MXPA99000970A - Fusion polypeptides com0prising an ige-binding domain and a hsa component, and their diagnostic and therapeutic uses - Google Patents

Abstract

Fusion polypeptides and salts thereof comprising at least one IgE-binding domain fused to at least one human serum albumin component, optionally via a peptide linker, and in particular, dimeric fusion polypeptides comprising HSA protein fused, at each of its amino and carboxy termini, to an extracellular domain of the&agr;-chain of the human high affinity receptor for IgE (Fc&egr;RI&agr;);process for the preparation thereof, functionally equivalent polypeptides which are intermediates in their preparation, and polynucleotide and oligonucleotide intermediates and vectors therefor. They are indicated for use in the prevention and/or treatment of IgE-mediated allergic diseases and related disorders such as atopic dermatitis, atopic asthma and chronic urticaria.

Description

FUSION POLYPEPTIDES THAT COMPRISE A GE LINK DOMAIN AND A COMPONENT OF HUMAN SOUTHERN ALB MINE. AND ITS DIAGNOSTIC AND THERAPEUTIC USES Field The invention relates to fusion polypeptides. Refers to fusion polypeptides comprising an IgE binding domain and a human serum albumin (HSA) component, and salts thereof. It also relates to polynucleotides and physiologically functional equivalent polypeptides which are intermediates in the preparation of these fusion polypeptides; to appropriate recombinant expression vectors for them, to the corresponding prokaryotic and eukaryotic expression systems, and to processes for synthesizing the fusion polypeptides.

Background The interaction between immunoglobulin E (IgE) and its receptors has an established role in the defense against parasitic infections in humans (M. Capron and A. Capron, Science 264 [1994] 1876-1877). However, in industrialized countries, with better hygiene conditions, the encounter with parasites is less frequent than the alteration of the IgE network due to overproduction of IgE in response to allergens from the environment, resulting in allergies and other conditions. of disease mediated by IgE or by the IgE receptor. IgE is the primary antibody involved in the initiation of an immediate allergic response, and is an important participant in the maintenance of the late phase response. IgE is synthesized in B lymphocytes and exerts its effects after binding to the high affinity receptor for IgE, ie FceRI, which is found on the surface of allergy-effector cells, such as mast cells, basophils , and eosinophils. IgE also exerts inducing functions through its binding to its receptor on antigen presenting cells, such as Langerhans cells, B cells, and monocytes (GC Mudde et al., All rgy 50 [1995] 193-199 ). An allergic response or condition is manifested when the IgE molecules bind to the surface of the allergy effector cells by means of the IgE receptor, FceRI, and become cross-linked by the allergen, thus effecting the signaling to initiate degranulation of the cytoplasmic granules in the cells, with the release accompanied by the allergy mediators, such as histamine, serotonin, prostaglandins and cytokines, and the consequent local tissue edema and the influx of inflammatory cells. An alternative means to stimulate allergy and associated conditions is the interaction of the IgE receptor, FceRI, with the circulating autoantibodies against FceRI. FceRI normally exists as a tetramer comprising a chain a, a chain ß, and two chains? (ie, "subunits"), although on the monocytes and Langerhans cells, the β subunit is absent. It has been shown that the IgE binding site of FceRI is contained entirely within its subunit (referred to as FceRIa (J. Hakimi et al., J. Biol. Chem. 2____5. [1990] 22079-22081; U. Blank et al. , J. Biol. Chem. 266 [1991] 2639-2646) It has been found that recombinant "unconscious" mice which have been genetically deleted from the entire subunit are unable to present an allergic response to the allergen challenge ( D. Dombrowicz et al., Cell _Zü [1993] 969-976) FceRIo is a highly glycosylated polypeptide with a molecular weight of approximately 60 kD, comprising a hydrophobic transmembrane domain, as well as extracellular domains. ("ecto-") and cytoplasmic hydrophilic, which are exposed to the outer surface of the cell. The binding capacity with IgE of FceRIc. it has also been located in its extracellular portion (J. Hakimi et al [1990], supra; Leder et al., United States Patent Number 4,962,035). It is possible to produce a secretable soluble molecule by separating the transmembrane portion and the downstream sequences therefrom (C. Ra et al., Int. Immunol. 5. [1993] 47-54); and the resulting truncation, consisting essentially of the human extracellular domain of FceRIa, has IgE binding activity in vi tro and in vivo (M. Haak-Frendscho et al., Immunol. 151 [1993] 351-358: amino acid residues 1 -204 of FceRIc, fused with a C region of H chain of truncated IgGl, C. Ra. Et al [1993] supra: residues 1-172 of FceRIat thereof, corresponding to residues 26-197 of SEQ ID N0: 1 of the present). The structural features of this fragment include two potential disulfide bridges, and seven potential glycosylation sites (M. Haak-Frendscho et al. [1993], supra). Accordingly, truncations of FceRIc, which essentially consist of the extracellular domain, can potentially be administered in a therapeutic manner to a mammal to bind to serum IgE, in order to prevent its binding to its high affinity receptor on the allergy effector cells, and also to suppress de novo IgE biosynthesis in human lymphocytes (Y. Yanagihara et al., J. Clin.Invest.94 [1994] 2162-2165). However, the effective use of an IgE-binding polypeptide, such as the extracellular domain of FceRIc. for the systemic treatment of allergic disorders mediated by IgE or by the IgE receptor in mammals, it has been hampered by its extreme transience in vivo, due to the rapid release of circulating plasma. Considering that diseases mediated by IgE or by the IgE receptor represents 10 to 20 percent of physician-patient contact, an effective treatment would constitute a significant benefit for patients suffering from these conditions, and an important advance in the treatment of disorders mediated by IgE or by the IgE receptor, such as allergy and allergy-related conditions. Accordingly, it would be beneficial to obtain an IgE-binding polypeptide having an effective and prolonged serum life, and therefore, a better clinical utility in the treatment of allergy, and in particular in the systemic treatment of atopic dermatitis, atopic asthma, and chronic urticaria, as well as a better activity in an efficient and effective way for the cost.

Compendium It has now been found that, by fusion of an IgE binding domain to a human serum albumin (HSA) component, fusion polypeptides having a prolonged serum half life relative to the binding domain of IgE alone, without losing the IgE binding activity, resulting in IgE-binding polypeptides indicated for use in the systemic treatment of allergy and other IgE-mediated disorders. The systemically administered IgE binding polypeptide will bind with serum IgE, as well as circulating autoantibodies, against the IgE receptor, FceRIc., Preventing them from binding to the FceRIa bound to the cell, and thus preventing and / or inhibiting an allergic reaction and its associated manifestations. In addition, it has been found that significant improvements in IgE binding activity can be obtained by utilizing the fusion polypeptides of the invention, which comprise more than one IgE binding domain per molecule. For example, it has been found that a "dimeric" molecule of the invention has a significantly increased IgE binding activity relative to a "monomer" of the invention. The term "dimer" herein refers to a fusion polypeptide of the invention that possesses two IgE binding domains. The term "monomer" refers to a fusion polypeptide of the invention that possesses an Ig ?. The monomer or dimer can be constructed from a single component or from multiple components of human serum albumin. For example, a monomeric fusion polypeptide of the invention may comprise an IgE binding domain fused at its amino or carboxyl terminus, with a component of human serum albumin. Alternatively, the monomer may comprise an IgE binding domain fused in both terms with the components of human serum albumin. A dimeric fusion polypeptide according to the invention may comprise, for example, two IgE binding domains fused via the carboxyl terminus of one, and the amino terminus of the other, with an intervening component of human serum albumin. Alternatively, the dimer may comprise, in addition to its two IgE binding domains, multiple components of human serum albumin. In addition, it has been found that a dimeric molecule of the invention possesses unexpectedly favorable activity. Accordingly, the invention relates to fusion polypeptides and salts thereof, which comprise at least one IgE binding domain fused to at least one component of human serum albumin; preferably, to monomeric fusion polypeptides, wherein a single IgE binding domain is fused to one or more components of human serum albumin, or to multimeric fusion polypeptides, wherein two or more IgE-binding domains are fused with at least one component of human serum albumin; more preferably, the dimers possess two IgE binding domains, and at least one component of human serum albumin. The invention also relates to polynucleotide intermediates therefor. The term "fused" or "fusion" herein refers to polypeptides wherein: (i) a given functional domain (ie, an IgE binding domain) is linked at its carboxyl terminus by a covalent bond (from preferably, peptide, ie amide), either to the amino terminus of another functional domain (i.e., a component of human serum albumin), or to a linker peptide that itself is linked by a covalent bond (preferably peptide) with the amino terminus of the other functional domain; and / or (ii) a given functional domain (i.e., an IgE binding domain) is linked at its amino terminus, by a covalent bond (preferably peptide, ie, amide), either to the carboxyl terminus of another functional domain (ie, a component of human serum albumin), or with a linker peptide that itself is linked by a covalent bond (preferably peptide), with the carboxyl term of the other functional domain. In a similar manner, "fused", when used in relation to the polynucleotide intermediates of the invention, means that the 3 '[or 5'] term of a nucleotide sequence encoding a first functional domain, is linked to the 5 '[or 3'] respective term of a nucleotide sequence encoding a second functional domain, either by a covalent bond, or indirectly by means of a nucleotide linker which itself is covalently linked preferably on its terms with the polynucleotide encoding the first functional domain, and the polynucleotide encoding the second functional domain. For reference, the fusion polypeptide or its salt is a monomer or dimer; when it is a dimer, it comprises two IgE binding domains fused to a human serum albumin component, such as a first IgE binding domain fused at its carboxyl terminus with the amino terminus of a human serum albumin component, and a second IgE binding domain fused at its amino terminus with the carboxyl terminus of that human serum albumin component. The fusion polypeptides of the invention may also comprise additional functional domains, in addition to the IgE binding domains and the human serum albumin components, e.g., full-length or truncated human proteins (e.g., soluble extracellular fragments of the same), such as cytokines or the amino-terminal fragment of urokinase. These additional functional domains themselves may serve as linker peptides, for example, to bind an IgE binding domain with another IgE binding domain, or with a human serum albumin component; in an alternative way, they can be located elsewhere in the fusion molecule, for example, in the amino or carboxyl terms thereof. Examples of the fusion polypeptides of the invention can be represented by the following formulas: I I. R- L I I I L - R- V. R L R-. L - R, where: R? is the amino acid sequence of an IgE binding domain, R2 is an amino acid sequence of a human serum albumin component, each L is independently a covalent bond (preferably peptide), or is a peptide linker that is linked by a covalent bond (preferably peptide) with a term of Rx and / or R2, whereby the above molecule fragments are read directionally, ie, the left side corresponds to the amino terminus, and the right side corresponds to the carboxyl terminus of the molecule. It has been found that IgE binding is achieved at high levels when the fusion polypeptide is driven by an IgE binding domain; that is, when the IgE binding domain constitutes the N-terminal portion of the mature fusion protein (such as in the compounds of formulas I, III, and IV above). A particularly preferred embodiment of the invention comprises a dimer of formula III above, that is, wherein the protein region that leads to expression is an IgE binding domain, fused at its carboxyl terminus with the amino terminus of a component of human serum albumin, which in turn is fused at its carboxyl terminus with the amino terminus of a second IgE binding domain. When there is more than one fraction R1 # or more than one fraction R2, or more than one fraction L, these fractions may be, but not necessarily identical. Preferably, the fusion polypeptides of the invention comprise at least one soluble secretable mammalian IgE binding domain, fused to at least one component of human serum albumin, and pharmaceutically acceptable salts thereof. The invention further relates to a method for the prevention and / or treatment of disorders mediated by IgE or by the IgE receptor, and in particular, to allergic reactions, such as atopic dermatitis, atopic asthma, and chronic urticaria, which comprises administering the fusion polypeptides of the invention, or pharmaceutically acceptable salts thereof, to a subject in need of such treatment; and pharmaceutical compositions comprising the fusion polypeptides of the invention, or pharmaceutically acceptable salts thereof, together with at least one pharmaceutically acceptable carrier or diluent.

The invention further relates to physiologically functional equivalents of the fusion polypeptides of the invention, which are intermediates in the synthesis of the polypeptides; to polynucleotides that are intermediates in the preparation of the fusion polypeptides or their physiologically functional equivalents, as defined above; to oligonucleotide intermediates for use in their construction; to the peptides encoded by these oligonucleotides; to recombinant vectors to express the fusion molecules; to prokaryotic or eukaryotic expression systems (especially mammalian ones), and to processes for the preparation of the fusion polypeptides or their physiologically functional equivalents using these expression systems.

Description of Sequences and Related Definitions SEQ ID NO; 1: Amino acid sequence of the dominant form of FceRIo? full-length native human, including the signal sequence. The term "pre-IgER" refers to the Metx-Leu204 residues of SEQ ID NO: 1. The term "IgER" refers to the mature form of pre-IgER, and constitutes residues Val26-Leu204 of SEQ ID NO: 1 (ie, the extracellular domain of FceRIa).

SEQ ID NO; 2; Amino acid sequence of the dominant form of native human serum prepro-albumin (referred to herein as "prepro HSA I"), which comprises the Met- ^ Leugog residues. The dominant form of the mature native protein (referred to herein as "HSA I") is represented by the residues Asp25-Leu609 of SEQ ID NO: 2. The term "prepro-HSA II" represents a truncation of the native sequence by an amino acid (Leu609) at the carboxyl terminus, and therefore, refers to the Met? -Gly608 residues of SEQ ID NO: 2. The mature form of prepro-HSA II, referred to herein as "HSA II ", is represented by the residues Asp25-Gly608 of SEQ ID NO: 2.

SEQ ID NO; 3; Amino acid sequence encoded by the EcoRI fragment of plasmid R-H-R / SK # 50. prepared in Example 5 comprising: the "pre-IgER" sequence in residues 1-204; the AlaSer linker (Gly4Ser (referred to herein as "LL") at residues 205-211; the sequence of HSA II at residues 212-795; the linker (Gly) 3Ser (hereinafter referred to as "L2" ) at residues 796-799, and the "IgER" sequence at residues 800-978. A mature dimeric fusion polypeptide of the invention, referred to herein as "IgER-L? -HSA II-L2-IgER". , or alternatively as "dimmer? IgER-HSA-IgER", expressed from Chinese hamster ovary cells in the manner described in Example 7, has the amino acid sequence Val2S-Leu978 of SEQ ID NO: 3.

