, 152, 171, 172 and 207; (xi) 171, 172, and 207. In additional preferred embodiments, Rl comprises Cys207Ser and a modification that is selected from the group consisting of (i) Leu67Tyr + Leu68Asn, (ii) Tyrl52Phe, (iii) Aspl71Tyr / Phe + Phel72Ser, (iv) Leu67Tyr + Leu68Asn + Tyrl52Phe, (v) Tyrl52Phe + Asnl7lTyr / Phe + Phel72Ser, (vi) Leu67Tyr + Leu68Asn + Aspl71Tyr / Phe + Phel72Ser, (vii) Tyrl52Phe + Leu67Tyr + Leu68Asn + Aspl71Tyr / Phe + Phel72Ser.

R2 The human wild-type IL-13Ral protein, normally present in nature, is a 427 amino acid protein having the sequence of SEQ ID NO: 3 including an extracellular domain of 343 amino acids. In one embodiment, R2 is a component of IL-13 binding polypeptide comprising amino acids 1-343 or 27-343 of SEQ ID NO: 3, optionally modified with one or more of the modifications defined in modification group II . In another embodiment, R2 is a component of the IL-13 binding polypeptide comprising amino acids 1-343 or 23-343 of SEQ ID NO: 4, optionally modified with one or more of the modifications defined in modification group III. .

Modification group II (a) amino acids 1-120 of SEQ ID NO: 3 are replaced with amino acids 1-123 of human gpl30 (SEQ ID NO: 5); (b) amino acids 338-343 of SEQ ID NO: 3 are deleted; (c) amino acids 1-26 of SEQ ID NO: 3 are replaced with a different signal sequence, for example, SEQ ID NO: 6, or (d) one or more of the amino acid (s) at position 46, 73, 143, 235, 293 and / or 329 of SEQ ID NO: 3 are replaced with a different amino acid. In more specific modalities, the preferred replacement is Cys at position 46 (of SEQ ID NO: 3) with either Ala, Gly, or Tyr (Cys46Ala / Gly / Tyr), preferably Ala, which in specific modalities could be desirable to reduce the formation of aberrant disulphide bridges and covalent aggregates; Lys73Gln; Lysl43Gln, which removes patches with a high amount of positive charges and could be desirable in specific ways to reduce aggregation and / or increase solubility. R2 may also be modified at one or more glycosylation sites to remove sites that are incompletely glycosylated and which may be desirable to improve the pharmacokinetics and / or production of consistency: Asn235Ser / His, Asn293Gly, Asn329Asp.

Modification group III (a ') amino acids 1-22 of SEQ ID NO: 4 are deleted. In specific embodiments in which it might be desirable to replace the suppressed amino acids with, for example, a signal sequence such as SEQ ID NO: 6, thereby removing Cys22 to reduce the formation of aberrant disulfide bonds; (b ') Cys252Ile of SEQ ID NO: 4; (c ') an amino acid changed at position 310 of SEQ ID NO: 4. In a specific embodiment, Ser310 is replaced with Cys, which may be desirable to stabilize the tertiary structure of the protein. The optional fusion component (F) is any component that increases the functionality of the fusion polypeptide. Thus, for example, a fusion component can increase the biological activity of the fusion polypeptide, aid in its production and / or recovery, or increase a pharmacological property or the pharmacokinetic profile of the fusion polypeptide, for example, by increasing its time of serum half-life, penetration capacity in tissues, absence of immunogenicity, or stability. In preferred embodiments, the fusion component is selected from the group consisting of a multimerization component, a serum protein or a molecule capable of binding to a serum protein. When the fusion component is a multimerization component, it includes any natural or synthetic sequence that can interact with another multimerization component to form a higher order structure, for example, a dimer, a trimer, etc. In specific embodiments, the multimerization component is selected from the group consisting of (i) an immunoglobulin-derived domain, (ii) a region susceptible to cleavage (region C), (ii) an amino acid sequence with a length between 1 to 500 amino acids, optionally comprising at least one cysteine residue, (iii) a leucine zipper, (iv) a helical loop motif, (v) a spiral-spiral motif (coil-coil motif). In a more specific embodiment, the immunoglobulin-derived domain is selected from the group consisting of the Fc domain of IgG or the heavy chain of IgG. In a more specific embodiment the Fc domain of IgG is Fc? L (a), an Fc molecule with a deletion of the region involved in the formation of the disulfide bridge with the light chain. When the fusion component is a serum protein, the serum protein can be any serum protein or fragment of a serum protein, such as alpha-1-microglobulin, AGP-1, albumin, vitamin D binding protein, hemopexin, afamin, or haptoglobin. When the fusion component is a molecule that can bind to a serum protein, it can be a small molecule, a nucleic acid, a peptide, or an oligosaccharide. This can also be a protein such as Fc gamma RI, ScFv, etc. In preferred embodiments, the fusion component is encoded by the nucleic acid, which encodes the fusion polypeptide of the invention. However, in some embodiments such as in the case when the fusion component is an oligosaccharide, the fusion component binds after translation to the expressed fusion polypeptide. The nucleic acid molecule of the invention may also optionally comprise a signal sequence (SS) component. When an SS is part of the polypeptide, any known SS may be used, including synthetic or natural sequences from any source, e.g., from a membrane-bound or secreted protein. In a preferred embodiment, an ROR signal sequence (SEQ ID NO: 6) is used. In a second related aspect, the invention features a vector comprising a nucleic acid molecule of the invention. In a third and fourth additional aspects, the invention encompasses vectors comprising the nucleic acid molecules of the invention, including expression vectors comprising nucleic acid molecules operably linked to an expression control sequence, and host systems -vector for the production of a fusion polypeptide comprising the expression vector, in an appropriate host cell; host-vector systems, in which the appropriate host cell is, without limitation, a bacterial, yeast, insect, mammalian or plant cell, such as tobacco or animal such as cows, mice, or rabbits. Examples of suitable cells include E. coli, B. Subtillis, BHK, COS and CHO cells. Additionally modified fusion polypeptides of the invention are encompassed by acetylation or polyethylene glycol. In a fifth related aspect, the invention features a method for producing a fusion polypeptide of the invention, which comprises culturing a host cell transfected with a vector comprising a nucleic acid molecule of the invention, under conditions appropriate for the expression of the protein from the host cell, and the recovery of the polypeptide produced in this way. In the sixth, seventh and eighth aspects, the invention features a fusion polypeptide at IL-4 and IL-13 comprising (R1) x- (R2) and F, in which R1, R2, F, xyy "are as In a preferred embodiment, xy "y" are 1-3, most preferred, xy "y" are each 1. In a ninth aspect, the invention features a multimeric polypeptide, comprising two or more fusion polypeptides of The invention In a more specific embodiment, the multimeric polypeptide is a dimer The dimeric IL-4/13-specific fusion polypeptides of the invention can inhibit both IL-4 and IL-13 with an IC50 of at least 10"10 molar, as determined using test methods known in the art. For example, the IC50 can be determined with the biological test TF1 described below. In general terms, the ability of the dimeric IL-4/13 fusion polypeptides to inhibit (e.g., block) the biological activity of hIL-4 and hIL-13 can be measured by biological test or ELISA for free ligand and / or united. Biological tests may include luciferase-based assays using a STAT6 promoter element, and / or stimulation with hIL-4 or hIL-13 from cell lines such as TF1 or human peripheral blood cells with readings such as growth or secretion from sCD23. In different embodiments of the dimeric IL-4/13 polypeptides of the invention, the Rl component is modified to have an increased or reduced capacity to block the activity of hIL-4 and / or the activity of hIL-13 and / or component R2 is modified to have an increased or reduced capacity to block IL-4 activity and / or IL-13 activity. In a tenth aspect, the invention features pharmaceutical compositions comprising a fusion polypeptide of the invention with a pharmaceutically acceptable carrier. Said pharmaceutical compositions may comprise a monomeric or multimeric polypeptide, or nucleic acids encoding the fusion polypeptide. The IL-4/13 specific polypeptides of the invention are therapeutically useful for treating any disease or condition, which is improved, reduced, or inhibited by removal, inhibition, or reduction of IL-4 and / or IL-13. These polypeptides are particularly useful for the treatment of conditions, such as asthma, which are improved, reduced, or inhibited by removal, inhibition, or reduction of IL-4 and IL-13. Accordingly, in a further aspect, the invention features a therapeutic method for the treatment of a disease or condition related to IL-4 and / or IL-13, which comprises administering a fusion polypeptide of the invention to an individual suffering from a disease or condition related to IL-4 and / or IL-13. Although any mammal can be treated by the therapeutic methods of the invention, the individual is preferably a human patient suffering from or at risk of suffering from a condition or disease that can be improved, reduced, inhibited or treated with a fusion polypeptide. of the invention. In a further aspect, the invention also features diagnostic and prognostic methods, as well as kits for detecting, quantifying, and / or monitoring IL-4 and / or IL-13 with the fusion polypeptides of the invention. Other objectives and advantages will be evident from a review of the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION Before the methods of the present invention are described, it should be understood that this invention is not limited to particular methods, and the experimental conditions described, because such methods and conditions may vary. It should also be understood that the terminology used in the present invention is for the purpose of describing particular embodiments only, and that it is not intended to be limiting, because the scope of the present invention is limited only by the appended claims. As used in this description and the appended claims, the singular forms "a (one)", "one (one)", and "the (the)" include plural references unless the context clearly dictates otherwise. Therefore, for example, a reference to "a method" includes one or more methods, and / or steps of the type described in the present invention and / or which will be apparent to those skilled in the art after reading this description etc. . Unless defined otherwise, all technical and scientific terms used in the present invention have the same meaning as that commonly understood by the person skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described in the present invention can be used in the practice or analysis of the present invention, preferred methods and materials are described below. All publications mentioned in the present invention are incorporated therein for reference to describe the methods and / or materials in connection with which the publications are cited.

