The present invention comprises a fusion molecule comprising a Fc.epsilon. fragment sequence including functionally active CH2, CH3 and CH4 domains of the constant region of an IgE heavy chain (CH.epsilon.2-CH.epsilon.3-CH.epsilon.4 sequence) linked at its C-terminus to the N-terminus of a second polypeptide including functionally active hinge, CH2 and CH3 domains of the constant region of an IgG.sub.1 heavy chain (.gamma.hinge-CH.gamma.2-CH.gamma.3 sequence), pharmaceutical compositions comprising the fusion molecule and methods of treatment using the fusion molecule.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides novel fusion compounds that have the ability to crosslink Fc.gamma. receptors with Fc.epsilon. receptors and block Fc.epsilon. receptor-mediated biological activities, as well as methods for using such compounds, and compositions and articles of manufacture comprising them. The invention also provides compositions and methods suitable for the prevention or treatment of immune-mediated diseases.

One aspect of the invention concerns an isolated fusion molecule comprising a Fc.epsilon. fragment functionally connected at its carboxy terminus to an Fc.gamma.1 fragment. It has been found that connecting an Fc.gamma.1 fragment to the carboxy terminus of an Fc.epsilon. fragment provides a fusion protein with enhanced properties.

In another embodiment, the Fc.gamma.1 fragment comprises an amino acid sequence having at least about 85% identity to the hinge-CH2-CH3 domain amino acid sequence of SEQ ID NO: 3, at least about 90% identity, at least about 95% identity, or at least about 98% identity. In another embodiment, the Fc.gamma.1 fragment comprises an amino acid sequence having at least about 85% identity to the CH1-hinge-CH2-CH3 domain amino acid sequence of SEQ ID NO: 2, at least about 90% identity, at least about 95% identity, or at least about 98% identity. In still other embodiments, the Fc.gamma.1 fragment comprises a least part of the CH2 and CH3 domains of a native human IgG.sub.1 constant region, or additionally comprises a least part of the hinge of a native human IgG.sub.1 constant region. Alternatively, the Fc.gamma. fragment sequence comprises at least part of the hinge, CH2 and CH3 domains of a native human IgG.sub.1 heavy chain constant region in the absence of a functional CH1 region, and alternatively still, the Fc.gamma.1 fragment comprises an amino acid sequence encoded by a nucleic acid hybridizing under stringent conditions to the complement of the IgG heavy chain constant region nucleotide sequence of SEQ ID NO: 1.

In another embodiment, the Fc.epsilon. fragment comprises an amino acid sequence having at least about 85% identity to the CH2-CH3-CH4 domain amino acid sequence of SEQ ID NO: 6, at least about 90% identity, at least about 95% identity, or at least about 98% identity. In still other embodiments, the Fc.epsilon. fragment comprises a least part of the CH2, CH3 and CH4 domains of a native human IgE constant region. Alternatively, the Fc.epsilon. fragment comprises at least part of the CH2, CH3 and CH4 domains of a native human IgE heavy chain constant region in the absence of a functional CH1 region, and alternatively still, the Fc.epsilon. fragment comprises an amino acid sequence encoded by a nucleic acid hybridizing under stringent conditions to the complement of the IgE heavy chain constant region nucleotide sequence of SEQ ID NO: 4.

In another embodiment, the fusion molecule comprises the polypeptides sequence CH.epsilon.2-CH.epsilon.3-CH.epsilon.4-.gamma.hinge-CH.gamma.2CH- .gamma.3. In another embodiment the fusion molecule comprises the sequence SEQ ID NO:19.

In some embodiments, the Fc.epsilon. and the Fc.gamma.1 polypeptide sequences may be functionally connected via a linker, e.g., a polypeptide linker. The length of the polypeptide linker typically is about 1 to 25 amino acid residues, or from about 2 to 25 amino acid residues. In one embodiment, the linker may replace the .gamma.hinge sequence. In another embodiment, the linker may functionally connect the CH.epsilon.3 to the .gamma.hinge. In other embodiments, the polypeptide linker sequence comprises at least one endopeptidase recognition motif. In other embodiments, the polypeptide linker sequence comprises a plurality of endopeptidase recognition motifs, and these endopeptidase motifs may include cysteine, aspartate or asparagine amino acid residues. In other embodiments the linker may comprise amino acids encoded by a nuecleic acid restriction enzyme site.

In other embodiments, the fusion molecule comprises at least one amino-terminal ubiquitination target motif.