SEQ ID NO: 4: Nucleotide sequence of the EcoRI fragment of plasmid R-H-R / SK # 50 of Example 5, comprising: a polynucleotide sequence encoding "pre-IgER" at positions 10-621; an oligonucleotide that encodes L? in positions 622-642; a polynucleotide encoding HSA II at positions 643-2394; an oligonucleotide encoding L2 at positions 2395-2406; and a polynucleotide encoding "IgER" at positions 2407-2943; with 2 stop codons at positions 2944-2949. The restriction sites at the ends of the coding proteins and in the linker regions are in positions 1-6; 622-627; 637-642; 2387-2393; 2401-2406; and 2950-2955. A Kozak sequence is at nucleotide positions 7-9. Point mutations different from the consensus HSA nucleotide sequence are at positions 804; 1239; 1290; 1446; 1815; 2064, and 2079. Because point mutations are in the wavy position, they do not affect the amino acid sequence.

SEQ ID NO: 5; Nucleotide sequence of the dominant form of native prepro-HSA, corresponding to Figure 12, and to the amino acid sequence of SEQ ID NO: 2.

SEQ ID NO; 6: Nucleotide sequence of the dominant form of FcßRIc. full length native human, including the signal sequence, corresponding to Figure 13, and to the amino acid sequence of SEQ ID NO: 1.

Description of the Figures In the following Figures, the indicated molecules are read directionally, that is, the left side corresponds to the amino terminus (or 5 '), and the right side corresponds to the carboxyl terminus (or 3').

FIGURES 1A-C: Serum half-life in mice: Concentration of protein in vivo (in picomoles of protein per milliliter of serum) over time (10 to 800 minutes after injection) of: (A) protein HSA I free, and (B) IgER protein (referred to as "free alpha chain"), and dimeric fusion polypeptide IgER - ¡1 - HSA II - L2 - IgER (referred to as dimer "IgE 7.R-HSA-IgE R,"?), prepared from Chinese hamster ovary cells, as described in Example 7.

(C) serum half life of free HSA I from: (A) compared to that of the dimeric fusion polypeptide ("IgE PR? - HSA - IgEpR?"), From (B) by normalization to 1 with respect to serum concentrations at 10 minutes after injection.

FIGURE 2; Extravasculation resulting from the passive cutaneous anaphylaxis reaction in mice given intravenous serial dilutions (10 micrograms / kilogram, 50 micrograms / kilogram, or 500 micrograms / kilogram) of the IgER-L polypeptide? - HSA II-L2-IgER ("IgER-HSA-IgER dimer"), prepared as described in Example 7 (the control group received 0 micrograms / kilogram, area in square millimeters, at intervals of 5, 15, or 30 minutes before sensitization by intradermal injection of anti-dinitrophenyl mouse IgE monoclonal antibody (DNP), and subsequent stimulation with a solution of DNP-bovine serum albumin containing 1 percent Evans blue.

FIGURE 3; Schematic representation of three fusion polypeptides of the invention (two monomers and one dimer), the polypeptide linkers also being shown: I. Monomers: (1.) Leading monomer of HSA comprising HSA II (referred to in the Figure as "HSA") fused by means of L2 ("GGGS") with IgER. The nucleotides encoding the Leu607Glyg0g positions of HSA II contain a single MstII site as indicated, and the nucleotides encoding GlySer of L2 contain a BamH1 site (the encoded amino acids are underlined); (2.) IgE binding domain leader monomer comprising IgER fused at its carboxyl terminus, by means of L-L (ie, "ASGGGGS"), with HSA II (referred to in the Figure as "HSA"). The oligonucleotide encoding Lx contains an Nhel site and a BamH1 site, respectively (the AlaSer and GlySer encoded amino acids of L-L are underlined). II. Dimer: IgE binding domain leader dimer comprising a first IgE fused at its carboxyl terminus by means of LL, with the amino terminus of HSA II (referred to in the Figure as "HSA"), whose carboxyl terminus is fused with a second IgE by means of L2, with restriction sites in the encoding polynucleotide, as described above for the monomer.

FIGURE 4; Polymerase chain reaction primers for truncating the FceRIo cDNA? Full length human (referred to as the "IgE receptor cDNA"), to obtain DNA encoding: (i) IgE? [The BamHI site is added to the 5 'end of the coding strand for FceRIce by oligonucleotide # 18; and the stop codon and the EcoRI and SalI sites are added to the 3 'end of the non-coding strand, by oligonucleotide # 19]; (ii) pre-IgER [Oligonucleotide # 20 adds the SstI, EcoRI, and Kozak sites to the 5 'end of the coding strand for FceRIc; Oligonucleotide # 31 adds Notl and Nhel (and deletes a second Nhel site) in the non-coding strand for FceRIc.]; (iii) pre-IgER [Oligonucleotides # 20 and # 19 are used as described above]: (A.) "Leader of HSA": subcloning of (i) above into the vector SK, providing the clone pEKl of the vector of TA cloning in the construction of the leader monomer of HSA II; (B.) "IgER leader": subcloning of (ii) into the SK vector, providing the IgER / TA # l construct used to prepare the IgER leader monomer; (C.) "IgER alone": subcloning of (iii) into the SK vector, providing the IgER FL / TA # 34 construct used in the expression of mature IgER, to be used as a standard. The double asterisks represent two stop codons.

FIGURE 5; Pairs of polymerase chain reaction primers to truncate the full-length human prepro-HSA I cDNA to produce cDNA encoding: (i) prepro-HSA II fused at the carboxyl terminus with L2 [oligonucleotides # 24 adding the Spel sequence, EcoRI, and Kozak at the 5 'end of the coding chain; Oligonucleotides # 28 and # 29 that add a linker encoding the MstII and HindIII sites in the 3 'terminus of HSA II]; (ii) L? fused with the 5 'terminus of HSA II [Oligonucleotide # 26 which adds Notl sites, Nhel, linker, and BamHl to the coding chain; Oligonucleotide # 27 containing the Ncol site in the non-coding strand]; (iii) prepro-HSA I [Oligonucleotide # 24 used as above; and Oligonucleotide # 25 that adds the stopping sites, EcoRI and HindIII to the 3 'end of the non-coding strand]: (A.) "HSA leader": subcloning (i) into the SK vector, providing pEK7 used for build the leading monomer of HSA II; (B.) "IgER leader": subcloning of (ii) into the SK vector, providing HSA / SK # 5 used to construct the IgER leader monomer; (C.) "HSA alone": subcloning (iii) into the SK vector, providing HSA / SK # 17 which encodes the mature native human serum albumin protein (i.e., SA I) to be used as a standard.

FIGURE 6; Human serum albumin sequencing oligonucleotides (HSA) # 1-10, 12, 16, 17, 22, and 30, used to sequence materials cloned in TA, and after binding to the IgE binding fragments.

FIGURE 7; IgE receptor sequencing oligonucleotides # 11, 13, and 23, used to sequence the fragments cloned in TA, and after binding to the HSA component.

FIGURE 8; (A) Mutagenic oligonucleotides # 14 and # 15 for human serum albumin; (B) Polymerase chain reaction and. linker oligonucleotides # 18-20, 24-29 and 31, to construct fusion polypeptides.

FIGURE 9; Construction of the vector HSA-IgER / SK # 49, comprising a polynucleotide encoding prepro-HSA II (referred to in the Figure as "HSA"), fused at the 3 'terminus by means of the oligonucleotide encoding the linker L2 (depicted in Figure 9 by "GGG"), with the 5 'terminus of the polynucleotide encoding IgER (referred to in the Figure as "IgER"), by ligating the BamHI fragment, leaving pEKl in pEK7 cut with Ba Hl. I left .

FIGURE 10: Construction of IgER-HSA / SK # 1 vector, comprising a polynucleotide encoding pre-IgER (referred to in the Figure as "IgER") fused at the 3 'terminus, by means of the oligonucleotide for linker L? (represented by "GGG"), with the 5 'terminus of the polynucleotide encoding HSA II ("HSA"), by ligation of the Sstl, Nhel fragment of IgER / TA # l in HSA / SK # 5 cut by Sstl, Nhel.

FIGURE 11: Construction of the HSA-IgER Pst Sal / SK # 37 vector, comprising a polynucleotide encoding HSA II (indicated by "HSA3"), which is fused at the 3 'terminus via the oligonucleotide for L2 (not shown) , with the 5 'terminus of the polynucleotide encoding IgER (indicated as "IgER"), by ligation of the pStl.SalI fragment of HSA-IgER / SK # 49 in the vector SK. Also shown is the construction of a RHR / SK # 50 vector comprising a polynucleotide encoding pre-IgER (referred to as "IgER") fused at its 3 'terminus, by means of the oligonucleotide for LL (not shown), with the term 5 'of the polynucleotide encoding HSA II ("HSA"), which in turn is fused by means of the oligonucleotide for L2 (not shown), with a polynucleotide encoding IgER ("IgER"). The vector is prepared by ligating the PstI.Kpnl fragment of HSA-IgER Pst Sal / SK # 37 in IgER-HSA / SK # l cut with PstI.Kpnl.

FIGURE 12: Nucleotide sequence of human serum albumin, and amino acid sequence, corresponding to SEQ ID NO: 2 and SEQ ID NO: 5.

FIGURE 13: IgER nucleotide sequence, and amino acid sequence corresponding to SEQ ID NO: 1 and SEQ ID NO: 6.

FIGURE 14: Nucleotide and amino acid sequence of the EcoRI fragment of R-H-R / SK # 50, corresponding to SEQ ID NO: 3 and SEQ ID NO: 4, which encode the dimeric fusion protein Rl-HSA-Rl. The HSA sequence is shown in italics. Linker sequences are shown in lowercase. Point mutations different from the consensus HSA nucleic acid sequence are shown in tiny bold letters. Because the point mutations are in the wavy position, they do not affect the amino acid sequence. The restriction sites at the ends of the fragments and in the linker region are underlined.

FIGURE 15: SDS-PAGE of the purified mature fusion polypeptide of Example 7: Total amounts applied to the gel: 8 micrograms (lane 1), 6 micrograms (lane 2), 4 micrograms (lane 3), and 2 micrograms (lane 4); molecular weight standards: 97.4, 66.2, 45, 31, 21.5, and 14.4 kDa (lane 5).