Definitions The term "affinity for" IL-4 and / or IL-13 means that the fusion polypeptide of the invention binds to the intended cytokine (s) with an affinity of at least 10-10 molar, preferably at least 10"11 molar, as determined by test methods known in the art, for example, BiaCore analysis The term" which can specifically block "or" which can inhibit the activity of "IL-4 and / or IL-13, means that the IL-4/13 fusion polypeptides of the invention inhibit the biological activity of the target cytokines, as measured, for example, by biological tests or ELISA for free and / or bound ligand.The biological tests may include tests based on luciferase using a STAT6 promoter element, and / or stimulation of IL-4 or IL-13 from cell lines such as TF1 or from human peripheral blood cells with readings such as growth or secretion of sCD23. define as the concentration n of fusion protein required to inhibit 50% of the response to IL-4 or IL-13 as measured in a bioassay. The fusion polypeptides of the invention can preferably inhibit the biological activity of IL-4 and / or IL-13 with an IC 50 of at least 1 x 10 ~ 10 M, even more preferred 10 ~ M (for IL-13) . The terms "treatment", "treating", and the like are used in the present invention to mean in general terms to obtain a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease, condition, or symptoms thereof, and / or may be therapeutic in terms of a partial or complete cure for a disease or condition and / or adverse effect attributable to the disease or condition. "Treatment" as used in the present invention covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in an individual who may be pre-disposed to the disease or condition but has not yet been diagnosed with it; (b) inhibit the disease or condition, that is, stop its development; or (c) alleviating the disease or condition, i.e., causing the regression of the disease or condition. The population of individuals treated with the method of the invention includes individuals suffering from the undesirable condition or disease., as well as individuals at risk of developing the condition or disease. By the term "therapeutically effective dose" is meant a dose that produces the desired effect for which it is administered. The exact dose depends on the purpose of the treatment, and can be determined by the person skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). As used in the present invention, a "condition or disease" generally encompasses a condition of a mammalian host, particularly a human host, that is undesirable and / or harmful to the host. Therefore, treating a condition or disorder with an IL-4/13 specific fusion polypeptide encompasses the treatment of a mammal, in particular, a human having symptoms that are reflective of IL-4 and / or IL-13. high or harmful, or who is expected to have such reduced activation in response to a disease, condition or treatment regimen. The treatment of a condition or disease related to IL-4 and / or IL-13 encompasses the treatment of a human individual in which reducing the levels of IL-4 and / or IL-13 with the fusion polypeptide of the invention gives as a result the alleviation of an undesirable symptom that results from the condition or disease related to IL-4 and / or IL-13.

General description Studies in animals lacking IL-4 and IL-13 have indicated that these cytokines perform both overlap and additive functions in the induction of Th2-type responses such as eosinophil infiltration, immunoglobulin E production and IL- production. 5 (McKenzie et al. (1999) J. Exp. Med. 189 (10): 1565-72). The present invention provides novel polypeptides, both monomers and multimers, which can act as specific fusion polypeptides of IL-4 and / or IL-13 or antagonists that can bind to IL-4 and / or IL-13 and block these biological actions.