In a further aspect, the present invention provides isolated nucleic acid molecules encoding a fusion molecule of the present invention. The invention also provides vectors and host cells comprising these nucleic acids.

In a further aspect, the invention concerns a pharmaceutical composition comprising a fusion molecule as hereinabove defined in admixture with a pharmaceutically acceptable excipient or ingredient. In a still further aspect, the invention concerns an article of manufacture comprising a container, a fusion molecule as hereinabove defined within the container, and a label or package insert on or associated with the container. The label or package insert comprises instructions for the treatment or prevention of an immune disease.

In a further aspect, the present invention concerns methods for the treatment or prevention of immune-mediated diseases, where the subject is administered a fusion polypeptide as-described herein.

In another aspect, the invention provides a method for the treatment or prevention of symptoms resulting from a type I hypersensitivity reaction in a subject comprising administering at least one fusion molecule of the present invention to the subject. In another embodiment, the type I hypersensitivity reaction is an anaphylactic response. In another embodiment of this method, the type I hypersensitivity symptoms being prevented comprise an anaphylactic response.

In one aspect of this method of the invention, the immunotherapy received by the subject is for the treatment of type I hypersensitivity-mediated disease or autoimmune disease. In various embodiments of this method, the fusion molecule is administered to the subject prior to the subject receiving immunotherapy, co-administered to the subject during immunotherapy, or administered to the subject after the subject receives the immunotherapy.

In yet another aspect, the invention provides a method for the prevention of a type I hypersensitivity disease in a subject receiving immunotherapy, comprising administering at least one fusion molecule of the present invention to the subject.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention is directed to an isolated fusion molecule comprising an Fc.epsilon. fragment functionally connected at the carboxy end of the Fc.epsilon. fragment to an Fc.gamma.1 fragment.

In one embodiment, the fusion molecules of the present invention comprise a Fc.epsilon. fragment sequence including functionally active CH2, CH3 and CH4 domains of the constant region of an IgE heavy chain (CH.epsilon.2-CH.epsilon.3-CH.epsilon.4 sequence) linked at its C-terminus to the N-terminus of a second polypeptide including functionally active hinge, CH2 and CH3 domains of the constant region of an IgG.sub.1 heavy chain (.gamma.hinge-CH.gamma.2-CH.gamma.3 sequence).

In one embodiment, the IgE heavy chain constant region sequence (or a homologous sequence) is fused C-terminally to the N-terminus of the IgG.sub.1 heavy chain constant region sequence (or a homologous sequence). The fusion molecules may also comprise repeats of identical or different IgG and/or IgE heavy chain constant region sequences. For example, a IgE heavy chain constant region sequence can be followed by two repeats of IgG.sub.1 heavy chain constant region sequences (EGG structure), or two repeats of identical or different IgG heavy chain constant region sequences may flank an IgE heavy chain constant region sequence (GEG structure), etc. Fusion molecules comprising more than one binding sequence for a target receptor (e.g. an Fc.gamma.RIIb receptor) are expected to have superior biological, e.g. anti-allergic properties.

In all embodiments, the two polypeptide sequences are functionally connected, which means that they retain the ability to bind to the respective native receptors, such as a native IgG inhibitory receptor, e.g. a low-affinity Fc.gamma.RIIb receptor, and to a native high-affinity IgE receptor, e.g. Fc.epsilon.RI or low-affinity IgE receptor, e.g. Fc.epsilon.RII as desired. As a result, the fusion molecules, comprising the Fc.epsilon. fragment and the Fc.gamma. fragment functionally connected to each other, are capable of cross-linking the respective native receptors, such as Fc.gamma.RIIb and Fc.epsilon.RI or Fc.gamma.RIIb and Fc.epsilon.RII. In order to achieve a functional connection between the two binding sequences within the fusion molecules of the invention, it is preferred that they retain the ability to bind to the corresponding receptor with a binding affinity similar to that of a native immunoglobulin ligand of that receptor.

In one embodiment, the Fc.epsilon. fragment present in the fusion molecules of the invention has at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity with the amino acid sequence of the CH2-CH3-CH3 region of a native IgE, e.g.native human IgE. In one embodiment, the sequence identity is defined with reference to the human CH.gamma.2-CH.gamma.3-CH.epsilon.4 sequence of SEQ ID NO: 6.