Detailed Description The invention relates to fusion polypeptides and salts thereof, which comprise at least one IgE binding domain fused to at least one HSA component. The term "IgE binding domain" refers to an amino acid sequence that can be linked or otherwise associated with mammalian IgE, eg, human, in a manner that prevents the binding of IgE to its receptor, FceRI. The cDNA and the deduced amino acid sequence of FceRIc. human are known (J. Kochan et al., Nucleic Acids Research 16 [1988] 3584; and Leder et al., United States Patent Number 4,962,035). The FceRio. human encodes a signal peptide with NH2 [amino acid residues (including the first Met) Met-L-Ala25], two extracellular immunoglobulin-like domains (residues Val26-Leu204), a hydrophobic transmembrane region (Gln205-Ile224), and a hydrophilic cytoplasmic tail (residues Ser225-Asn257) (with reference to SEQ ID NO: 1). The signal peptide is dissociated during intracellular processing. The full-length amino acid sequence of the dominant form of FceRIc. native human is included herein as SEQ ID NO: l. "IgER" refers herein to the amino acid sequence Val26-Leu204 of SEQ ID NO: 1 (which encodes the extracellular domain); and the term "pre-IgER" refers to residues Met1-Leu204 of SEQ ID NO: 1 (which encodes the signal sequence upstream of the extracellular domain). The IgE binding domain is, for example, an amino acid sequence having at least 80 percent, more preferably at least 85 percent, most preferably at least 95 percent, and still more preferably at least 99 percent. percent homology with IgER as defined above (homology is calculated as the percent identity between the native sequence and the altered sequence, as a function of the total sequence length of the native molecule, after aligning the sequences and introducing hollows, without need, to reach the maximum percentage of sequence identity). In a subgroup of fusion polypeptides of the invention, the IgE binding domain component is: (Xa) wherein (Xa) is: (a) IgER; or (b) an allele that occurs naturally from IgE, or (c) a truncation of (a) or (b) at the carboxyl terminus by 1-12 (for example 1-10, 1-7, or 1-4) amino acids; or (d) a variant of (a), (b), or (c). "Variant" means: the sequence of (a), (b), or (c) as modified by one or more deletions (which are different from the truncations at the carboxyl terminus) of up to 10 (for example 1-7 or 1-5) amino acids; - insertions of a total of up to 10 (for example 1-5) amino acids internally in the amino acid sequence, or up to a total of 100 amino acids in any term; or conservative substitutions of a total of up to 15 (for example 1-5) amino acids. The IgE binding domain is more preferably: (Xb) where (Xb) is: (a) IgER; (b) truncation of IgER at the carboxyl terminus by 1-7 amino acids; or (c) a variant of (a) or (b). The IgE binding domain is still more preferably: (Xc) where (Xc) is: (a) IgER; or (b) truncation of IgER at the carboxyl terminus by 1-7 amino acids (eg, the Val26-Ala197 sequence with reference to SEQ ID NO: 1). The IgE binding domain is more preferably igER. Preferably, any homologue, truncation, or variant, is prepared in a recombinant manner as a "secretable" polypeptide, i.e., the sequence of the mature peptide encoding the IgE binding domain can be secreted from the host cell where it is synthesize it (or a precursor form, such as a pre-form) from a polynucleotide. The other major constituent of the recombinant fusions of the invention, human serum albumin (HSA), constitutes the most abundant plasma protein, contributing 60 percent, on a weight basis, of the total protein content of the plasma. A human serum albumin molecule consists of a single non-glycosylated polypeptide chain of 585 amino acids with a molecular weight of 66.5 kDa. A specific feature of albumin is its complex disulfide bond pattern (F.F. Clerc et al., J. Chromatogr., 662 [1994] 245-259). Human serum albumin is widely distributed throughout the body, particularly in the intestinal and blood compartments where it is mainly involved, as the most abundant protein in serum, in the maintenance of osmolarity and plasma volume. In addition, it is released slowly by the liver, and exhibits a half-life in vivo of 14 to 20 days in humans (T. A. Waldamann, "Albumin Structure, Function and Uses", Pergamon Press [1977] 255-275). Human serum albumin has no enzymatic or immunological function. It is a natural carrier involved in endogenous transport and the supply of different natural as well as therapeutic molecules (H. Lu et al, FEBS Lett 356 [1994] 56-59, Patents Numbers WO 93/15199, EP 648499, P. Yeh and collaborators, PNAS USA 89 [1992] 1904-1908; EP 413622). Human cells synthesize serum albumin initially in the form of a prepro-polypeptide. A signal sequence of 18 amino acids (including the first Met) is removed when the protein passes through the lumen of the endoplasmic reticulum, still leaving 6 amino acids in the N-terminus (in the predominant form: Arg-Gly-Val-Phe-Arg) -Arg), which are then subjected to proteolytic separation during or immediately following transport through the secretory apparatus. It is well known that human serum albumin is polymorphic (D.C. Carter and J.X. Ho, Adv. Prot. Chem. 45 [1994] 153-203). For example, Naskapi albumin has Lys372 in place of Glu372, and pro-albumin Christchurch has an altered pro-sequence, ie, Glu24 in place of Arg24 (Latta et al., U.S. Patent No. 5,100,784). The complete amino acid sequence of the dominant form of the naturally occurring protein, referred to herein as "prepro-HSA I", is known (A. Dugaiczyk et al., PNAS USA 79 [1982] 71-75) (SEQ. ID NO: 2). The dominant form of the mature protein ("HSA I") is represented by Asp25-Leu609 of SEQ ID NO: 2. The HSA carrier of the fusion polypeptide of the invention can be an amino acid sequence having at least 80 percent, more preferably at least 85 percent, still more preferably at least 95 percent, or most preferably at least 99 percent homology with residues 25-609 of SEQ ID NO: 2 (ie, HSA I) (the homology is calculated as the percent identity between the native sequence and the altered sequence, as a function of the total sequence length of the native molecule, after aligning the hollow-insertion sequences, if necessary, to reach the maximum percentage of sequence identity). In a subgroup of fusion polypeptides of the invention, the HSA component is: (Ya) wherein (Ya) is: (a) HSA I; or (b) an allele that occurs naturally from (a); or (c) a truncation of (a) or (b) at the carboxyl terminus thereof by 1-10 (eg, 1-5, or 1 or 2) amino acids), - or (d) a truncation of (a) ending at amino acid residue n, where n is from 369 to 419, and especially where n is 373, 387, 388, 389, 390, and 407 (as described in U.S. Pat. 5,380,712); (e) a variant of (a), (b), (c), or (d). "Variant" means: the sequence of (a), (b), (c), or (d) modified by one or more deletions (which are different from truncations at the carboxyl terminus) of up to 10 (for example 1-7 or 1-5) amino acids; - insertions of a total of up to 10 (for example 1-5) amino acids internally in the sequence, or up to a total of 100 amino acids in any term; or conservative substitutions of a total of up to 15 (for example 1-5) amino acids. The HSA component is more preferably (Yb) where (Yb) is: (a) HSA I; or (b) an allele that occurs naturally from (a); or (c) a truncation of (a) or (b) at the carboxyl terminus thereof by 1-10 (for example 1-5, or 1 or 2) amino acids; or (d) a variant of (a), (b), or (c). The HSA component is still more preferably: (Ye) where (Ye) is: (a) HSA I; or (b) a truncation of HSA I at the carboxyl terminus thereof by 1-10 (for example 1) amino acids (an example of this truncation is "HSA II", which is the amino acid sequence represented by Asp25-GlyS08 of the SEQ ID NO: 2). In a still more preferred subgroup, the HSA component is HSA I or HSA II, particularly HSA II. Especially preferred is the fusion polypeptide of Example 7, especially without leader sequence, ie, the Val26-Leu978 polypeptide of SEQ ID NO: 3. Preferably, any homologue, truncation, or variant of the native HSA that is used for preparing a fusion protein of the invention will have no enzymatic function; and will not prevent or inhibit the binding of the IgE binding domain to serum IgE. In addition, it is preferred that any homologue or variant have a serum half-life of at least 14 days. With respect to the IgE binding domain or the HSA component, the term "conservative substitutions" refers to the replacement of one or more amino acids by others having similar properties, such that one would expect at least the secondary structure, and preferably the tertiary structure of the polypeptide will be substantially unchanged. For example, typical substitutions include alanine or valine for glycine, asparagine for glutamine, serine for threonine, and arginine for lysine. All amino acids (with the exception of glycine) are preferably L-amino acids that occur naturally. Preferably, each IgE binding domain is at least 95 percent homologous with IgER, and each component of HSA is at least 95 percent homologous with HSA I. In other preferred subgroups: each IgE binding domain is preferably (Xa), and each HSA component is (Ya), or more preferably (Yb), or even more preferably (Ye), as defined above; each IgE binding domain is more preferably (Xb), and each HSA component is (Ya), or more preferably (Yb), or even more preferably (Ye), as defined above; each IgE binding domain is still more preferably (Ye), and each component of HSA is (Ya), or more preferably (Yb), or even more preferably (Ye). In a further preference, the IgE binding domain is IgER, and the HSA component is (Ya), or more preferably (Yb), or even more preferably (Ye), as defined above, and is most preferably HSA I or HSA II, in particular HSA II. The preferences expressed above also apply in particular to the polypeptides of each of the formulas I, II, III, IV, and V of the present. Preferred polypeptides of the invention are those comprising a first IgER molecule that is fused, by its carboxyl terminus, with the amino terminus of an HSA II molecule, whose HSA is fused, at its carboxyl terminus, with the amino terminus of a second IgER molecule (IgER being the residues Val2g-Leu204 of SEQ ID NO: 1; HSA II residues Asp25-Gly608 of SEQ ID NO: 2). Particularly preferred polypeptides of the invention comprise dimers of formula III above, wherein each R? is IgE, and R2 is HSA II. Especially preferred are the dimers of formula III, wherein each L is from 1 to 25 amino acids. Any peptide linker (expressed as "L" in the formulas I-V of the present) preferably allows a fold and activity independent of the IgE binding domain; it is free from propensity to develop an ordered secondary structure, which could interfere with the IgE binding domain, or cause an immunological reaction in the patient, and has minimal hydrophobic or charge characteristics that could interact with the IgE binding domain. The peptide linker is preferably from 1 to 500 amino acids; more preferably from 1 to 250; and still more preferably from 1 to 100 (eg, from 1 to 25, from 1 to 10, from 1 to 7, or from 1 to 4) amino acids. The linker is preferably linear, ie unbranched. In general, peptide linkers comprising Gly, Ala, and Ser can be expected to satisfy the criteria for a linker. For example, the linkers of the present invention include: GlyGlyGlySer ("L-," in the present), and AlaSerGlyGlyGlyGlySer ("L2" of the present). Linkers of other different lengths and sequence composition can also be used. The invention also includes oligonucleotides that encode the peptide linkers of the invention. These oligonucleotides must be "fused in frame" with the polynucleotides encoding the IgE binding domain and the HSA component, and preferably include unique restriction sites in the molecule. "Fused in frame" means that: (1) there is no change in the reading frame of the IgE binding domain or the HSA component caused by the linker oligonucleotide; and (2) there is no translation termination between the reading frames of the IgE binding domain and the HSA component. The invention further encompasses physiologically functional equivalents of novel fusion polypeptides that are typically intermediates in the synthesis of novel polypeptides. The term "physiologically functional equivalent" preferably refers to a larger molecule comprising the fusion polypeptide of the invention to which the amino acid sequence has been added, as is necessary or desirable for the effective expression and secretion of the mature recombinant fusion of the invention from a particular host cell. This aggregated sequence is usually at the amino terminus of the mature polypeptide, and usually constitutes a leader (ie, signal) sequence which serves to direct them towards the secretory pathway, and normally dissociates after or prior to secretion of the polypeptide from the cell . The signal sequence can be derived from the natural N-terminal region of the relevant polypeptide, or it can be obtained from host genes that encode secreted proteins, or it can be derived from any sequence known to increase secretion of the polypeptide of interest, including synthetic sequences and all combinations between a "pre" region and a "pro" region. The binding between the signal sequence and the sequence encoding the mature polypeptide, must correspond to a dissociation site in the host. In fusion polypeptides wherein an IgE binding domain "drives" the expression, i.e., is upstream from other coding sequences in the fusion molecule, it is convenient to use the leader sequence of FceRIc. native (ie, Met? + Ala2-Ala25 of SEQ ID NO: 1), and this has been effectively employed to obtain the mature polypeptide from mammalian expression systems (eg, CHO, COS), as well as from yeast (Pichia pastoris). An example of a physiologically functional equivalent of the mature dimeric fusion polypeptide IgER-L? - HSA II - L2 - IgER is: pre-IgER - L? - HSA II - L2 - IgER. However, the additional leader sequence is not necessarily that of FceRIc. human, and can be obtained from any suitable source, provided it is suitable for effecting the expression / secretion of the mature polypeptide from the particular host cell. For example, the HSA prepro-sequence can also be used to prepare the above dimeric fusion polypeptides, particularly for their expression in yeast. In the fusion polypeptides of the invention wherein an HSA component drives expression, a suitable leader sequence can comprise the native leader sequences. For example, the native prepro region of HSA can be used to perform the secretion of the mature heterologous polypeptide from mammalian (eg, CHO, COS) or yeast (Pichia pastoris) cells. An example of a physiologically functional equivalent of the mature fusion polypeptide represented by HSA II-L2-IgER is: prepro HSA II-L2-IgER. However, other leader sequences, not necessarily native to HSA or host cell, can provide effective expression of the mature fusion protein in certain hosts (US Patents USP No. 5,100,784; USP 5,330,901). The invention also comprises polynucleotides that are intermediates in the preparation of the recombinant fusion polypeptides of the invention, including polynucleotides that encode the fusion polypeptides or physiologically functional equivalents thereof, and oligonucleotides that encode linker peptides, for example as illustrated in Figure 8. As a further aspect, there is provided a process for the preparation of a recombinant fusion polypeptide as defined above, or a salt thereof, which comprises: (a) transforming a host cell with a vector comprising DNA encoding a fusion polypeptide as defined above, or a physiologically functional equivalent thereof. (b) expressing the fusion polypeptide or its physiologically functional equivalent in that cell, whereby the physiologically equivalent functional polypeptide is modified (e.g., by dissociating a signal sequence) to produce a fusion polypeptide as defined previously; and (c) recovering the resulting polypeptide from the host cell, preferably as a secreted product, optionally in the form of a salt thereof.

Yet a further aspect of the present invention provides vectors, preferably plasmids, for use in the expression of fusion polypeptides. These vectors comprise DNA encoding the polynucleotides defined above, or physiologically functional equivalents thereof. In general, appropriate vectors that can transform microorganisms capable of expressing the fusion polypeptides include expression vectors comprising nucleotide sequences encoding the fusion polypeptides linked with transcriptional and translational regulatory sequences, such as promoters, which together constitute an expression cassette. The promoters can be derived from genes of the particular host used, or these control regions can be modified, for example, by site-directed mutagenesis in vi tro, by the introduction of additional control elements or synthetic sequences. The expression cassette specifically used in the present invention, therefore, also includes a transcription and translation termination region that is functional in the intended host, and which is positioned at the 3 'end of the sequence encoding the hybrid macromolecule. In addition to the expression cassette, the vector will include one or more markers that make it possible for the transformed host to be selected. These markers include markers that confer resistance to antibiotics, such as G418. These resistance genes will be placed under the control of the appropriate transcription and translation signals, which allow expression in a given host. More particularly, the preparation of the recombinant fusion polypeptides of the invention can be carried out, for example, as follows: A. Construction of Expression Vectors of the Fusion Protein The first step in the construction of the recombinant fusion polypeptides is to subclone portions of the fusion polypeptides into cloning vectors. In this context, a "cloning vector" is a DNA molecule, such as a plasmid, cosmid, or bacteriophage, that can replicate autonomously in a prokaryotic host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites where foreign DNA sequences can be inserted in a determinable manner without losing an essential biological function of the vector, as well as a marker gene which is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide resistance to tetracycline or resistance to ampicillin. Suitable cloning vectors are described in J. Sambrook et al. (Eds), Molecular Cloning: A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press [1989]), and can be obtained, for example, from different sources. The clone pGEM3-110B-l of the FceRIx cDNA is described in A. Shimizu et al., Proc. Nat. Acad. Sci. USA £ 5. [1988] 1907-1911, and can be obtained from the American Type Tissue Collection (ATCC, supply # 67566). The single-stranded human liver cDNA can be obtained from Clontech (Quick Clone cDNA ready for polymerase chain reaction, Cat. D # 7113-l). The sequence of the HSA is available in GenBank under the access numbers: V00495, J00078, L00132, L00133. The HSA cDNA can be obtained by amplification with polymerase chain reaction using the oligonucleotides numbers 24 and 25, as described in Example 2. A polynucleotide encoding a suitable IgE binding domain or a component DNA can be prepared. of HSA using the polymerase chain reaction (PCR). The polymerase chain reaction uses a single-stranded cDNA template, and a mixture of oligonucleotide primers. The procedure of the polymerase chain reaction is carried out by means of a well-known methodology (see, for example, C.M.R. Bangham, "The Polymerase Chain Reaction: Getting Started" in Protocols in Human Molecular Genetics, Human Press [1991], Chapter I, pages 1-8). The polymerase chain reaction kits and the material to be used in the kits are also commercially available in different sources. The kits and methods for using them are further described, for example, in U.S. Patent No. 5,487,993). DNA sequences encoding signal peptides can be added by polymerase chain reaction, or if necessary, using synthetic oligonucleotides that encode known signal peptide sequences. The DNA sequences encoding a heterologous signal peptide are subcloned in frame with DNA sequences encoding the N terminus of the fusion polypeptide. The fusion of the polynucleotides can be carried out by subcloning into intermediate vectors. Alternatively, a gene can be cloned directly into a vector that contains the other gene. Linkers and adapters can be used to join the DNA sequences. The subcloning is performed according to conventional techniques, such as by the use of restriction enzyme digestion, to provide appropriate terms, the use of an alkaline phosphatase treatment to avoid undesirable binding of DNA molecules, and ligation with appropriate ligases. The techniques for this manipulation are described in the literature and are known in this field.