Nucleic acid constructs and their expression The present invention provides for the construction of nucleic acid molecules that encode specific IL-4/13 polypeptides. As described above, the nucleic acid molecules of the invention encode modified fragments of the wild type IL-4Ra and / or IL-13 Ra proteins (or that normally occur in nature). Accordingly, nucleic acid molecules can be termed "recombinant", "artificial", or "synthetic" because these are not nucleic acid molecules found in nature, for example they are not sequences that occur in nature, but they are sequences built using recombinant DNA technology. These nucleic acid molecules are inserted into a vector that can express the fusion polypeptides of the invention when introduced into an appropriate host cell. Suitable host cells include, but are not limited to, bacteria, yeast, insect, and mammalian cells. Any of the methods known to the person skilled in the art can be used for the insertion of DNA fragments into a vector to construct expression vectors that code for the fusion polypeptides of the invention under control of transcription control signals and / or translation. The expression of the nucleic acid molecules of the invention can be regulated by a second nucleic acid sequence such that the molecule is expressed in a host transformed with the recombinant DNA molecule. For example, the expression can be controlled by any promoter / enhancer element known in the art. Promoters that can be used to control the expression of chimeric polypeptide molecules include, but are not limited to, a long terminal repeat (Squinto et al. (1991) Cell 65: 1-20); SV40 early promoter region, M-MuLV thymidine kinase promoter, CMV, regulatory sequences of the metallothionin gene; prokaryotic expression vectors such as the beta-lactamase promoter, or the tac promoter (see also Scientific American (1980) 242: 74-94); promoter elements from yeasts or other fungi such as the promoter Gal 4, ADH, PGK, alkaline phosphatase, and tissue-specific transcription control regions obtained from genes such as elastase I. Expression vectors are used which can be replicated in a bacterial or eukaryotic host comprising the nucleic acid molecules of the invention for transfecting the host and thereby directing the expression of said nucleic acids to produce the fusion polypeptides of the invention. The transfected cells can express transiently or, preferably, constitutively and permanently, the polypeptides of the invention. When the polypeptide expressed in this manner comprises a fusion component such as a multimerization component that can be associated with a multimerization component of a second polypeptide, the monomers expressed in this way are multimerized due to the interactions between the multimerization components to form a multimeric polypeptide (WO 00/18932). The fusion polypeptides of the invention can be purified using any technique known in the art. When the polypeptides of the invention comprise a multimerization component, which spontaneously forms a multimer with another polypeptide, the purification techniques used allow the subsequent formation of a stable, biologically active multimeric polypeptide, also known as a "fusion polypeptide". For example, and not by way of limitation, the factors can be recovered from cells either as soluble proteins or as inclusion bodies, from which they can be extracted quantitatively using 8M guanidinium hydrochloride and dialysis (see, for example, U.S. Patent No. 5,663,304). In order to further purify the factors, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration can be used.

Fusion Components The fusion polypeptides of the invention comprise a fusion component (F) which, in specific embodiments, is selected from the group consisting of a multimerization component, a serum protein, or a molecule that can be bound to a serum protein When F is a multimerization component, it includes any natural or synthetic sequence that can interact with another multimerization component to form a higher order structure, for example, a dimer, a trimer, etc. The multimerization component may be selected from the group consisting of (i) a multimerization component comprising a region susceptible to shear, (ii) a truncated multimerization component, (iii) an amino acid sequence with a length between 1 up to 500 amino acids, (iv) a leucine zipper, (v) a helix loop motif, and (vi) a spiral-spiral motif. When F is a multimerization component comprising from about 1 to 500 amino acids in length, the sequence may contain one or more cysteine residues that can form a disulfide bridge with a corresponding cysteine residue in another fusion polypeptide comprising an F with one or more cysteine residues. In a preferred embodiment, the multimerization component comprises one or more immunoglobulin-derived domains derived from human IgG, IgM or IgA. In specific embodiments, the immunoglobulin derived domain is selected from the group consisting of the Fc domain of IgG or the heavy chain of IgG. The Fc domain of IgG can be selected from the isotypes IgGl, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group. In a specific embodiment, F is the Fc domain of IgG4 with Ser 228 (Cabot numbering) mutated to Pro to stabilize the formation of covalent dimer (Mol.

Immunol. (1993) 30: 105-108) and / or Leu235- »Glu which eliminates residual effector functions (Reddy et al (2000) J. Immunol. 164: 1925-1933). In a preferred embodiment, F is the Fc domain of IgGl, or a derivative thereof which may be modified for specifically desired properties (see, for example, Armor et al. (2003) Mol.Immunol.40-585-593; Shields et al (2001) J. Biol. Chem. 276: 6591-6604).

In specific embodiments, the IL-4 / IL-13 specific polypeptide of the invention comprises one or two Fc domains of IgGl. In one embodiment, F is a serum protein or fragment thereof, which is selected from the group consisting of a-1-microglobulin, AGP-1, orosomucioid, oc-1 acid glycoprotein, vitamin D binding protein (DBP), hemopexin, human serum albumin (hsA), transferrin, ferritin, afamin, haptoglobin, α-fetoprotein thyroglobulin, -2-HS-glycoprotein, β-2-glycoprotein, hyaluronan binding protein, syntaxin, ClR, chain a of Clq, galectin3-Mac2 binding protein, fibrinogen, polymeric Ig receptor (PIGR), -2-macroglobulin, urea transport protein, haptoglobin, IGFBPs, macrophage scavenger receptors, fibronectin, gigantin, Fc, - 1-antichyrotropin, a-1-antitrypsin, antithrombin III, apolipoprotein AI, apolipoprotein B, β-2-microglobulin, ceruloplasmin, complement component C3 or C4, Cl esterase inhibitor, C reactive protein, cystatin C, and protein C In a more specific embodiment, F is selected from the group consisting of a-1-microglobulin, AGP-1, orosomucioid, α-1-acid glycoprotein, vitamin D binding protein (DBP), hemopexine, serum albumin human (HsA), afamin, and haptoglobin. The inclusion of an F component can extend the serum half-life of the IL-4/13 specific polypeptide of the invention when desired. See, for example, US Patents Nos. 6,423,512, 5,876,969, 6,593,295, and 6,548,653, for examples of serum albumin fusion protein. When F is a molecule that can bind to a serum protein, the molecule can be a synthetic small molecule, a lipid or liposome, a nucleic acid, including a synthetic nucleic acid such as a tablet, a peptide, or an oligosaccharide. The molecule can also be a protein, such as, for example, Fc? RI, Fc? R2, Fc? R3, polymeric Ig receptor (PIGR), ScFv, and other antibody fragments specific for a serum protein.