In one embodiment, the Fc.gamma. fragment present in the fusion molecules of the invention has at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity with the amino acid sequence of the hinge-CH2-CH3 region of a native IgG.sub.1, e.g. native human IgG.sub.1. In one embodiment, the Fc.gamma. fragment present in the fusion molecules of the invention has at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity with the amino acid sequence of the CH1-hinge-CH2-CH3 region of a native IgG.sub.1, e.g. native human IgG.sub.1. In one embodiment, the sequence identity is defined with reference to the human .gamma.hinge-CH.gamma.2-CH.gamma.3 sequence of SEQ ID NO: 3.

It is required that Fc.epsilon. fragment and the Fc.gamma.1 fragment retain the ability to bind to the corresponding native receptor, such as a native high-affinity IgE receptor (e.g. Fc.epsilon.RI) or native low-affinity IgE receptor (Fc.epsilon.RII, CD23) and a native IgG inhibitory receptor (e.g. Fc.gamma.RIIb), respectively. The receptor binding domains within the native IgG and IgE heavy chain constant region sequences have been identified. It has been reported that the CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors, including the Fc.gamma.RIIb low-affinity receptor (Wines et al., J. Immunol. 164(10):5313-5318 (2000)). Based on Fc.epsilon.RI binding studies, Presta et al., J. Biol. Chem. 269:26368-26373 (1994) proposed that six amino acid residues (Arg-408, Ser-411, Lys-415, Glu-452, Arg-465, and Met-469) located in three loops, C-D, E-F, and F-G, computed to form the outer ridge on the most exposed side of the human IgE heavy chain CH3 domain, are involved in binding to the high-affinity receptor Fc.epsilon.RI, mostly by electrostatic interactions. Helm et al., J. Cell Biol. 271(13):7494-7500 (1996), reported that the high-affinity receptor binding site in the IgE molecule includes the Pro343-Ser353 peptide sequence within the CH3 domain of the IgE heavy chain, but sequences N-- or C-terminal to this core peptide are also necessary to provide structural scaffolding for the maintenance of a receptor binding conformation. In particular, they found that residues, including His, in the C-terminal region of the .epsilon.-chain make an important contribution toward the maintenance of the high-affinity of interaction between IgE and Fc.epsilon.RI. The Fc.epsilon. and Fc.gamma.1 polypeptide sequences within the fusion molecules of the invention are designed to bind to residues within such binding regions.

Based on this knowledge, the amino acid sequence variants may be designed to retain the native amino acid residues essential for receptor binding, or to perform only conservative amino acid alterations (e.g. substitutions) at such residues.

In making amino acid sequence variants that retain the required binding properties of the corresponding native sequences, the hydropathic index of amino acids may be considered. For example, it is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score without significant change in biological activity. Thus, isoleucine, which has a hydrophatic index of +4.5, can generally be substituted for valine (+4.2) or leucine (+3.8), without significant impact on the biological activity of the polypeptide in which the substitution is made. Similarly, usually lysine (-3.9) can be substituted for arginine (-4.5), without the expectation of any significant change in the biological properties of the underlying polypeptide.

Other considerations for choosing amino acid substitutions include the similarity of the side-chain substituents, for example, size, electrophilic character, charge in various amino acids. In general, alanine, glycine and serine; arginine and lysine; glutamate and aspartate; serine and threonine; and valine, leucine and isoleucine are interchangeable, without the expectation of any significant change in biological properties. Such substitutions are generally referred to as conservative amino acid substitutions, and, as noted above, are one type of substitutions within the polypeptides of the present invention.

Alternatively or in addition, the amino acid alterations may serve to enhance the receptor binding properties of the fusion molecules of the invention. Variants with improved receptor binding and, as a result, superior biological properties can be readily designed using standard mutagenesis techniques, such as alanine-scanning mutagenesis, PCR mutagenesis or other mutagenesis techniques, coupled with receptor binding assays, such as the assay discussed below or described in the Example.

The fusion molecules of the present invention are typically produced and act as homodimers or heterodimers, comprising two of the fusion molecules hereinabove described covalently linked to each other. The covalent attachment may be achieved via one or more disulfide bonds. For example, the prototype protein designated E2G is produced as a homodimer composed of the two CH.epsilon.2-CH.epsilon.3-CH.epsilon.4-.gamma..sub.1hinge-CH .gamma..sub.12-CH .gamma..sub.13-chains connected to each other by interchain disulfide bonds, to provide an immunoglobulin-like structure. It is also possible to produce heterodimers, in which two different fusion molecules are linked to each other by one or more covalent linkages, e.g. disulfide bond(s). Such bifunctional structures might be advantageous in that they are able to cross-link the same or different Ig.epsilon.R(s) with different inhibitory receptors.