B__ Cloning of Expression of Fusion Polypeptides Then the cloned fusion protein is dissociated from the cloning vector, and inserted into an expression vector. Suitable expression vectors typically contain: (1) prokaryotic DNA elements that encode a bacterial replication origin and a resistance marker to the antibiotic to provide growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control the initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of the transcripts, such as a transcription termination / polyadenylation sequence. Accordingly, another aspect of the present invention relates to vectors, preferably plasmid vectors, for use in the expression of the fusion polypeptides of the invention, which contain the polynucleotide sequences described herein, which encode the polypeptides of fusion of the invention. Suitable expression vectors that can transform prokaryotic or eukaryotic cells include expression vectors comprising nucleotide sequences encoding the fusion molecules linked to transcriptional and translational regulatory sequences, which are selected according to the host cells used. For expression in E. coli, vectors such as those described by M.W. Robertson, J. Mol. Chem. 268 [1993] 12736-12743. The product is expected to be bound by disulfide, but not glycosylated. For expression in yeast, an expression vector, such as pHIL-D2 supplied with the Pichia pastoris Invitrogen expression kit (San Diego, CA, USA) (catalog # K1710-01) can be used. The protein product is expected to be linked by disulfide and glycosylated. Other suitable yeast expression systems include Saccharomyces cerevisiae and Kluveromyces lactis. For expression in baculovirus, vectors, such as pAC360, pVL1392, and pVL1393 (Invitrogen, San Diego, CA, USA) are useful for infection of insect cells, which would be expected to secrete a glycosylated and disulfide linked product. The fusion polypeptide of the invention is usually a glycoprotein, particularly when expressed in mammalian cells, and the invention includes fusion polypeptides in any glycosylation or disulfide bridge state. In particular, mutation analysis suggests that glycosylation linked to N in the first, second, and seventh positions of the glycosylation sites linked to L of the o chain. of the IgE receptor (a.Shimizu et al., PNAS USA 85 [1988] 1907-1911, Figure 2) (which corresponds to amino acid residues 46, 67, and 191 of SEQ ID NO: 1), promotes activity of the IgER molecule, and the monomers and dimers that comprise it. The most preferred expression system is one wherein any added sugars will be more similar to those of the native molecule from which the polypeptide is derived. It is known that yeast and insect cells modify glycoproteins differently from mammalian cells, whereas E. coli does not add sugar molecules after secretion. Accordingly, although expression from any of these expression systems can produce a protein product that is useful for diagnostic applications (e.g., detection of an allergic condition), the most preferred way to express this product for use as a therapeutic molecule, it is the expression in mammalian cells, or possibly expression in the milk of transgenic mammals. For expression in a mammalian host, transcriptional and translational regulatory signals used in an expression cassette can be derived from viral sources, such as adenovirus, bovine papilloma virus or simian virus, in which the regulatory signals are associated with a particular gene that has a high level of expression. Suitable transcriptional and translational regulatory sequences can also be obtained from mammalian genes, such as the actin, collagen, myosin, and metallothionein genes. Transcriptional regulatory sequences include a sufficient promoter region to direct the initiation of RNA synthesis in a mammalian cell. Examples of typical mammalian cells are Chinese hamster ovary (CHO) cells, monkey kidney cells transformed with SV40 (COS), mouse embryo cells HeLa, BHK, Swiss NIH, rat fibroblasts, of monkey, or humans, or rat hepatoma cells. For expression in mammalian cells, the preferred method is secretion from Chinese hamster ovary cells. Neither Chinese hamster ovary cells nor human cells add the galactose residues linked with c.1,3- which are typical of expression in murine cells, such as C127 and SP2 / 0 cells. Antibodies to this sugar bond are present in human serum [C.F. Goochee et al., BioTechnol. 3. [1991] 1347-1355], and may affect the half-life, accessibility, and release of recombinant products expressed from these murine cells. Chinese hamster cells, dhfr cells, are mutants for dihydrofolate reductase (DHFR), and therefore, are unable to synthesize purines, thymidine, and de novo glycine. The number of copies of the chromosomally integrated plasmids carrying wild type copies of the dihydrofolate reductase gene can be increased or amplified, exposing the transformed cells to these plasmids, in order to increase the levels of methotrexate, an analogue of folate that competes for the folate link in the active site of the enzyme. A suitable vector for expression in Chinese hamster ovary cells, dhfr cells, is pMT2 (R. J. Kaufman et al. EMBO J. £ [1987] 187-193). The pMT2 vector has a wild-type copy of the dihydrofolate reductase gene that is transcribed as a single mRNA with the foreign gene. Accordingly, on the treatment of Chinese hamster ovary cells, the transformed dhfr cells, with increasing concentrations of methotrexate, co-amplify both the foreign gene and the dihydrofolate reductase gene. It is expected that the secreted product of this cell is disulfide and glycosylated in a mammalian pattern. An expression vector can be introduced into host cells using a variety of techniques, including calcium phosphate transfection, liposome-mediated transfection, electroincorporation, and the like. Preferably, transfected cells are selected and propagated, wherein the expression vector is stably integrated into the genome of the host cell to produce stable transformants. The cells can be cultured, for example, in a DMEM medium. The polypeptide secreted in the medium can be recovered by conventional biochemical approaches, followed by transient expression 24 to 72 hours after transfection of the cells, or after the establishment of stable cell lines following selection, for example. , resistance to antibiotics. Conventional methods for recovering a polypeptide expressed from a culture include fractionation of the portion containing the polypeptide from the culture, using well-known biochemical techniques. For example, gel filtration, gel chromatography, ultrafiltration, electrophoresis, ion exchange, or affinity chromatography methods, such as are known for protein fractionation, can be used to isolate the expressed proteins found. in the crop. In addition, conventional immunochemical methods can be performed, such as immunoaffinity or immunosorption. The techniques for transformation, cultivation, amplification, selection, and production and purification of the product are also well known (see, for example, J. Sambrook et al.

[1989] = lipra; R.J. Kaufman, Genetic Engineering: Principies and Methods, Plenum Press, Volume 2. [1987] 156-198). The fusion polypeptides of the invention are indicated to be used therapeutically for the purpose of treating mammalian patients, and in particular humans, suffering from allergies. The IgE binding domain of the fusion polypeptides competes for IgE with the IgE receptor naturally present in the mast cells, in the basophils, and in the Langerhans cells, so that the IgE binds to the protein administered, and is unable to bind to these allergy effector cells to mediate the allergic response. The IgE binding domain also competes with the IgE receptor, FceRI, by its binding to auto-antibodies to FceRI. Accordingly, the present invention provides a pharmaceutical composition for competitively binding IgE (and / or antibodies to FceRI, and / or inhibiting the production of IgE, and consequently, for the suppressive and / or preventive treatment of IgE-mediated or by the IgE receptor, and more specifically, the allergy and conditions associated with allergy, such as atopic dermatitis, atopic asthma, and chronic urticaria. "IgE or IgE receptor-mediated disorders" means the disorders associated with the binding of the IgE receptor bound to the cell, FceRI, IgE, or auto-antibodies to FceRI, for example, allergic-type reactions ("hyper-IgE syndrome"), such as bronchial asthma, atopic asthma, fever of hay, allergy to pollen, allergic rhinitis, atopic dermatitis, eczema, anaphylaxis, as well as chronic urticaria, and also non-allergic Kimura disease, and other pulmonary, dermatological, or autologous diseases In particular, atopic dermatitis, one of the most dramatic manifestations of atopy, is a chronic inflammatory skin disease associated with high levels of serum IgE, and sensitization to different allergens in the environment (M.-A , Morren et al., J. Am. Acad. Dermatol. 31 [1994] 467-473). Current treatment of atopic dermatitis concentrates on the use of steroid-containing creams, and severe cases of atopic dermatitis have been successfully treated with cyclosporin A (H. Granlund et al., Br. J. Dermatol. 132 [1995] 106-112), but side effects restrict this treatment to a minority of patients. An agent that inhibits IgE functions, such as degranulation of mast cells and the presentation of IgE-mediated antigen by B cells and other antigen-presenting cells, would be superior to any currently known treatment for atopic dermatitis, and in addition , it would also be useful for other lighter forms of allergy. In addition to atopic dermatitis and atopic asthma, the polypeptides of the invention can be used to treat or prevent chronic urticaria (CU), where the degranulation of mast cells through the activation of FceRIa has a role. The fusion polypeptides of the invention can release the circulating autoantibodies against FceRIc, in contrast to the anti-IgE monoclonal antibodies alternatively proposed for the treatment of the disease. The polypeptides may be administered in the form of a pharmaceutical composition comprising a polypeptide of the invention, preferably an unmodified polypeptide, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier or diluent. The term "salt" refers in particular to pharmaceutically acceptable salts prepared from pharmaceutically acceptable non-toxic acids, to form acid addition salts, for example, of an amino group of the polypeptide chain, or from non-toxic bases pharmaceutically acceptable to form basic salts, for example, of a carboxyl group of the polypeptide chain. These salts can be formed as internal salts, and / or as salts of the amino or carboxylic acid term of the polypeptide of the invention. Suitable pharmaceutically acceptable acid addition salts are those of pharmaceutically acceptable organic acids, polymeric acids, or non-toxic inorganic acids. Examples of suitable organic acids include acetic, ascorbic, benzoic, benzenesulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isothionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, oxalic, pamoic acids , pantothenic, phosphoric, salicylic, succinic, sulfuric, tartaric, and p-toluenesulfonic, as well as polymeric acids, such as tannic acid or carboxymethyl cellulose. Suitable inorganic acids include mineral acids, such as hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids. Examples of suitable inorganic bases for forming salts of a carboxyl group include the alkali metal salts, such as sodium, potassium, and lithium salts; alkaline earth salts, such as calcium, barium, and magnesium salts; and the ammonium, copper, ferrous, ferric, zinc, manganese, aluminum, and manganese salts. The ammonium, calcium, magnesium, potassium, and sodium salts are preferred. Examples of pharmaceutically acceptable organic bases to form salts of a carboxyl group include organic amines, such as amine ethylic tri amines, trietílica amine, tri (normal propílica) amine December ic lohexí 1 ica, ß - (tell you 1 amino) et anol, tris- (hydroxymethyl) aminomethane, trietanólica amine, beta - (diethylamino) ethanol, arginine, lysine, histidine, piperidine N-ethylic, hydrabamine, choline, betaine, ethylenic diamine, glucosamine, methyl glucamine, theobromine, purines, piperazines , piperidines, caffeine, and procaine. Acid addition salts of the polypeptides can be prepared in a conventional manner by contacting the polypeptide with one or more equivalents of the desired inorganic or organic acid, such as hydrochloric acid. They may conventionally be prepared salts of the carboxyl groups of the peptide, by contacting the peptide with one or more equivalents of a desired base such as a base metal hydroxide, for example sodium hydroxide; a metal carbonate or bicarbonate base, such as sodium carbonate or sodium bicarbonate; or an amine base such as triethyl amine or trietanolic amine. The invention, therefore, also relates to pharmaceutical compositions comprising a novel fusion polypeptide as defined above, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or diluent. The vehicle is preferably a sterile liquid, free of pyrogen. parenterally acceptable. Preferred liquid carriers or diluents are water, physiological saline, dextrose accus, and glycols, particularly (when they are isotonic) for injectable solutions. The composition can be a lyophilized conventionally prepared. The compositions can be administered systemically, i.e. parenterally (eg, intramuscularly, intravenously, subcutaneously, or intradermally), or by intraperitoneal administration. For parenteral administration, it is preferred that the fusion polypeptides are essentially soluble in the serum or plasma of the patient, for example, that at least 1 milligram of the polypeptide is soluble in 1 milliliter of serum or plasma. The compositions can also be administered by known techniques for topical administration. Examples of suitable dosage forms include sprays, ophthalmic solutions, nasal solutions, and ointments. For example, a spray may be made by dissolving the peptide in an appropriate solvent, and placing it in a spray to serve as an aerosol for a commonly used inhalation therapy. An ophthalmic or nasal solution can be made by dissolving the active ingredient in distilled water, adding any required auxiliary agent, such as a regulator, isotonizing agent, thickener, preservative, stabilizer, surfactant, or antiseptic, and adjusting the mixture to a pH of 4. to 9. Ointments can be obtained, for example, by preparing a composition from a polymer solution, such as 2 percent aqueous carboxyvinyl polymer, and a base, such as 2 percent sodium hydroxide, mixed to obtain a gel, and to mix with the gel a quantity of the purified fusion polypeptide. The composition is preferably administered subcutaneously or intravenously, and more usually, subcutaneously. The invention also comprises a method of treating allergic conditions, which comprises administering a therapeutically effective amount of a fusion polypeptide of the invention, or a pharmaceutically acceptable salt thereof, to a patient in need of such treatment. The method of treatment is practiced during the course of an allergic disease state (ie, when relief of symptoms is specifically required); or as a continuous or prophylactic treatment (ie, prior to the establishment of an IgE-mediated or anticipated IgE receptor disorder, such as an allergic reaction). The effective dosage according to the present can vary over a wide range, taking into consideration, for example, the degree of severity of the condition being treated, the age, sex and condition of the subject, the duration of the treatment , and the potency of the particular fusion protein, factors that can be determined by conventional methods. Since individual subjects vary widely in their IgE content (as well as antibody to FceRI), a therapeutically effective dosage according to the present may best be described as between 5 x 102 and 1 x 104 times the total content of IgE in serum and antibodies to FceRI, on a molar scale. The patient can be treated on a daily basis in a single or multiple administration. The composition can also be administered on a monthly basis (or at weekly intervals as appropriate), also in single or multiple administrations. For an average subject (70 kilograms), a suitable monthly dosage can be from a lower dosage of approximately 0.5 milligrams per month to a higher dosage of approximately 500 milligrams per month, preferably from approximately 1 milligram to approximately 300 milligrams, more preferably from about 20 milligrams to about 250 milligrams per month, of the fusion polypeptide of the invention, such as the dimer of Example 7, conveniently administered in divided dosages once or twice a month. Higher dosage ranges may be indicated, for example, from 500 milligrams per month to 2 grams per month, in patients with high serum IgE concentrations, or in early treatment phases. The dosage and the time of administration may vary. Initial administrations of the composition may be at higher dosages within the above ranges, and may be administered more frequently than later administrations in the treatment of the disease. Appropriate dosages can be determined by measuring the IgE content and antibodies to FceRI in the patient's serum, as indicated above. For example, early in the course of the disease, the fusion polypeptide of the invention, such as the dimer of Example 7, can be administered in weekly doses of 200 to 500 milligrams of the polypeptide in the average patient (70 kilograms). After being freed of the serum IgE antibody to FceRI, the treatment regimen can be reduced to weekly treatments, or treatments every third week, with dosages of 50 micrograms to 100 milligrams of the polypeptide per treatment. The compositions of the present invention can be administered alone or in combination with other compounds of the present invention, or additional pharmaceutical agents, such as antihistamines or corticosteroids. The invention also comprises the use of the fusion polypeptides of the invention in a diagnostic assay in vi tro of any conventional format, e.g. ELISA, to determine the level of IgE or autoantibodies for FceRI (e.g., in patients of chronic urticaria), in a biological sample obtained from a human patient, for example blood or tissue samples. The HSA component conveniently facilitates the binding and detection of IgE or autoantibodies to FceRI in an assay formatted in ELISA. The amount of IgE or autoantibodies present in the sample can serve as a measure of the patient's allergic response to a substance that has been exposed to the patient. Levels of IgE or auto-antibodies can also be measured to determine the efficiency of anti-allergy therapies, and to monitor a patient's allergic state over time. The invention further comprises a method for performing gene therapy in humans, using a polynucleotide encoding a fusion polypeptide of the invention, for the treatment of disorders mediated by IgE or by the IgE receptor. The method of gene therapy comprises modifying the cells of a patient, by introducing thereto a polynucleotide encoding a fusion polypeptide of the invention, and expressing the polypeptide from these cells. For example, first the somatic cells of a patient can be removed, then they are genetically modified in culture by the insertion of the polynucleotide, and the resulting modified cells are reintroduced into the patient, whereby, a polypeptide of the invention is expressed by the patient's cells. Alternatively, the cells can be modified in vivo by direct insertion of the vector DNA encoding the polypeptide. Cells suitable for modification include endothelial cells or leukocytes. For gene therapy applications, the polynucleotide of the invention is preferably under the control of an adjustable (eg, inducible) promoter. In this way, the expression of the polypeptide can be made to depend on the patient's exposure to an exogenous factor using known regulatable promoter systems. The invention also includes using the methods of gene therapy to prepare non-human somatic transgenic or recombinant animals that express the fusion polypeptides of the invention, for example in milk. These modified non-human animals are also part of the invention. Examples of useful animals include mice, rats, rabbits, pigs, sheep, goats, and cattle, all of which have been made transgenic using conventional techniques (eg, DR Hurwitz et al., Transgenic Research 3. [1994] 365- 375). The animals can be used for modeling purposes or for the actual production of a protein. In particular, the invention relates to a mouse, goat, cow, or transgenic pig, which expresses a fusion polypeptide of the invention. The methods for making these animals are well known. A polynucleotide encoding the fusion protein of the invention can be introduced into the somatic cells of the animals, living in vivo, to produce somatic recombinant animals, which are genetically modified but can not pass the modification genetics to the progeny. Alternatively, the polynucleotide can be inserted into the cells of the embryos for the production of transgenic animals capable of passing on to their progeny the ability to express the proteins of the invention. Transfection of the cells for gene therapy can be performed in a conventional manner, for example, by electroincorporation, calcium phosphate precipitation, a lipofectin-based procedure, intramuscular injection, microinjection, or through the use of a "gene gun". " Plasmid-based vectors preferably contain a marker, such as the neomycin gene, for the selection of stable transfectants with the cytotoxic aminoglycoside G418 in eukaryotic cells. The infection is carried out by incorporating the genetic sequence for the fusion polypeptide into a retroviral vector. Various methods are known in the art for this incorporation. This method, which has been widely used, employs a defective murine retrovirus to package the retrovirus, and an amphotrophic packaging cell line to prepare the infectious amphotrophic virus for the purpose of being used in the infection of the target donor cells. An additional characterization can be carried out, for example, as follows: In vivo determination of serum half-life General procedure: Female Charles River SKH1 / hr / hr mice, weighing approximately 25 grams, are injected intravenously with test proteins diluted in serum regulated with Ca + 2 -free, sterile Mg2 + phosphate. The mice are divided into groups as follows: - Group (i), which receives 130 micrograms (1 nanomole) of fusion polypeptide, for example, the dimer IgER-LL-HSA II-L2-IgER, prepared as in Example 7 , or Group (ii) that receives 60 micrograms of IgER (2 nanomoles), or - Group (iii) that receives 65 micrograms of HSA I (1 nanomole). 100 microliters of blood are taken from each mouse at 10 minutes, at 30 minutes, at 3 hours, at 6 hours, and at 12 hours after injection. The serum is prepared by centrifugation. The serum concentration levels of the different proteins are determined by: a) ELISA binding assay of the IgE receptor; b) Inhibition ELISA; or c) HSA Sandwich ELISA, as follows: a) IgER ELISA binding assay: The IgE serum concentration is determined by detection of the IgE binding, by the following Sandwich ELISA: 200 nanograms of human IgE are immobilized on a COSTAR 8-slot fringe plate ( Cambridge, MA, USA), in 100 microliters of coating buffer, in a humidified chamber at 4 ° C overnight. Each cavity is washed twice with 300 microliters of phosphate-regulated serum free of Ca2 +, Mg2 +, pH 7.2. The plates are blocked for 1 hour at room temperature with 200 microliters of serum regulated with Ca 2+ -free phosphate, Mg 2+ containing 5 percent bovine serum albumin (Sigma). After washing twice with 300 microliters of serum regulated with Ca 2+ -free phosphate, Mg 2+ containing 0.05% Tween 20 (PBST), samples diluted in 100 microliters of mouse serum diluted 1:10 (in phosphate buffer) free of Ca2 +, Mg2 +), are added and incubated for 1 hour at room temperature. The wells are washed twice with 300 microliters of phosphate-buffered serum, and incubated with 100 microliters (1 nanogram) of a monoclonal anti-human IgE receptor antibody, such as 5H5 / F8 for 1 hour at room temperature.