Optional component spacers The components of the fusion polypeptides of the invention can be directly connected to each other or can be connected through spacers. Generally speaking, the term "spacer" (or linker) means one or more molecules, for example, nucleic acids or amino acids, or non-peptide portions, such as polyethylene glycol, which can be inserted between one or more component domains. For example, spacer sequences may also be used to provide a desirable site of interest between components for ease of handling. A spacer can also be provided to increase the expression of the fusion protein from a host cell, to reduce the spherical hindrance such that the component can assume its optimal tertiary structure and / or interact appropriately with its target molecule . For spacers and methods for identifying desirable spacers, see, for example, George et al. (2003) Protein Engineering 15: 871-879. A spacer sequence can include one or more amino acids naturally connected to a component of the receptor, or it can be an aggregated sequence used to increase the expression of the fusion protein, to provide specifically desired sites of interest, to allow the domains of the component form optimal tertiary structures and / or to increase the interaction of a component with its target molecule. In one embodiment, the spacer comprises one or more peptide sequences between one or more components that are (are) between 1-100 amino acids, preferably 1-25. In a specific embodiment, the spacer is a sequence of 3 amino acids; more specifically, the 3-amino acid sequence of Gly Ser Gly.

Inhibition of the biological activity of IL-4 and / or IL-13 The fusion polypeptides of the invention can inhibit the biological activity of IL-4 and / or IL-13 with an IC0 (concentration of fusion protein required to inhibit the 50% of the response to IL-4 or IL-13) of at least 1 x 10 ~ 10 M (for both); even more preferred 10-11 M (for IL-13). The data presented in the following Tables 1-7 are determined in a biological test of TF1 for growth stimulated by IL-4 or IL-13, as described below. Other biological tests useful for determining IC50 are known in the art, including for example, luciferase-based assays using a STAT6 promoter element, and / or stimulation with hIL-4 or hIL-13 from human peripheral blood cells with a reading such as secretion of sCD23. The data shown below in Tables 1-7 are presented as times of difference from the precursor molecule (IC 50 value of the variant fusion polypeptide divided by the IC 50 value of the precursor molecule 1132). As set forth in the experiments below, the variant fusion polypeptides may have an improved ability 1.5 to 3.0 times or even higher to block IL-4 and / or IL-13 relative to the precursor molecule. In specific embodiments, the variant fusion polypeptide of the invention has an improvement of at least 2.0 times or greater, at least an improvement of 2.5 times or greater, or even at least a 3-fold or greater improvement in capacity. to block IL-4 and / or IL-13.

Therapeutic uses The fusion polypeptides of the invention are therapeutically useful for treating any disease or condition that is improved, reduced, inhibited or prevented by removal, inhibition, or reduction of IL-4 and / or IL-13. IL-4 and IL-13 both independently and jointly have been implicated in a variety of clinical conditions, such as eosinophilic infiltration, IgE production and IL-5 production, which are characterized by a controlled response per Th2 cell. Accordingly, blockage of these responses by the fusion polypeptide will be useful for the treatment of any disease or condition in which there is an increased occurrence of helper cells of the TH2 type. In a modality, the IL-4/13 fusion polypeptide is used to treat asthma. The data obtained from animal experiments and the examination of asthmatic humans implicates IL-4 and IL-13 as the critical initiators of the atopic condition and as agents that perpetuate the chronic inflammatory state that typifies the asthmatic lung. IL-4 and IL-13 induce effects that are associated with the asthmatic phenotype, including isotype exchange for IgE production, eosinophilia, mastocytosis, mucus formation, increased vascular permeability, hyper-responsiveness of airways, hyperplasia of smooth muscle, and sub-epithelial fibrosis (Hogan et al. (1997) Pharmacol. Ther. 74 (3): 259-283; Mckenzie et al. (2000) Pharmacol. Ther. 88 (2): 143-151; Wills-Karp (2001) J. Allergy Clin. Immunol., 107 (1): 9-18). In effect, IL-4 and IL-13 signaling in mice is required for the development of an IgE response to an allergen, and the development of an asthma response against ovalbumin is attenuated in mice deficient in IL-4Ra. Likewise, the transgenic expression of IL-4 or IL-13 in the lungs of mice leads to an asthmatic phenotype, which can be mimicked by the direct administration of IL-4 or IL-13 protein in the murine lung. Therefore, it is expected that blocking IL-4 and IL-13 will lead to a reduction in some or all of the aforementioned parameters. Also, because of the ability of either IL-4 or IL-13 to independently initiate the signaling cascade and induce the asthma phenotype, inhibiting both molecules at the same time can lead to more potent anti-asthma effectiveness. A non-exclusive list of specific conditions enhanced by inhibition of IL-4 and / or IL-13 includes atopic dermatitis, allergic conditions of immune complex disease (such as lupus, nephritis, and Grave's disease), hyper-IgE syndrome , immune deficiencies, idiopathic pulmonary fibrosis, liver fibrosis, HIV, pulmonary "remodeling", COPD, ulcerative colitis, cancer, Hodgkin's lymphoma, bullous pemphigoid, transplant and graft-versus-host disease, viral, parasitic, bacterial and fungal infection . (U.S. Patent No. 6,328,954 issued on Nov. 11, 2001. Idzerda, R.J., et al., 1990 J Exp. Med. 171: 861-873). In alternative embodiments, the fusion polypeptide is used as an adjuvant with a vaccine to boost the immune response toward one of cell-mediated immunity, which is often accompanied by changes in the Ig isotypes as well as a CTL response (lymphocyte). Cytotoxic T). CTLs are primarily CD8 positive T cells, which aid in the destruction of virally infected cells or with intracellular bacteria and tumor cells.

Suitable Individual for Treatment A suitable individual for treatment is a human who has been diagnosed as suffering from specific conditions improved by the inhibition or reduction of IL-4 and / or IL-13 including atopic dermatitis, allergic conditions of the complex disease immune (such as lupus, nephritis, and Grave's disease), hyper-IgE syndrome, immune deficiencies, idiopathic pulmonary fibrosis, liver fibrosis, HIV, pulmonary "remodeling", COPD, ulcerative colitis, cancer, Hodgkin's lymphoma, bullous pemphigoid , transplant disease and graft against host, viral disease, parasitic, bacterial and fungal infection.

Combination Therapies In numerous embodiments, the fusion polypeptides of the invention can be administered in combination with one or more additional compounds or therapies. Combinations include inhaled short-acting beta 2 agonists, oral beta 2 agonists, inhaled anti-cholinergics, oral corticosteroids, inhaled corticosteroids, cromolyn sodium (Gastrocrom ™ / Celltech), nedocromil, long-acting beta 2 agonists, leukotriene modifications, theophylline, inhibitors of calcinerin, picrolimus, sirolimus, anti-IgE (Zolair ™, Genentech), inhibitors of NFKB, p38 MAP kinase inhibitors (VX-702), ICE inhibitors (VX-765), IL-1 inhibitors (IL-1 specific fusion polypeptide, Regeneron; anakinra, Amgen), inhibitors of TNFa (Remicade ™, Centocor, Enbrel ™, Amgen, Humira ™, Abbott), inhibitors of IL-5, inhibitors of IL-18, inhibitors of IFN gamma, IFN alpha blockers. For example, multiple fusion polypeptides can be co-administered, or a polypeptide can be administered in conjunction with one or more therapeutic compounds. When a polypeptide of the invention removes IL-4 and / or IL-13, said one or more other therapeutic agents is one that is used to prevent or treat a condition associated with the presence of IL-4 and / or IL-13. . A benefit of the combined use of the fusion polypeptide of the invention with a second therapeutic agent is that it provides improved efficacy and / or reduced toxicity of any therapeutic agent.

Methods of administration The invention provides methods of treatment comprising administering to an individual an effective amount of a fusion polypeptide of the invention. In a preferred aspect, the fusion polypeptide is substantially pure (eg, substantially free of substances that limit its effect or produce undesired side effects). The individual of preference is a mammal, and a human is more preferred. Various delivery systems are known and can be used to administer an agent of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells that can express the compound, receptor-mediated endocytosis (see, for example. , Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432) construction of a nucleic acid as part of a retroviral vector or other vector, etc. The methods of introduction may be enteral or parenteral and include, but are not limited to routes of intradermal, intramuscular, intra-articular administration, peritoneal infusion (polypeptideeritoneal infusion), intravenous, subcutaneous, intranasal, intraocular, and other routes. The compounds can be administered using any convenient route, for example by infusion or bolus injection, by absorption through epithelial or muco-cutaneous inner linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. The administration can be systemic or local. The administration can be acute or chronic (for example, daily, weekly, monthly, etc.) or in combination with other agents. Pulmonary administration can also be used, for example, by the use of an inhaler or nebulizer, and formulation with an aerosol agent. In another modality, the active agent can be delivered in a vesicle, in particular a liposome, in a controlled release system, or in a pump. In another embodiment, in which the active agent of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote the expression of its encoded protein, constructing it as part of an acid expression vector. nucleic acid and administering it in such a way that it becomes intracellular, for example, through the use of a retroviral vector (see, for example, US Patent No. 4,980,286), by direct injection, or by the use of micro-particle bombardment , or by coating with lipids or cell surface receptors or transfection agents, or by administration thereof in conjunction with a homeotic box-like peptide which is known to enter the nucleus (see, for example, Joliot et al., 1991, Proc. Nati, Acad. Sci. USA 88: 1864-1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated into the host cell DNA for expression, by homologous recombination. Systemic expression can also be achieved by injection of plasmid (intradermally or intramuscularly) and electroporation into the cells. In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment.; this can be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, for example, by injection, by means of a catheter, or by means of an implant, the implant being a porous material , non-porous or gelatinous, including membranes, such as sialastic membranes, fibers or commercial skin substitutes. A composition useful for practicing the methods of the invention may be a liquid comprising an agent of the invention in solution, in suspension or both. The term "solution / suspension" refers to a liquid composition in which a first portion of the active agent is present in solution and a second portion of the active agent is present in the form of particles, suspended in a liquid matrix. A liquid composition also includes a gel. The liquid composition can be aqueous or be in the form of an ointment. In one embodiment, the pharmaceutical composition of the invention is a sustained release composition. Sustained release formulations for the delivery of biologically active peptides are known in the art. For example, US 6,740,634 describes a sustained release formulation containing a hydroxynaphthoic acid salt of a biologically active substance and a biodegradable polymer. US 6,699,500 describes a sustained release formulation that can release a physiologically active substance over a period of at least 5 months.

Diagnostic and evaluation methods The fusion polypeptides of the invention can be used in diagnostic manner and / or in evaluation methods. For example, the fusion polypeptide can be used to monitor the levels of IL-4 and / or IL-13 during a clinical study to evaluate the efficacy of the treatment. In another embodiment, the methods and compositions of the present invention are used to evaluate individuals for admission to a clinical study to identify individuals who have, for example, a very high or very low level of IL-4 and / or IL-13. . The fusion polypeptides of the invention can be used in methods known in the art which relate to the localization and activity of IL-4 and / or IL-13, for example, imaging, measuring their levels in samples physiological, in diagnostic methods, etc. The fusion polypeptides of the invention can be used in an in vivo and in vitro evaluation test to quantify the amount of unbound IL-4 and / or IL-13 present, for example, in an evaluation method to identify agents of proof that they can reduce the expression of IL-4 and / or IL-13. More generally, the fusion polypeptides of the invention can be used in any test or procedure in which the quantification and / or isolation of 11-4 and / or IL-13 is desired.

Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising a fusion polypeptide of the invention. Said compositions comprise a therapeutically effective amount of one or more fusion polypeptides, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State government or listed in the Pharmacopoeia of the United States of North America or other Pharmacopoeia generally recognized for use in animals, and more particularly, in humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Said pharmaceutical vehicles can be sterile liquids, such as water and oils, including those of petrochemical, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting agents or emulsifiers, or agents for pH regulation. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Examples of pharmaceutically suitable vehicles are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The fusion polypeptide of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those which are formed with free amino groups such as those obtained from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those obtained from sodium, potassium , ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. The amount of the fusion polypeptide that can be effective for its intended therapeutic use can be determined using standard clinical techniques based on the present disclosure. In addition, in vitro tests can optionally be used to help identify optimal dosage ranges. In general terms, appropriate dosage ranges for intravenous administration are generally about 0.02-10 mg of active compound per kg body weight. Appropriate dosage ranges for intranasal administration are generally from about 0.01 pg / kg of body weight to 10 mg / kg of body weight. Effective doses can be extrapolated from dose-response curves derived from test systems in animal or in vitro models. Of course, the amount of compound administered will depend on the individual being treated, the weight of the individual, the severity of the affliction, the manner of administration, and the judgment of the attending physician. Therapy can be repeated intermittently as long as the symptoms are detectable or even when they can no longer be detected.