Again, the cavities are washed twice with 300 microliters of PBST, and incubated with 100 microliters of goat anti-mouse IgG serum peroxidase (Biorad, diluted 1: 2000 in serum regulated with Ca 2+ -free phosphate, Mg 2 * ) for 1 hour at room temperature. Plates are washed three times with 300 microliters of PBST, and horseradish peroxidase conjugates (HRP) are detected with 100 microliters of ABTS substrate (Biorad). The reaction is stopped after 5 minutes with 100 microliters of 3 percent oxalic acid. The color intensities are measured with an EASY READER photometer at 405 nanometers. b) Inhibition ELISA: 100 nanograms of FceRIa are immobilized in Nunc 96-well immunoplates (F96 cert Maxisorb) in 100 microliters of coating buffer (0.1 M NaHCO3, 0.01% NaN3, pH of 9.6) in a humidified chamber at 4 ° C during the night. Each cavity is washed four times with 300 microliters of phosphate regulated serum, 0.05 percent Tween 20 (wash buffer). To different sets of cavities, either a negative control is added (50 microliters of mouse serum diluted to 1:25 in phosphate-regulated serum, 0.05 percent Tween 20, 2 percent calf fetal serum), a dilution series of the fusion polypeptide standard, for example the standard IgER-Lx-HSA II - _L fgE (dilutions from 400 nanograms / milliliter to 1.6 nanograms / milliliter), or series of dilution of the samples. The standard and samples are diluted in mouse serum diluted 1:25 in phosphate buffered serum, 0.05 percent Tween 20, 2 percent fetal calf serum. Immediately after, 50 microliters of 400 nanograms / microliter of the human IgE-biotin conjugate are added and mixed in dilution buffer. The dilution of the final mouse serum in the incubation mixture is 1:50. After incubation for 2 hours at 37 ° C, the plates are washed four times with 300 microliters of wash buffer, and 50 microliters of streptavidin-alkaline phosphatase conjugate (Gibco) diluted 1: 1000 in a buffer is added. dilution, and incubate for 1 hour at 37 ° C. The plates are washed four times with 300 microliters of wash buffer, and after the addition of 100 microliters of substrate (1 milligram / milliliter of p-nitrophenyl phosphate buffer in dietanolic amine, pH 9.8 [BIORAD]), the reaction is stopped after incubation for 30 minutes at 37 ° C, with 50 microliters of 2 M NaOH. Optical densities are measured with a BIOMEK-1000 workstation photometer at 405 nanometers. The quantitative evaluation based on the standard curves (adjustment of the 4-parameter logistic curve) is done with the IMMUNOFIT ELISA Beckman evaluation program. Calculation: Link% = [OD (sample or standard value) / OD (regulator value)] x 100 c) HSA Sandwich ELISA: 500 nanograms of monoclonal anti-mouse HSA (HSA-9) are immobilized in 96-well immunoplates Nunc (F96 cert Maxisorb), in 100 microliters of coating buffer (0.1 M NaHCO3, 0.01% NaN3, pH 9.6), in a humidified chamber at 4 ° C overnight. Each cavity is washed four times with 300 microliters of wash buffer (serum regulated with phosphate, 0.05% Tween 20). Add 100 microliters of mouse serum diluted 1: 100 (phosphate buffered serum, 0.05 percent Tween 20, 2 percent calf fetal serum = dilution buffer) as a negative control, or 100 microliters of the standard ( 1 microgram / milliliter-2 nanograms / milliliter of human serum albumin, KABI), or 100 microliters of sample diluted in mouse serum diluted 1: 100. After incubation for 2 hours at 37 ° C, the plates are washed four times with 300 microliters of wash buffer, and 100 microliters of 1 nanogram / microliter of rabbit anti-HSA-biotin conjugate diluted in the regulator is added. of dilution. The anti-HSA-biotin conjugate is purified by immunoaffinity chromatography with CH-Seph4B-HSA, and the cross-reactivities are removed with the mouse serum by immunosorption with mouse-agarose serum. After incubation for 2 hours at 37 ° C, the plates are washed four times with 300 microliters of wash buffer, and 50 microliters of streptavidin-alkaline phosphatase conjugate (Gibco) diluted 1: 1000 in the buffer is added. dilution, and incubate for 1 hour at 37 ° C. The plates are washed four times with 300 microliters of wash buffer, and after addition of 100 microliters of substrate (1 milligram / milliliter of p-nitrophenyl phosphate buffer in dietanolic amine, pH of 9.8, BIO-RAD), the reaction is stopped after incubation for 15 minutes at 37 ° C, with 50 microliters of 2 M NaOH. Optical densities are measured with a BIOMEK-1000 workstation photometer at 405 nanometers. The quantitative evaluation from the standard curve (adjustment of the 4-parameter logistic curve) is done with the IMMUNOFIT ELISA Beckman evaluation program.

Results: The concentrations for the dimeric fusion polypeptide, IgER-L-_-HSA II-L2-IgER, free IgER (referred to as "free alpha chain"), or HSA I (referred to as "HSA"), as a function of time elapsed after injection, they are given in picomoles / milliliter of serum in Figure 1 (A) and (B). Figure 1 (B) shows that the free receptor can only be detected in mouse serum for about 10 minutes, while the dimeric fusion polypeptide can still be detected approximately 12 hours after administration. Figure 1 (C) shows the relative release kinetics for HSA, and the dimeric fusion polypeptide. After the stabilization of the blood-tissue distribution in the first 10 minutes, the dimer and the HSA show an almost identical release curve. This confirms that the serum half-life of an IgE binding domain can be substantially prolonged by fusion with HSA.

Inhibition of passive cutaneous anaphylaxis (PCA) in mice General Procedure: (a) Administration of the Polypeptide: Serial dilutions of 10, 50, or 500 micrograms / kilogram of the fusion polypeptide are injected intravenously, for example, IgER-LL-HSA II-L2-IgER obtained from ovarian cells of Chinese hamster in Example 7, in Charles River SKHl / hr / hr female mice weighing approximately 25 grams, at varying intervals before sensitization. Three groups of mice are established, depending on the amount of polypeptide injected prior to sensitization (i.e., 10 micrograms / kilogram, 50 micrograms / kilogram, or 500 micrograms / kilogram). A fourth group of control mice receive 200 micrograms / kilogram of phosphate-regulated serum intravenously in place of the polypeptide. The four groups of mice are each divided into three subgroups, which differ by the interval between intravenous injection of the compound, and intracutaneous sensitization with IgE [step (b) below]. The intervals tested are: 5 minutes, 15 minutes, and 30 minutes. (b) Intracutaneous Sensitization: Mice are anesthetized and sensitized on the skin of the back by four intradermal injections of 5 nanograms each, of anti-dinitrophenyl mouse IgE monoclonal antibody (DNP) in 10 milliliters of phosphate-buffered serum ( BioMakor, Rehovot, Israel). In the control group, saline is injected intradermally at one site. (c) Stimulus with Allergen: 90 minutes after sensitization, the mice are again anesthetized, and stimulated by intravenous injection of a solution containing 50 micrograms of dinitrophenyl-bovine serum albumin (DNP-BSA) (Calbiochem-Behring , San Diego, USA) containing 1 percent Evans blue. The response to PCA was quantified by measuring the diameter of the stained test site due to extravascularization.

Results: These are illustrated in the bar graph of Figure 2 for the mature fusion polypeptide of Example 7 (Val26-Leu978 amino acids of SEQ ID NO: 3). At a concentration of "500 micrograms / kilogram, the dimeric fusion polypeptide completely blocks the PCA at all points of the application time." At 50 micrograms / kilogram, the treatment at 30 minutes before the stimulus, gives a statistically significant reduction 36% At 15 minutes before the stimulus, a tendency is seen with the administration of both 10 and 50 micrograms / kilogram of the dimer.Therefore, the dimeric fusion polypeptide is effective in preventing PCA.

The methods and techniques for carrying out the present invention are known in the art. As far as its preparation in the present is not particularly described, the compounds, reagents, vectors, cell lines, etc., which are to be used, are known and readily available, or can be obtained in a conventional manner from known materials. and readily available, or equivalent materials can be prepared in a conventional manner, from known and readily available materials. The following non-limiting examples illustrate the invention. All temperatures are in degrees centigrade.

Materials and methods Amplification with polymerase chain reaction; The de novo chemical synthesis of the primers of the invention can be conducted using any suitable method, such as, for example, phosphotriester or phosphodiester methods. Methods and systems for amplifying a specific nucleic acid sequence are described in US Pat. Nos. 4,683,195, and in USP. 4,683,202; and in Polymerase Chain Reaction, H.A. Erlich - et al., Eds., Cold Spring Harbor Laboratory Press [1989]. Following the polymerase chain reaction, the DNA fragments are separated and purified using the QiaEx protocol (Qiagen, Inc., Chatsworth, CA, USA), then subcloned into a TA vector [TA Cloning R Kit (Invitrogen) (product literature, Version 2.2)]. The primers used in the generation of nucleic acids amplified with polymerase chain reaction are stipulated in Figure 8. DNA is sequenced using the Sequenase method (USB, Cleveland, OH, USA).