Cell Transfection and Gene Therapy The present invention encompasses the use of nucleic acids encoding the fusion polypeptides of the invention for the transfection of cells in vi tro and in vivo. These nucleic acids can be inserted into any of a number of well-known vectors for transfection of target cells and organisms. The nucleic acids are transfected into cells ex vivo and in vivo, through the interaction of the vector and the target cell facilitated by lipid mixtures or electroporation. The compositions are administered (eg, by injection into a muscle) to an individual in an amount sufficient to induce a therapeutic response. An amount adequate to accomplish this is defined as "a therapeutically effective dose or amount". In another aspect, the invention provides a method for reducing the levels of IL-4 and / or IL-13 in a human or other animal that comprises transfecting a cell with a nucleic acid encoding a polypeptide of the invention, in which the nucleic acid comprises an inducible promoter linked in operable form to the nucleic acid encoding the polypeptide. For gene therapy procedures in the treatment or prevention of human diseases, see, for example, Van Brunt (1998) Biotechnology 6: 1149-1154.

Cases The invention also provides a package or pharmaceutical case comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally, a note in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceutical or biological products, whose note reflects (a) approval by the manufacturing agency, may be associated with said container or containers. use or sale for administration to humans, (b) instructions for use or both.

EXAMPLES The following example is indicated to provide those skilled in the art with a description and full disclosure of the manner in which to make and use the methods and compositions of the invention, and is not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to numbers used (eg, quantities, temperature, etc.) but certain errors and experimental deviations must be taken into account. Unless otherwise indicated, the parts are parts by weight, the molecular weight is average molecular weight, the temperature is in degrees Centigrade, and the pressure is atmospheric pressure or is close to atmospheric pressure.

EXAMPLE 1 Construction of variant IL-4/13 fusion polypeptides To create the precursor fusion polypeptide IL-4/13, 1132 (SEQ ID NO: 8), nucleic acids coding for the extracellular domain of human IL-4Ra (SEQ ID NO: 1-2) and the extracellular domain of human IL-13Ral are amplified (27-343 of SEQ ID NO: 3) using standard PCR techniques, and they are ligated into an expression vector which contains the human Fc sequence, thereby creating a fusion protein consisting of the IL regions. -Ra and / or IL-13R, and the hinge, CH2 and CH3 regions of human IgG1 from the N-terminal to the C-terminus. The substitutions Cys207-Ser of SEQ ID NO: 8 and Cys251? Ala of SEQ ID NO: 8 (Cys251 corresponds to Cys46 of SEQ ID NO: 3) by site-directed mutagenesis using standard techniques known in the art. All sequences are confirmed using standard techniques. The appropriate coding sequence is sub-cloned into a eukaryotic expression vector using standard molecular biology techniques. The IL-4/13 fusion polypeptide variants are created by site-directed mutagenesis of the precursor fusion polypeptide 1132 using techniques known in the art, and are confirmed by sequence determination. IL-4/13 variant fusion polypeptides are produced as small-scale supernatants by transiently transfecting CHO cells, using Lipofectamine / LIPO Plus ™ (Life Technologies), with DNA constructs that code for variant proteins. Supernatants are harvested after 72 hours and protein expression is measured using Western blot analysis with antibody conjugated to HRP anti-human Fc (Promega) and visualized by ECL (Pierce). For large-scale purification of IL / 4/13 variant fusion polypeptides, the DNA encoding the fusion protein is transfected into CHO cells either for transient expression or to create stable lines using FASTR technology (publication of US patent application 20020168702). Culture medium is collected from 1-2 liters of the cells expressing the fusion protein and passed through a protein A column to capture the fusion protein containing Fc. The purification of protein A is carried out in accordance with the manufacturer's protocol (Amersham). After concentrating, the fusion protein is characterized relative to the percentage of contaminating aggregates and further purified using size exclusion chromatography (SEC) using a Superdex 200 column (Amersham) or similar column. The stability of IL-4/13 variant fusion polypeptides is evaluated using standard methods, including analysis by SEC and Western blot after 20 cycles of freezing / thawing, incubating the protein at 37 ° C for 7 days in regulatory solutions with low salt content (10 mM sodium phosphate buffer solution) and medium salt content (PBS), or by incubating the protein in a PBS solution regulated at a variety of pH values for two hours. The pharmacokinetics of the molecules is determined by injecting mice or rats with 1 mg / kg of the IL-4/13 fusion polypeptide variant intravenously or subcutaneously, collecting the blood at various time points, and isolating the serum. The serum samples are analyzed for the amount of variant fusion polypeptide using an ELISA with an anti-IL-13Ra monoclonal antibody to capture the fusion protein, a biotin-modified anti-IL-4Ra monoclonal antibody to form a complex, and a streptavidin-HRP conjugate to detect the complex. The fusion polypeptide concentrations are determined by comparison of the OD from the serum samples with the ODs obtained from a standard curve that is produced using the purified fusion protein. The quality of the fusion polypeptide is also monitored by Western blot analysis of 1 ul of serum using one of three antibodies, anti-IL-13Ra, anti-IL-4Ra or anti-human Fc antibodies and a secondary antibody conjugated with HRP for the detection.

EXAMPLE 2 Inhibition of bioactivity of hIL-4 and IL-13 using variant fusion polypeptides Biological test of TF1 TF1 cells that have been stably transfected with hIL-13Ral are maintained in growth medium (10 ng / ml GM-CSF, RPMl 1640, 10% FBS, L-glutamine, penicillin, streptomycin) . For the biological test, the cells are washed 3 times in test medium (as above but without GM-CSF) and then seeded at a density of 2 x 10 4 cells in 50 μl of test medium. The purified fusion polypeptides are serially diluted in the test medium. 25 ul of each of the IL-4/13 variant fusion polypeptides is added to the cells. Then 25 μl of either IL-13 (15 pM) or IL-4 (20 or 40 pM) are added to the cavities containing the cells and the fusion polypeptides. The cells are then incubated at 37 ° C, 5% C02 for about 70 hours. The degree of TF1 cell proliferation is measured using the CCK-8 test in accordance with the manufacturer's protocol (Doj indo Laboratories). All biological tests for Tables 1-7 include the precursor IL-4/13 fusion polypeptide, 1132 (SEQ ID NO: 8 shown with the signal sequence which is cut from the mature polypeptide) (encoded by SEQ. ID NO: 7), which consists of a signal sequence (amino acids 1-23) + a component of IL-4Ra (amino acids 24-231 of SEQ ID NO: 8 with Cys207- »Ser) (corresponding to 24- 231 of SEQ ID NO: 2) + a component of IL-13Ral (amino acids 232-548 of SEQ ID NO: 8 with Cys251? Ala) (Cys251 corresponds to Cys46 of SEQ ID NO: 3) (corresponding to 27-343 of SEQ ID NO: 3) + a multimerization component (IgG1 Fc) (549-766 of SEQ ID NO: 8). All variant fusion polypeptides, except those specifically described (table 1) contain the Cys207- »Ser precursor mutation, and all variant fusion polypeptides contain the mutation Cys251-» Ala (Cys 46 of SEQ ID NO: 3) in the IL-13Ral component. See, for example, SEQ ID NO: 10 (construction 2674, coded by SEQ ID NO: 9), SEQ ID NO: 12 (construction 2681, coded by SEQ ID NO: 11), SEQ ID NO: 14 (construction 2795; encoded by SEQ ID NO: 13), and SEQ ID NO: 16 (construct 2796; encoded by SEQ ID NO: 15) which include a signal sequence finally cut from the mature fusion polypeptide). Table 1 shows the IC50 data (the concentration at which 50% of cell growth is inhibited) for the precursor trap (parental trap), 1132, and two example variant fusion proteins, as well as the difference in from the IC50 value for the IL-4/13 fusion polypeptide precursor 1132 (IC50 of the precursor fusion polypeptide divided by IC50 of the variant fusion polypeptide).

TABLE 1 Tables 2 to 6 show the difference in times of the IC50 values of the biological test of the variant fusion polypeptides analyzed using transient CHO supernatants, whose concentrations are determined by Western blot analysis. Table 2 shows the difference in times in the IC 50 values with respect to the ability of the variants having cysteine mutations to block the activity of hIL-13 or hIL-4; Table 3 shows the ability of fusion polypeptide variants having site stabilizing and active site mutations to block the activity of hIL-13 or hIL-4; Table 4 shows the variants of charge change and combinations to block the activity of hIL-13 or hIL-4; Table 5 shows the hydrophobic patch variants and combinations for blocking the activity of hIL-13 or hIL-4; Table 6 shows the activity of the variant fusion polypeptides with N-terminal and C-terminal deletions of IL-4Ra (all expressed relative to precursor fusion polypeptide 1132).

TABLE 2 TABLE 3 Capacity of stabilization variants of active site and center to block the activity of IL-13 and IL-4 TABLE 4 Loading variants and combination variants to inhibit the activity of IL-4 and IL-13 TABLE 4 (cont.) TABLE 5 TABLE 5 (cont.) TABLE 6 EXAMPLE 3 Characterization of purified fusion polypeptide variants with respect to bioactivity Table 7 shows the ability of variant fusion polypeptides to block the activity of IL-4 and IL-13. The results are shown as the difference in times from the CI5o value for the precursor fusion polypeptide (the IC50 of the precursor fusion polypeptide divided by the IC50 of the variant fusion polypeptide) IL-4/13 1132 fusion polypeptide ( SEQ ID NO: 8). The precursor molecule consists of a signal sequence (amino acids 1-23) + one component of IL-4Ra (amino acids 24-231 with Cys207Ser) (corresponding to 24-231 of SEQ ID NO: 2) + one component of IL-13Ral (amino acids 232-548 with Cys251Ala) (corresponding to 27-343 of SEQ ID NO: 3) + a multimerization component (IgGl Fc) (549-776). Standard errors are provided for those that are analyzed three or more times. All variant fusion polypeptides, except those with other specified Cys207 substitutions, contain the Cys207- »Ser mutation, and all variant fusion polypeptides contain the Cys251-Ala mutation in the IL-13Ral component.

TABLE 7 EXAMPLE 4 Determination of the binding affinity of IL-4/13 of the variants using BIAcore The affinity of IL-4/13-specific polypeptides for human IL-4 and IL-13 is measured using a BIAcore 2000 or BIAcore 3000, as described in WO 00/75319. The BIAcore test evaluates the precursor construction 1132 (SEQ ID NO: 8) in relation to the Rl-R2-Fc variants, all of which consist of a signal sequence, an IL-4Ra component, followed by an IL component. -13Ral and a multimerization component (IgGl Fc). IL-4/13 fusion polypeptide variants are captured on the surface of the chip using anti-human Fc antibodies. Various concentrations of human IL-4 and / or IL-13 are injected onto the surface and the time course of association and dissociation is monitored. The kinetic analysis is carried out using the BIA evaluation software to obtain the rate constants of association and dissociation. The results are shown in table 8.

TABLE 8 Binding Affinity of IL-4 / IL-13 Measured Using BIAcore Construction # IL-4 IL -13 Variant KD - ^ asoc. -K-sep. ? D - ^ asoc. K Sep 1132 Precursor 1.04 X 10 '"8.75 x 10' 9.09 x 10" 4 4.56 X 10"" 2.36 X 10 ° 1.08 X 10"s 4.11 X 10" 12 3.11 X 106 1.28 X 10"5 2547 L67Q, 68S 4.79 X 10"" 7.12 x 10 '3.41 x 10"J 2.73 X 10" "3.01 X 106 3.41 X? OJ 2549 D171Y, F172S, 1.09 X 10" 11 7.46 x 10' 8.13 x? O-4 1.09 X 10"" 7.46 X 10 '8.23 X lO "" Y175H 2558 D171Y, F172S, 3.18 X 10"11 2.5 x 10' 7.96 x 10" 4 1.63 X 10"" 3.39 X 10b 5.52 X 10"b Y175H, R198S 2560 Y152F 6.62 X 10"" 8.8 x 10 '5.87 x lO'4 2.09 X 10"" 2.21 X 10"4.61 X 10" b 2576 S207C 3.13 X 10"" 5.37 x 10' 1.66 x 10"J 3.15 X 10 '" 4.02 X 10b 1.26 X 10 ~ s 2586 Y152F, D171Y, 2.07 X 10"" 1.06 x 10B 2.18 x 10"J 2.17 X 10" "6.12 X 10b 1.33 X 10" * cp Cp F172S, Y175H 2594 A228C 3.45 X 10"" 7.92 x 10 '2.73 x 10"J 9.71 X 10'" 5.28 X 10"5.13 X? Ob 2602 L67Y, L68N 1.45 X 10" "6.90 x 10 '1.00 x 10" J 3.01 X 10"" 1.12 X 10 '3.36 X 10 ~ s 2647 D171Y 6.87 X 10"12 4.80 x 10' 3.30 x 10'4 5.09 X 10" "1.07 X 10 '5.44 X 10" s F172S 2651 L67Y, L68N , 1.08 X 10"" 5.15 x 10 '5.57 x 10"" 4.26 X? Xv? 7.83 X 10b 3.34 X 10"3 Y152F 2674 67Y, L68N, 1.78 X 10" "1.94 x 10" 3.46 x 10"J 5.99 X 10"" 3.33 X 10b 2.00 X lo-5 D171Y, F172S 2681 67Y, L68N, 9.54 X 10"" 6.32 x 10 '6.03 x 10"" 4.96 X 10"" 3.18 X 10b 1.58 X 10 ~ s 15 Y152F, D171Y , F172S EXAMPLE 5 Ability of the component variants to inhibit the activity of IL-4 and IL-13 Table 9 shows the differences in times of the biological IC50 test values of the fusion polypeptides analyzed using the transient CHO supernatants as described above. These fusion polypeptides contain alternative component arrays, for example R2-R1-Fc (SEQ ID NOS: 3 and 2) and are compared with the precursor molecule 1132. The variants evaluated are constituted by the ROR signal sequence (amino acids 1- 29) + the component of IL-13Ral (amino acids 30-346 with Cys46Ser corresponding to 27-343 of SEQ ID NO: 3) + a component of IL-4Ra (amino acids 347-554 with Cys207Ser corresponding to 24-231 of SEQ ID NO: 2) + a multimerization component (IgGl Fc, amino acids 555-784).