Plasmids and Reagents: The cDNA clone of FceRIa, pGEM-3 -110B-l (A. Shimizu et al., Proc. Nat. Acad. Sci. USA 85 [1988] 1907-1911), is obtained in the American Type Tissue Collection (ATCC, supply # 67566). The single-stranded human liver cDNA is obtained in Clontech (Quick Clone cDNA ready for polymerase chain reaction, Cat. D # 7113-l). The HSA sequence is available in GenBank, Accession Number V00495, J00078, L00132, and L00133. Restriction enzymes are obtained from Boehringer-Mannheim or Gibco / BRL. The Taq DNA polymerase is obtained in Perkin-Elmer Cetus (PECI), or in Boehringer-Mannheim. The vector SK is obtained in Stratagene. pHIL-D2 is available from Invitrogen (San Diego, CA, USA: Catalog Number K1710- 01 [1994]) (see "Pichia Expression Kit - Protein Expression - A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris - Version 3 .0"[December 1994]) (hereinafter referred to as the" Invitrogen Manual "). The vector pXMT3 is derived from pMT2 (Sambrook et al. (Eds.) [1989] supra), by cloning the Pst I linker with EcoRI from pUC8 (Pharmacia) at the Pst I and EcoRI sites of pMT2 ( R. J. Kaufman et al. [1987] supra). Conventional techniques that are used below are described in Sambrook et al. (Eds.) [1989] supra.

Example A: Cloning vector TA The plasmid pCR2 is ligated separately with each of the products of the amplification with polymerase chain reaction, obtained in Examples 1 and 2. The ligation reactions are performed using T4 DNA ligase in the following reaction mixture: M Tris-HCl (pH of 7.8) 10 mM MgCl 2 1 mM DTT 1 mM ATP 50 nanograms of the DNA of the vector 100-200 nanograms of the polymerase chain reaction products (unpurified). 4 units of T4 DNA ligase (New England Biolabs).

Each reaction mixture is maintained at 15 ° C for 18 hours before being transformed into competent cells.

Example 1: Amplification with polymerase chain reaction, and cloning of the FceRIo cDNA. The FceRIa cDNA is purified from pGEM-3-110B-l using the Qiagen method. Then: (A) for the preparation of a leading HSA construct, amplification is carried out with a polymerase chain reaction of the FceRIo cDNA. with oligonucleotides # 18 and # 19 (Figures 4, 8), using the following reaction mixture: 1 microliter (50 nanograms) of FceRIa cDNA; 50 picomoles of each of the oligonucleotides # 18 and # 19 microliters of polymerase chain reaction regulator at percent (PECI). 0. 5 microliters of 20 mM dNTP delivery solution = final concentration of 200 μM dNTPs. 0.5 microliters (2.5 Units) of Taq DNA polymerase (PECI). water up to 50 microliters.

The reaction mixture is covered with mineral oil to prevent evaporation, and thermocycled in a Perkin-Elmer Cetus model 480 DNA thermal cycler. Cycling conditions for this reaction are: heating at 95 ° C for 5 minutes, then 30 cycles of 94 ° C for 1.5 minutes, 53 ° C for 2 minutes. minutes, 72 ° C for 3 minutes, followed by an extension of 3 minutes at 72 ° C, and soaking overnight at 4 ° C. Following electrophoresis, an amplified product of approximately 550 base pairs is confirmed by staining with ethidium bromide. The fragment is subcloned into the PCR2 vector to give pEKl encoding IgER (Figures 4, 8B). (B) For the preparation of a leading construction of XgE., The amplification with polymerase chain reaction of the FceRIa cDNA is performed according to the procedure of (A) above, except that the oligonucleotides # 20 and # 31 are used. (Figure 8B). Following electrophoresis on 0.7 percent agarose gel, an amplified product of approximately 600 base pairs is confirmed by staining with ethidium bromide. The fragment is subcloned into the PCR2 vector to form IgER / TA # l encoding pre-IgER (Figure 4). (C) For the preparation of a construct that encodes pre-IgER, in order to express the mature truncated protein to be used as a control in the assays, the amplification with polymerase chain reaction of the cDNA of FACE as in (A) above, with oligonucleotides # 20 and # 19 (Figure 8B). Following electrophoresis, an amplified product of approximately 620 base pairs is confirmed by staining with ethidium bromide. The fragment of the polymerase chain reaction is separated and subcloned into the PCR II vector, to provide IgERFL / TA # 34, which encodes pre-IgER, followed by a stop codon (Figure 4).

Example 2: Amplification with polymerase chain reaction and cloning of human serum albumin cDNA To obtain a sequence encoding prepro-HSA II, the amplification is carried out with polymerase chain reaction of the full-length human serum albumin cDNA, with oligonucleotides # 24 and # 25 (Figure 8B), following the general procedure of Example 1 (A). The resulting clone HSA / TA # 1 had the sequence that most closely resembled the GenBank sequence. In this clone, seven mutations were detected in the wavy position, and a mutation that resulted in a change from lysine to glutamic acid at base 1333. The seven mutations in the wavy position were: base 309: A to T; base 744: A to G; base 795; G to A; base 951: G to A; base 1320: C to T; base 1569: A to C; base 1584: G to A (with reference to HSA / TA # 1). The mutation of lysine to glutamic acid was corrected back to the sequence in GenBank, using Site-directed mutagenesis, with the oligonucleotides shown in Figure 8A, and the in-vitro mutagenesis kit Bio-Rad Mutagene Phagemid (Biorad, Cat. # 170-3581). This method relies on the incorporation of uracil residues in the parenteral chain and its subsequent removal in the mutagenized chain (T.A. Kunkel, Proc. Nat. Acad. Sci. USA 82 [1985] 488-492). Because the point mutations in the wavy position do not alter the native amino acid sequence of the encoded protein, the seven previous mutations were not corrected. Following the electrophoresis, an amplified product of approximately 1.8 kb was confirmed by staining with ethidium bromide. The 1.8 kb product is subcloned into the pCR2 vector to form HSA / TA mut # 16, which is verified by DNA sequencing, and contains the complete sequence of prepro-HSA II. HSA / TA # 16 is subcloned in Bluescript SK as a Spel fragment, HindIII, producing HSA / SK # 17 (Figure 5). (A) For the preparation of a leading HSA construct, HSA / SK # 17 is digested with MstII and HindIII to remove the nucleotide sequence encoding the 3'-terminal amino acid (Leu609); and an oligonucleotide encoding the L2 linker, as defined above, is introduced into the 3 'terminus of the linearized HSA / SK # 17, kinassing the oligonucleotides # 28 and # 29 (Figure 8B), and quenching and ligating these fragments into the MstII / HindIII sites of linearized HSA / SK # 17, to produce pEK7 encoding prepro-HSA II fused to L2 (Figure 8B). Miniprep DNA is verified by digestion with BamHl and by sequencing. (B) For the preparation of an IgE leader construct, HSA / SK # 17 encoding prepro-HSA II is amplified by polymerase chain reaction with oligonucleotides # 26 and # 27 (Figures 5, 8). Oligonucleotide # 26 removes the prepro sequence from HSA, and adds an oligonucleotide encoding L-L at the 5 'end of HSA. Oligonucleotide # 27 terminates at a unique Ncol site that occurs naturally, approximately at the position of nucleotide 800 of the HSA coding sequence. The amplification with polymerase chain reaction is carried out according to the procedure of Example 1 (A). A fragment of approximately 800 base pairs isolated is subcloned by gel electrophoresis and Qia in a pCR2 vector, to provide the NSA / TA # 13 HSA clone, whose sequence is verified by DNA sequencing. The Ncol to Notl fragment from this clone is subcloned into the Ncol and Notl cut # 17 HSA / SK DNA, producing HSA / SK # 5 (Figure 5), which encodes LL linked to the 5 'terminus of the cDNA encoding HSA II.

(C) To express HSA alone, the HSA / SK # 17 construction prepared above is employed.

EXAMPLE 3 Construction of fusion HSA-IqER / SK # 22 crue encodes prepro-HSA + linker + IsER pEK7 (containing the cDNA of prepro-HSA II + the oligonucleotide for L2) and pEKl (containing IgER) are digested with BamHl and I left. The 1.8 kb fragment obtained from pEK7 phosphatase, and then ligated with the 550 kb fragment obtained from pEKl. A positive minipreparation, # 23, was prepared, but it was contaminated with another unknown plasmid that prevented the recovery of the HSA-IgER band. Accordingly, the Spel to Sali fragment of 2.4 kb containing the HSA-IgER fusion, and the 2.9 kb fragment containing the vector DNA, were purified from miniprep # 23, and ligated together, resulting in clone HSA-IgE / SK # 49 (Figure 9) [it was found that vector sites were missing from either end of the subcloned region, and according to the above, the Spel to Sali fragment of 2.4 kb from of HSA-IgE / SK # 49 was subcloned into Spel + Sall-Bluescript SK cut, resulting in HSA-IgE / SK # 22 (not shown in the Figures)]. The sequence of the binding of HSA and the linker with the IgE receptor DNA and the sequence of the ends of the fusion with the vector DNA were as expected in HSA-IgE / SK # 22, as verified by analysis of DNA sequence.

Example 4: IsE-HSA / SK # l fusion construct encoding IctER + prepro-HSA II HSA / SK # 5 and IgER / TA # 1 were digested with Sstl and Nhel. The 600 base pair fragment from IgE / TA # 1 is purified by gel electrophoresis, and ligated in HSA / SK # 5 cut and treated with alkaline phosphatase, producing the IgE-HSA / SK # l clone.

(Figure 10).

Example 5: Fusion construction R-H-R / SK # 50 that codes Pi * ep-IgER - Lj - HSA II - 2 - IsER The PstI site, unique for the HSA region of IgER-HSA / SK # 1 and HSA-IgER / SK # 49, is used to bind IgER and prepro-HSA II by means of the oligonucleotide for L2. HSA / IgER / SK # 49 and Bluescript SK are digested with PstI and Sali. A 1.2 kb fragment containing the 3 'portion of HSA II, the linker, and the IgER sequence, are ligated into the cut PstI + Sali-Bluescript DNA, resulting in HSA-IgER Pst Sal / SK # 37 ( Figure 11), which is digested with PstI and Kpnl, and the 1.2 kb fragment is prepared. The IgER-HSA / SK # l DNA is digested with PstI and KpnI, and a 4.8 kb fragment containing the vector, IgER, is isolated to the linker, and the 5 'half of HSA is treated with phosphatase, and ligates with the 1.2 kb fragment from HSA-IgER Pst Sal / SK # 37. The resulting dimeric construction R-H-R / SK # 50 is obtained (Figure 11).

Example 6: Monomeric fusion polypeptides of HSA II - 2 - IqER, by transfection and culture of Pichia pastoris Plasmid MB # 2, which encodes HSA II - L2 - IgER, is prepared by cutting the plasmid HSA / SK # 49 with EcoRI , and isolating the 2.4 kb fragment encoding the fusion protein. This fragment is ligated into the unique EcoRI site of the Pichia pastoris expression vector, pHIL-D2 (Invitrogen), after digestion of the plasmid pHIL-D2 with EcoRI, and treatment with alkaline phosphatase. The resulting MB # 2 plasmid is linearized by Nopl digestion, and transformed into his4 GS115 cells, as described in the "Invitrogen Manual." His + transformants are selected to determine growth on methanol. Strains that exhibit slow growth on methanol, are cultured in a minimum glycerol medium, as specified in the "Invitrogen Manual", until the stationary phase, are transferred by centrifugation to a regulated complex methanol medium, and are cultured for 4 days. The supernatant of the cells is then assayed by ELISA to determine the presence of HSA and the binding capacity of IgE. The IgE binding capacity of the obtained product secreted from P. pastoris was equivalent, on a molar basis, to the concentration of HSA, and completely biologically active.

Example 7: Dimeric fusion polypeptide IqER-HSA II-2-IqER, by transfection and culture of Chinese hamster ovary cells Plasmid pXMT3-RIa-HSA-RIc. (which contains the polynucleotide of SEQ ID N0: 4, which encodes pre-IgE HSA II-L2-IgER), is prepared by digestion of R-H-R / SK # 50 (see Example 5) with EcoRI, and isolation of the 3 kb EcoRI fragment encoding the dimeric fusion polypeptide. This fragment is ligated into the unique EcoRI site of pXMT3 after digestion of pXMT3 with EcoRI, and treatment with alkaline phosphatase. The plasmid is transfected into CHO DUKX Bll cells. These cells lack a functional dhfr gene (dihydrofolate reductase) required for the synthesis of the nucleoside. Accordingly, the cells are maintained in an Alpha-i medium (MEDIO MEM ALPHA with ribonucleosides and deoxyribonucleosides / Gibco) containing 10 percent fetal calf serum (FCS). For transfection, the cells are washed twice in phosphate-regulated serum free of Ca ++-Mg ++ (CMF-PBS), and the cell concentration is adjusted to 2 × 10 6 cells / milliliter in CMF-PBS. 0.8 milliliters of the cell suspension is added to 15 micrograms of the plasmid DNA. The transfection is done by electroincorporation, using a BIO RAD Generator (voltage = 1000 volts, capacitor = 25 μF). After transfection, the cells are cultured in 15 milliliters of Alpha + medium with 10 percent fetal calf serum for 3 days. The dhfr gene located in the plasmid pXMT3 allows the selection of recombinant cells in a depleted nucleotide medium. Three days after transfection, the cells are placed in an alpha medium (MEDIO MEM ALPHA, without ribonucleosides and deoxyribonucleosides / Gibco), containing 10 percent dialyzed fetal calf serum (FCSD). After 2 weeks of culture, the recombinant cell colonies are visible. Cells are maintained in the Alpha-medium containing 10 percent dialyzed fetal calf serum for 4 additional steps before gene amplification is initiated. In the presence of methotrexate (MTX), the dihydrofolate reductase and the genes bound thereto are amplified, resulting in increased expression of the transgene. Accordingly, selection for recotnbinant cells in a nucleoside depleted medium is followed by culture in the presence of 20 nM methotrexate in an Alpha-medium containing 10 percent dialyzed fetal calf serum. Further amplification is achieved by steps increments in methotrexate up to 100 nM, and 500 nM methotrexate. The protein is produced by seeding the Tl / 3-500 nM group in an Alpha-medium containing 10 percent fetal calf serum (Gibco), in a density of 9x103 / square centimeter, in rolling bottles. The first supernatant is collected 5 days after sowing, followed by a change to Alpha-serum exempt. The second harvest is collected 3 days later, producing a total of 1 liter of supernatant for purification. A second batch is purified from 2 liters of supernatant derived from the Tl / 3-500 nM group, adapted to serum-free culture conditions. The cells are seeded in 5 x 10 4 cells / milliliter, and the supernatant is collected 6 days after sowing.