TABLE 9 Component array variants It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which results from the present description of the invention.

Claims (30)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. - A fusion (Rl) x- (R2) y-F polypeptide, characterized in that: Rl is chosen from sequences 1-231 and 24-231 of SEQ ID NO: 2, whose sequences may comprise one to ten modifications; R2 is chosen from the sequences SEQ ID NO: 3 and 4, whose sequences may comprise one to three modifications; F is a fusion component, and "x" and "and" are each independently a positive integer > 1.

The fusion polypeptide according to claim 1, characterized in that Rl is modified by substitution of one or more amino acids in the position or positions 67, 68, 71, 152, 164, 171, 172, 175, 198 , I 207 with a different amino acid.

3. The fusion polypeptide according to claim 2, characterized in that the amino acid at position 67 is replaced with Tyr.

4. - The fusion polypeptide according to claim 2, characterized in that the amino acid at position 68 is replaced with Asn.

5. The fusion polypeptide according to claim 2, characterized in that the amino acid at position 171 is replaced with Tyr or Phe.

6. The fusion polypeptide according to claim 2, characterized in that the amino acid at position 172 is replaced with Ser.

7. The fusion polypeptide according to claim 2, characterized in that the amino acid at position 152 is replaced with Phe.

8. - The fusion polypeptide according to claim 2, characterized in that the amino acid at position 198 is replaced with Ser.

9. The fusion polypeptide according to claim 2, characterized in that the amino acid at position 207 is replaces with Ser.

10. The fusion polypeptide according to claim 2, characterized in that Rl is modified by substitutions at positions 67, 68 and 207.

11. The fusion polypeptide according to claim 10, characterized in that the amino acid at position 67 is replaced with Tyr.

12. The fusion polypeptide according to claim 10, characterized in that the amino acid in position 68 is replaced with Asn.

13. The fusion polypeptide according to claim 11, characterized in that the amino acid in position 68 is replaced with Asn.

14. The fusion polypeptide according to claim 10, characterized in that Rl is further modified by substitutions at positions 171 and 172.

15. The fusion polypeptide according to claim 14, characterized in that the amino acid in the position 171 is replaced with Tyr or Phe.

16. The fusion polypeptide according to claim 15, characterized in that the amino acid at position 172 is replaced with Ser.

17. The fusion polypeptide according to claim 14, characterized in that Rl is also modified by a substitution at position 152.

18. The fusion polypeptide according to claim 17, characterized in that the amino acid at position 152 is replaced with Phe.

19. The fusion polypeptide according to any of the preceding claims, characterized in that R2 is selected from the group consisting of (i) 1-343 or 27-343 of SEQ ID NO: 3, and (ii) 1 -343 or 23-343 of SEQ ID NO: 4.

20. The fusion polypeptide according to claim 19, characterized in that (i) is modified (a) by replacing amino acids 1-120 of SEQ ID NO: 3 with amino acids 1-123 of SEQ ID NO: 5; (b) deleting amino acids 338-343 of SEQ ID NO: 3; (c) replacing amino acids 1-26 of SEQ ID NO: 3 with SEQ ID NO: 6, or (d) substituting one or more amino acids at position 46, 73, 143, 235, 293 and / or 329 with a different amino acid.

21. The fusion polypeptide according to claim 19, characterized in that (ii) is modified (a ') by deletion of amino acids 1-22 of SEQ ID NO: 4; (b ') Cys251Ile; or (c ') by substitution of the amino acid of position 310 of SEQ ID NO: 4.

22. The fusion polypeptide according to any of the preceding claims, characterized in that F is selected from the group consisting of a component of multimerization, a serum protein, or a molecule that can bind to a serum protein.

23. The fusion polypeptide according to claim 22, characterized in that F is a multimerization component that is selected from the group consisting of (i) a domain derived from immunoglobulin, (ii) a region susceptible to cutting ( region C), (ii) an amino acid sequence with a length between about 1 to 500 amino acids, optionally comprising at least one cysteine residue, (iii) a leucine zipper, (iv) a helix loop motif, (iv) v) a spiral-spiral motif.

24. The fusion polypeptide according to claim 23, characterized in that F is an immunoglobulin-derived domain that is selected from the group consisting of the Fc domain of IgG or the heavy chain of IgG.

25.- A multimeric protein characterized by porgue comprises two or more of the fusion polypeptides according to any of claims 1-24.

26. The multimeric protein according to claim 25, characterized in that it is a dimer comprising two fusion polypeptides.

27. A nucleic acid molecule characterized in that it encodes the fusion polypeptide according to any of claims 1-24.

28. - A vector characterized in that it comprises the nucleic acid molecule according to claim 27.

29.- A host-vector system characterized in that it comprises the vector according to claim 28, in an appropriate host cell.

30. A method for producing a fusion polypeptide, characterized in that it comprises culturing the host-vector system according to claim 29, under conditions suitable for the expression of the protein from the host cell, and the recovery of the polypeptide produced in this way.