Example 8: Purification of the fusion protein The culture supernatants of Example 7 are purified by immunoaffinity chromatography on immobilized anti-FceRI monoclonal antibodies (e.g., 5H5-F8; mouse IgG1), produced and purified according to the techniques conventional: a) Preparation of the chromatographic support: The monoclonal antibodies are coupled with Sepharose 4B activated with CNBr (Pharmacia, Uppsala, Sweden), in a density of 10 milligrams of antibodies / milliliter of gel, according to the manufacturer's instructions. Subsequently, excessive reactive groups with ethanolic amine are blocked, and the resin stored in phosphate regulated serum is supplemented with 0.02 percent NaN3 until its use. b) Affinity chromatography: The clear culture supernatant is applied to a 5 milliliter antibody column equilibrated in phosphate buffered serum, at a flow rate of 0.5 milliliters / minute. The absorbed material is eluted in 50 mM citric acid, 140 mM NaCl, pH 2.70. The fractions containing protein are immediately adjusted to a pH of 7.0 (NaOH), followed by sterile filtration. c) Quantification / Characterization: The concentration of the dimeric fusion polypeptide is determined by absorption at 280 nanometers in its native conformation, in 30 mM (3- [N-morpholino] propan) sulfonic acid (MOPS), pH 7.0, and in the denatured form (guanidine 6 M «HC1). The corresponding molar absorption coefficient is calculated from the number of residues of tryptophan, tyrosine, and cystine, using the tabulated absorption coefficients of these amino acids in the model compounds, and corrected to take into account the difference in optical density between the protein folded and unfolded. The fusion protein contains 17 tryptophan, 40 tyrosine, and 21 cystine, which results in a theoretical extinction coefficient of 150.840 M_1cm ~ 1. The quality of the purified material is evaluated by conventional SDS-PAGE, and by sequencing of Edman degradation in gas phase automated at the N-terminus, and mass spectrometry. After SDS-PAGE (Figure 15), the polypeptide migrates with an apparent molecular weight of about 140 kDa, reflecting a glycosylation of about 28 percent (theoretical molecular weight without glycosylation: 108.863.61 Da).

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Novartis AG (B) STREET: Schwarzwaldallee 215 (C) CITY: Basel (E) COUNTRY: Switzerland (F) POSTAL CODE: CH-4058 (G) TELEPHONE: 61-324 5269 ( H) TELEFAX: 61-322 7532 (ii) TITLE OF THE. INVENTION: FUSION POLYPEPTIDES (iii) SEQUENCE NUMBER: (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Relay # 1.0, Version # 1.30 (EPO) (v) DATA OF THE CURRENT APPLICATION: NUMBER OF APPLICATION: WO PCT / EP97 / ... (vi) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER US 08/690216 (B) DATE OF SUBMISSION: July 26, 1996 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 257 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Met Ala Pro Ala Met Glu Ser Pro Thr Leu Leu Cys Val Ala Leu I read. 1 5 10 '15 Phe Phe Wing Pro Asp Gly Val Leu Wing Val Pro Gln Lys Pro Lys Val 20 25 30 Ser Leu Asn Pro Pro Trp Asn Arg lie Phe Lys Gly Glu Asn Val Thr 35 40 45 Leu Thr Cys Asn Gly Asn Asn Phe Phe Glu Val Ser Ser Thr Lys Trp 50 bb 60 Phe His Asn Gly Ser Leu Ser Glu Glu Thr Asn Ser Ser Leu Asn lie 65 70 75 80 Val Asn Ala Lys Phe Glu Asp Ser Gly Glu Tyr Lys Cys Gln His Gln 85 t 90 95 Glr. Val Asn Glu Ser Glu Pro Val Tyr Leu Glu Val Phe Ser Asp Trp 100 105 110 Leu Leu Leu Gln Wing Be Wing Glu Val Val Met Glu Gly Gln Pro Leu 115 120 125 Phe Leu Arg Cys His Gly Trp Arg Asn Trp Asp Val Tyr Lys Val lie 130 135 140 Tyr Tyr Lys Asp Gly Glu Wing Leu Lys Tyr Trp Tyr Glu Asn His Asn 145 150 155 160 lie Ser lie Thr Asn Wing Thr Val Glu Asp Ser Gly Thr Tyr Tyr Cys 165 170 175 Thr Gly Lys Val Trp Gln Leu Asp Tyr Glu Ser Glu Pro Leu Asn lie 180 185 190 Thr Val lie Lys Wing Pro Arg Glu Lys Tyr Trp Leu Gln Phe Phe lie 195 200 205 Pro Leu Leu Val Val lie Leu Phe Wing Val Asp Thr Gly Leu Phe lie 210 215 220 Be Thr Gln Gln Gln Val Thr Phe Leu Leu Lys lie Lys Arg Thr Arg 225 230 235 240 Lys Gly Phe Arg Leu Leu Asn Pro Pro Pro Lys Pro Asn Pro Lys Asn 245 250 255 Asn 257 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 609 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Me: Lys Trp Val Thr Phe lie Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Wing His Lys Ser Glu Val Wing 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Wing Leu Val Leu 35 40 45 lie Wing Phe Wing Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Be Wing Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Wing Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Wing Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu lie Wing Arg Arg His Pro Tyr Phe Tyr Wing Pro 165 170 175 Glu Leu Leu Phe Phe Wing Lys Arg Tyr Lys Wing Wing Phe Thr Glu Cys 180 185 190 Cys Gln Wing Wing Asp Lys Wing Wing Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Be Ala Lys Gln Arg Leu Lys Cys 210 215 220 Wing Arg Leu Ser Gln Arg Phe Pro Lys Wing Glu Phe Wing Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu 'Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Wing Asp Asp Arg Wing Asp Leu Wing Lys Tyr lie 275 280 285 Cys Glu Aen Glr. Asp Ser lie Be Ser Lys Leu Lys Glu Cys Cys Glu 290 295 3uu Lys Pro Leu Leu Glu Lys Ser His Cys lie Wing Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Wing Asp Leu Pro Ser Leu Wing Wing Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 .-. Et Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355, 360 365 Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu lie Lys Gln Asn Cys 405 410 415 Glu Leu Phe Lys Gln Leu Gly Glu Tyr Lys Phe Gln Asn Wing Leu Leu 420 425 430 Vai Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 535 510 Be Wing Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Wing 515 520 525 Glu Thr Phe Thr Phe His Wing Asp lie Cys Thr Leu Ser Glu Lys Glu 530 535 540 Ara Gln lie Lys Gln Thr l »1 Glu Leu Val Lys His Lys 545 550 = .55 560 Pro Lys Wing Thr Lys Glu Gln Leu Lys Wing Val Met Asp Asp Phe Wing 565 570 575 Wing Phe Val Glu Lys Cys Cys Lys Wing Asp Asp Lys Glu Thr Cys Phe 580 585 590 Wing Glu Glu Gly Lys Lys Leu Val Wing Wing Gln Ala Ala Gl Leu 595 600 605 Leu 6G9 (2) IMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 978 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 Met Ala Pro Ala Met Glu Ser Pro Thr Leu Leu Cys Val Ala Leu Leu 1 5 10 15 Phe Phe Wing Pro Asp Gly Val Leu Wing Val Pro Gln Lys Pro Lys Val 20 25 30 Ser Leu Asn Pro Pro Trp Asn Arg He Phe Lys Gly Glu Asn Val Thr 35 40 45 Leu Thr Cys Asn Gly Asn Asn Phe Phe Glu Val Ser Ser Thr Lys Trp 50 55 60 Phe His Asn Gly Ser Leu Ser Glu Glu Thr Asn Ser Ser Leu Asn He 65 70 75 80 Val Asn Ala Lys Phe Glu Asp Ser Gly Glu Tyr Lys Cys Gln His Gln 85 90 95 GIn Val Asn Glu Ser Glu Pro Val Tyr Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gin Wing Be Wing Glu Va! . -1. Met Glu Gly Gln Pro Leu 115 120 125 Phe Leu Arg Cys His Gly Trp Arg C *. • - r Asp Val Tyr Lys Val He 130 135 140 Tyr Tyr Lys Asp Gly Glu Ala Leu Lys • i * • Trp Tyr Glu Asn His Asn 145 150 155 160 Thr Gly Lys Val Trp Gln Leu Asp Tyr Glu Ser Glu Pro Leu Asn He 180 185 190 Thr Val He Lys Wing Pro Arg Glu Lys Tyr Trp Leu Wing Ser Gly Gly • 195 200 205 Gly Gly Ser Asp Wing His Lys Ser Glu Val Wing His Arg Phe Lys Asp 210 215 220 Leu Gly Glu Gl? A < = - * Phe Lys Ala Leu Val Leu He Ala Phe Ala Gln 225 230 235 240 Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 245 250 255 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asr. 260 265 270 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 275 280 285 Wing Thr Leu Arg Glu Thr Tyr Gly Glu Met Wing Asp Cys Cys Wing Ly = 290 295 300 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asr. 305 310 315 320 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 325 330 335 Wing Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 340 345 350 He Wing Arg Arg His Pro Tyr Phe Tyr Wing Pro Glu Leu Leu Phe Phe 355 360 365 Wing Lys Arg Tyr Lys Wing Wing Phe Thr Glu Cys Cys Gln Ala Ala Asp 370 375 380 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 385 390 395 40C Lys Ala Ser Be Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 405 410 415 Phe Gly Glu Arg Wing Phe Lys Wing Trp Wing Val Wing Arg Leu Ser Glp 420 425 430 Arg Phe Pro Lys Wing Glu Phe Wing Glu Val Ser Lys Leu Val Thr Asp 435 440 445 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 450 455 460 Wing Asp Asp Arg Wing Asp Leu Wing Lys Tyr He Cys Glu Asn Gln Asp 465 470 475 480 Be He Be Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 485 490 495 Leu Pro Ser Leu Ala Wing Asp Phe Val Glu Ser Lys Asp Val Cys Lys 515 520 525 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 530 535 540 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 545 550 555 560 Ala Lys Thr Tyr Glu Thr Thr Lev *.? t.ys Oys Cys Ala Ala Ala Asp 565 \ 570 575 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 580 585 590 Glu Glu Pro Gln Asn Leu He Lys Gln Asn Cys Glu Leu Phe Lys Gln 595 600 605 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 610 615 620 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 525 630 635 640 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 645 650 655 Met Pro Cys Wing Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 660 665 670 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 675 680 685 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Wing Leu Glu Val 690 695 700 Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Wing Glu Thr Phe Thr Phe 705 710 715 720 His Wing Asp He Cys Thr Leu Ser Glu Lys Glu Arg Gln He Lys Lys 725 730 735 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 740 745 750 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 755 760 765 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 770 775 780 Lys Leu Val Wing Wing Gln Ala Wing Leu Gly Gly Gly Gly Ser Val 785 790 795 800 Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg He Phe 805 SiO 815 Lys Gly Glu Asn Val Thr Leu T..r Cys A-.n Gly Asn Asn Phe Phe Glu 820 --.- 830 Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu Thr 835 840 845 Asn Be Ser Leu Asn He Val Asn Wing Lys Phe Glu Asp Ser Gly Glu 850 855 860 Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr Leu 865 870 875 880 Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Wing Be Wing Glu Val Val 885 890 895 Met Glu Gly Gln Pro Leu Phe Leu Arg yp Hiß Gly Trp Arg Asn Trp 900 y05 910 Asp Val Tyr Lys Val He Tyr Tyr Lys Asp Gly Glu Wing Leu Lys Tyr 915 920 925 Trp Tyr Glu Asn His Asn He Ser He Thr Asn Ala Thr Val Glu Asp 930 935 940 Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr Glu 945 950 955 960 Ser Glu Pro Leu Asn He Thr Val He Lys Wing Pro Arg Glu Lys Tyr 965 970 975 Trp Leu 978 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2955 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (Üi) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 < i 'Á' "TCA C? TGGCTCCTGC CATGGAATCC CCTACTCTAC TGTGTGTAGC CTTACTGTTC 60 . 1 '.'ji- 1 * -. /. > ATGGCGTGTT AGCAGTCCCT CAGAAACCTA AGGTCTCCTT GAACCCTCCA 120 TJu? ATAGA? TATTTAAAGG AGAGAATGTG ACTCTTACAT GTAATGGGAA CAATTTCTTT 180 GAAGTCAGTT CCACCAAATG GTTCCACAAT GGCAGCCTTT CAGAAGAGAC AAATTCAAGT 240 TTGAATATTG TGAATGCCAA ATTTGAAGAC AGTGGAGAAT ACAAATGTCA GCACCAACAA 300 GTTAATGAGÁ GTGAACCTGT GTACCTGGAA GTCTTCAGTG ACTGGCTGCT CCTTCAGGCC 360 TCTGCTGAGG TGGTGATGGA GGGCCAGCCC CTCTTCCTCA GGTGCCATGG TTGGAGG AC 420 TGGGATGTGT ACAAGGTGAT CTATTATAAG GATGGTGAAG CTCTCAAGTA CTGGTATGAG 480 AACCACAACA TCTCCATTAC AAATGCCACA GTTGAAGACA GTGGAACCTA CTACTGTACG 540 GGCAAAGTGT GGCAGCTGGA CTATGAGTCT GAGCCCCTCA ACATTACTGT AATAAAA CT 600 CCGCGTGAGA AGTACTGGCT TGCTAGCGGT GGAGGTGGAT CCGATGCACA CAAGAGTGAG 660 GTTGCTC? TC GGTTTAAAGA TTTGGGAGA? GAAAATTTCA AAGCCTTGGT GTTGATTGCC 720 TTTGCTCAGT? TCTTCAGCA GTGTCCATTT GAAGATCATG TAAAATTAGT GAATGAAGTA 780 ACTGAATTTG CAAAAACATG TGTTGCTGAT GAGTCAGCTG AA? ATTGTGA CAAATCACTT 840 CATACCCTTT TTGGAGACAA ATTATGCACA GTTGCAACTC TTCGTGAAAC CTATGGTGAA 900 ATGGCTGACT GCTGTGCAAA ACAAGAACCT GAGAGAAATG AATGCTTCTT GCAACACAAA 960 GATGACAACC CAAACCTCCC CCGATTGGTG AGACCAGAGG TTGATGTGAT GTGCACTGCT 1020 TTTCATGACA ATGAAGAGAC ATTTTTGAAA AAATACTTAT ATGAAATTGC CAGAAGACAT 1080 CCTTACTTTT ATGCCCCGGA ACTCCTTTTC TTTGCTAAAA GGTATAAAGC TGCTTTTACA 1140 GAATGTTGCC AAGCTGCTGA TAAAGCTGCC TGCCTGTTGC CAAAGCTCGA TGAACTTCGG 1200 GATGAAGGGA AGGCTTCGTC TGCCAAACAG AGACTCAAGT GTGCCAGTCT CCAAAAATTT 1260 GGAGAAAGAG CTTTCAAAGC ATGGGCAGTA GCTCGCCTGA GCCAGAGATT TCCCAAAGCT 1320 G? GTTTGCAG AAGTTTCCAA GTTAGTGACA GATCTTACCA AAGTCCACAC GGAATGCTGC 1380 CATGGAGATC TGCTTGAATG TGCTGATGAC AGGGCGGACC TTGCCAAGTA TATCTGTGAA 1440 ? ATCAAGATT CGATCTCCAG TAAACTGAAG GAATGCTGTG AAAAACCTCT GTTGGAAAAA 1500 TCCCACTGCA TTGCCGAAGT GGAAAATGAT GAGATGCCTG CTGACTTGCC TTCATTAGCT 1560 GCTGATTTTG TTGAAAGTAA GGATGTTTGC AAAAACTATG CTGAGGCAAA GGATGTCTTC "l620 CTGGGCATGT TTTTGTATGA ATATGCAAGA AGGCATCCTG ATTACTCTGT CGTGCTGCTG 1680 CTGAGACTTG CCAAGACATA TGAAACCACT CTAGAGAAGT GCTGTGCCGC TGCAGATCCT 1740 CATGAATGCT ATGCCAAAGT GTTCGATGAA TTTAAACCTC TTGTGGAAGA GCCTCAGAAT 1800 TTAATCAAAC AAAATTGTGA GCTTTT AAG CAGCTTGGAG AGTACAAATT CCAGAATGCG 1860 CTATTAGTTC GTTACACCAA GAAAGTACCC CAAGTGTCAA CTCCAACTCT TGTAGAGGTC 1920 TCAAGAAACC TAGGAAAAGT GGGCAGCAAA TGTTGTAAAC ATCCTGAAGC AAAAAGA? TG 1980 CCCTGTGCAG AAGACTATCT ATCCGTGGTC CTGAACCAGT TATGTGTGTT GCATGAG? AA 2040 ACGCCAGTAA GTGACAGAGT CACCAAATGC TGCACAGAAT CCTTGGTGAA CAGGCGACCA 2100 TGCTTTTCAG CTCTGGAAGT CGATGAAACA TACGTTCCCA AAGAGTTTAA TGCTGAAACA 2160 TTCACCTTCC ATGCAGATAT ATGCACACTT TCTGAGAAGG AGAGACAAAT CAAGAAACAA 2220 ACTGCACTTG TTGAGCTTGT GAAACACAAG CCCAAGGCAA CAAAAGAGCA ACTGAAAGCT 2280 GTTATGGATG ATTTCGCAGC TTTTGTAGAG AAGTGCTGCA AGGCTGACGA TAAGGAGACC 2340 TGCTTTGCCG AGGAGGGTAA AAAACTTGTT GCTGCAAGTC AAGCTGCCTT AGGTGGAGGT 2400 GGATCCGTCC CTCAGAAACC TAAGGTCTCC TTGAACCCTC CATGGAATAG AATATTTAAA 2460 GGAGAGAATG TGACTCTTAC ATGTAATGGG AACAATTTCT TTGAAGTCAG TTCCACCAAA 2520 TGGTTCCACA ATGGCAGCCT TTCAGAAGAG ACAAATTCAA GTTTGAATAT TGTGAATGCC 2580? AATTTGAAG ACAGTGGAGA ATACAAATGT CAGCACCAAC AAGTTAATGA GAGTGAACCT 2640 GTGTACCTGG AAGTCTTCAG TGACTGGCTG CTCCTTCAGG CCTCTGCTGA GGTGGTGATG 2700 GAGGGCCAGC CCCTCTTCCT CAGGTGCCAT GGTTGGAGGA ACTGGGATGT GTACAAGGTG 2760 ATCTAT TATA AGGATGGTGA AGCTCTCAAG TACTGGTATG AGAACCACAA CATCTCCATT 2820 ACAAATGCCA CAGTTGAAGA CAGTGGAACC TACTACTGTA CGGGCAAAGT GTGGCAGCTG 2880 GACTATGAGT CTGAGCCCCT CAACATTACT GTAATAAAAG CTCCGCGTGA GAAGTACTGG 2940 CTATAGTAAG AATTC 2955 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1827 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: ATGAAGTGGG TAACCTTTAT TTCCCTTCTT TTTCTCTTTA GCTCGGCTTA TTCCAGGGGT 60 GTGTTTCGTC GAGATGCACA CAAGAGTGAG GTTGCTCATC GGTTTAAAGA TTTGGGAGAA 120 GAAAATTTCA AAGCCTTGGT GTTGATTGCC TTTGCTCAGT ATCTTCAGCA GTGTCCATTT 180 GAAGATCATG TAAAATTAGT GAATGAAGTA ACTGAATTTG CAAAAACATG TGTAGCTGAT 240 GAGTCAGCTG AAAATTGTGA CAAATCACTT CATACCCTTT TTGGAGACAA ATTATGCACA 300 GTTGCAACTC TTCGTGAAAC CTATGGTGAA ATGGCTGACT GCTGTGCAAA ACAAGAACCT 360 GAGAGAAATG AATGCTTCTT GCAACACAAA GATGACAACC CAAACCTCCC CCGATTGGTG 420. AGACCAGAGG TTGATGTGAT GTGCACTGCT TTTCATGACA ATGAAGAGAC ATTTTTGAAA 480 AAATACTTAT ATGAAATTGC CAGAAGACAT CCTTACTTTT ATGCCCCGGA ACTCCTTTTC 540 TTTGCTAAAA GGTATAAAGC TGCTTTTACA GAATGTTGCC AAGCTGCTGA TAAAGCTGCC 600 TGCCTGTTGC CAAAGCTCGA TGAACTTCGG GATGAAGGGA AGGCTTCGTC TGCCAAACAG 660 GCTCGCCTGA GCCAGAGATT TCCCAAAGCT GAGTTTGCAG AAGTTTCCAA GTTAGTGACA 780 GATCTTACCA AAGTCCACAC GGAATGCTGC CATGGAGATC TGCTTGAATG TGCTGATGAC 840 AGGGCGGACC TTGCCAAGTA TATCTGTGAA AATCAGGATT CGATCTCCAG TAAACTGAAG 900 GAATGCTGTG AAAAACCTCT GTTGGAAAAA TCCCACTGCA TTGCCGAAGT GGAAAATGAT 960 GAGATGCCTG CTGACTTGCC TTCATTAGCT GCTGATTTTG TTGAAAGTAA GGATGTTTGC 1020 AAAAACTATG CTGAGGCAAA GGATGTCTTC CTGGGCATGT TTTTGTATGA ATATGCAAGA 1080 AGGCATCCTG ATTACTCTGT CGTGCTGCTG CTGAGACTTG CCAAGACATA TGAAACCACT 1140 CTAGAGAAGT GCTGTGCCGC TGCAGATCCT CATGAATGCT ATGCCAAAGT GTTCGATGAA 1200 TTTAAACCTC TTGTGGAAGA GCCTCAGAAT TTAATCAAAC AAAACTGTGÁ GCTTTTTAAG 1260 CAGCTTGGAG AGTACAAATT CCAGAATGCG CTATTAGTTC GTTACACCAA GAAAGTACCC 1320 CAAGTGTCAA CTCCAACTCT TGTAGAGGTC TCAAGAAACC TAGGAAAAGT GGGCAGCAAA 1380 TGTTGTAAAC ATCCTGAAGC AAAAAGAATG CCCTGTGCAG AAGACTATCT ATCCGTGGTC 1440 CTGAACCAGT TATGTGTGTT GCATGAGAAA ACGCCAGTAA GTGACAGAGT CACAAAATGC 1500 TGCACAGAGT CCTTGGTGAA CAGGCGACCA TGCTTTTCAG CTCTGGAAGT CGATGAAACA 1560 TACGTTCCC A AAGAGTTTAA TGCTGAAACA TTCACCTTCC ATGCAGATAT ATGCACACTT 1620 TCTGAGAAGG AGAGACAAAT CAAGAAACAA ACTGCACTTG TTGAGCTTGT GAAACACAAG 1680 CCCAAGGCAA CAAAAGAGCA ACTGAAAGCT GTTATGGATG ATTTCGCAGC TTTTGTAGAG 1740 AAGTGCTGCA AGGCTGACGA TAAGGAGACC TGCTTTGCCG AGGAGGGTAA AAAACTTGTT 1800 GCTGCAAGTC AAGCTGCCTT AGGCTTA 1827 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 773 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal [xi) DESCRIPTION OF SEQUENCE: SEQ] CD NO: 6: ATGGCTCCTG CCATGGAATC CCCTACTCTA CTGTGTGTAG CCTTACTGTT CTTCGCTCCA 60 GATGGCGTGT TAGCAGTCCC TCAGAAACCT AAGGTCTCCT TGAACCCTCC ATGGAATAGA 120 ATATTTAAAG GAGAGAATGT GACTCTTACA TGTAATGGGA ACAATTTCTT TGAAGTCAGT 180 TCCACCAAAT GGTTCCACAA TGGCAGCCTT TCAGAAGAGA CAAATTCAAG TTTGAATATT 240 GTGAATGCCA AATTTGAAGA CAGTGGAGAA TACAAATGTC AGCACCAACA AGTTAATGAG 300 AGTGAACCTG TGTACCTGGA AGTCTTCAGT GACTGGCTGC TCCTTCAGGC CTCTGCTGAG 360 GTGGTGATGG AGGGCCAGCC CCTCTTCCTC AGGTGCCATG GTTGGAGGAA CTGGGATGTG 420 TACAAGGTGA TCTATTATAA GGATGGTGAA GCTCTCAAGT ACTGGTATGA GAACCACAAC 480 ATCTCCATTA CAAATGCCAC AGTTGAAGAC AGTGGAACCT ACTACTGTAC GGGCAAAGTG 540 TGGCAGCTGG ACTATGAGTC TGAGCCCCTC AACATTACTG TAATAAAAGC TCCGCGTGAG 600 AAGTACTGGC TACAATTTTT TATCCCATTG TTGGTGGTGA TTCTGTTTGC TGTGGACACA 660 GGATTATTTA TCTCAACTCA GCAGCAGGTC ACATTTCTCT TGAAGATTAA GAGAACCAGG 720 AAAGGCTTCA GACTTCTGAA CCCACATCCT AAGCCAAACC CCAAAAACAA CTG 773

Claims (10)

1. A fusion polypeptide, or salt thereof, comprising at least one immunoglobulin E binding domain (IgE) fused to at least one component of human serum albumin (HSA), or a physiologically functional equivalent thereof.

2. A polypeptide according to claim 1, or a salt thereof, wherein: the IgE binding domain is IgER or pre-IgER (SEQ. ID NO: 1); Or the HSA component is HSA-I, prepro-HSA-I, or HSA-II (SEQ ID NO: 2).

3. A polypeptide according to claim 1, or a salt thereof, which is the polypeptide of Example 7, including its leader sequence (SEQ ID NO: 3), or the polypeptide of the Example 7 in the mature form (residues Val26-Leu978 of SEQ ID NO: 3).

4. A polynucleotide that is an intermediate in the preparation of a polypeptide or a salt thereof according to claim 1.

5. A process for the preparation of a polypeptide or a salt thereof, according to claim 1, which comprises: (a) transforming a host cell with a vector comprising DNA encoding a polypeptide according to claim 1, or a physiologically functional equivalent thereof; (b) expressing the polypeptide or its physiologically functional equivalent in that cell, whereby, the physiologically equivalent functional polypeptide is modified to produce a fusion polypeptide as defined in claim 1; and (c) recovering the resulting polypeptide from the host cell, optionally in the form of a salt thereof.

6. A vector comprising DNA encoding a polypeptide or a salt thereof, according to claim 1, or encoding a physiologically functional equivalent thereof; or a non-human somatic recombinant or transgenic animal that expresses a polypeptide or a salt thereof according to claim 1, or a physiologically functional equivalent thereof.

7. A polypeptide according to claim 1, or a pharmaceutically acceptable salt thereof, for use as a medicament.

8. A pharmaceutical composition comprising a polypeptide according to claim 1, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or diluent.

9. The use of a polypeptide or salt thereof according to claim 1, in an in vitro diagnostic assay, to determine the level of IgE or autoantibodies for FceRI in a biological sample obtained from a patient human . A method for performing gene therapy in humans, which comprises removing the somatic cells of a patient, then genetically modifying them in culture by inserting a polynucleotide according to claim 4, and reintroducing the resulting modified cells into the patient , whereby, the cells of the patient express a polypeptide according to claim 1. SUMMARY Fusion polypeptides and salts thereof comprising at least one IgE binding domain fused to at least one component of human serum albumin, optionally by means of a peptide linker, and in particular, dimeric fusion polypeptides comprising proteins of human serum albumin fused, in each of its amino and carboxyl terms, with an extracellular domain of the a chain of the human high affinity receptor for IgE (FceRIc.); a process for the preparation thereof, functionally equivalent polypeptides which are intermediates in their preparation, and polynucleotide and oligonucleotide intermediates and vectors therefor. These are indicated for use in the prevention and / or treatment of IgE-mediated allergic diseases and related disorders, such as atopic dermatitis, atopic asthma, and chronic urticaria. * *