Receptor binding can be tested using any known assay method, such as competitive binding assays, direct and indirect sandwich assays. Thus, the binding of Fc .gamma..sub.1 polypeptide included in the fusion molecules herein to a low-affinity IgG inhibitory receptor, or the binding of Fc.epsilon. polypeptide included herein to a high-affinity or low-affinity IgE receptor can be tested using conventional binding assays, such as competitive binding assays, including RIAs and ELISAs. Ligand/receptor complexes can be identified using traditional separation methods as filtration, centrifugation, flow cytometry, and the results from the binding assays can be analyzed using any conventional graphical representation of the binding data, such as Scatchard analysis. The assays may be performed, for example, using a purified receptor, or intact cells expressing the receptor. One or both of the binding partners may be immobilized and/or labeled. A particular cell-based binding assay is described in the Example below.

In one embodiment, the IgE constant region sequence is directly functionally connected to the .gamma.hinge sequence of the Fc.gamma.1 constant region.

In another embodiment, the Fc.epsilon. and the Fc.gamma.1 polypeptide sequences may be connected by a polypeptide linker replacing the hinge region of the IgG1 fragment or in addition to the hinge region. The polypeptide linker functions as a "spacer" whose function is to separate the Fc.gamma. receptor binding domain and the Fc.epsilon. receptor binding domain so that they can independently assume their proper tertiary conformation. The polypeptide linker usually comprises between about 1 and about 25 residues or from about 2 to about 25 residues. The polypeptide linker may contain at least about 10, or at least about 15 amino acids. The polypeptide linker may be composed of amino acid residues which together provide a hydrophilic, relatively unstructured region. Linking amino acid sequences with little or no secondary structure work well. The specific amino acids in the spacer can vary, however, cysteines should be avoided. Suitable polypeptide linkers are, for example, disclosed in WO 88/09344 (published on Dec. 1, 1988), as are methods for the production of multifunctional proteins comprising such linkers.

The IgG1 and IgE constant region sequences may connected by a non-polypeptide linker. Such linkers may, for example, be residues of covalent bifunctional cross-linking agents capable of linking the two sequences without the impairment of the receptor (antibody) binding function. The bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g. amino, sulfhydryl, guanidino, indole, carboxyl specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied. A majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group (for review, see Ji, T. H. "Bifunctional Reagents" in: Meth. Enzymol. 91:580-609 (1983)).

In a further specific embodiment, the two polypeptide sequences (including variants of the native sequences) are dimerized by amphiphilic helices. It is known that recurring copies of the amino acid leucine (Leu) in gene regulatory proteins can serve as teeth that "zip" two protein molecules together to provide a dimer. For further details about leucine zippers, which can serve as linkers for the purpose of the present invention, see for example: Landschulz, W. H., et al. Science 240:1759-1764 (1988); O'Shea, E. K. et al., Science 243: 38-542 (1989); McKnight, S. L., Scientific American 54-64, April 1991; Schmidt-Dorr. T. et al., Biochemistry 30:9657-9664 (1991); Blondel, A. and Bedouelle, H. Protein Engineering 4:457-461 (1991), and the references cited in these papers.

In a different approach, the two polypeptide sequences (including variants of the native sequences) are linked via carbohydate-directed bifunctional cross-linking agents, such as those disclosed in U.S. Pat. No. 5,329,028.

The cross-linking of an inhibitory receptor expressed on mast cells and/or basophils, such as IgG inhibitory receptors, e.g. Fc.gamma.RIIb to a high-affinity IgE receptor, e.g. Fc.epsilon.RI or low-affinity IgE receptor, e.g. Fc.epsilon.RII, inhibits Fc.epsilon.R mediated biological responses. Such biological responses are the mediation of an allergic reactions or autoimmune reactions via Fc.epsilon.R, including, without limitation, conditions associated with IgE mediated reactions, such as, for example, asthma, allergic rhinitis, food allergies, chronic urticaria and angioedema, allergic reactions to hymenophthera (e.g. bee and yellow jacket) stings or medications such as penicillin up to and including the severe physiological reaction of anaphylactic shock.

2. Preparation of the Fusion Molecules

When the fusion molecules are polypeptides, in which the Fc.epsilon. and Fc.gamma.1 polypeptide sequences are directly fused or functionally connected by a polypeptide linker, they can be prepared by well known methods of recombinant DNA technology or traditional chemical synthesis. If the polypeptides are produced by recombinant host cells, cDNA encoding the desired polypeptide of the present invention is inserted into a replicable vector for cloning and expression. As discussed before, the nucleotide and amino acid sequences of native immunoglobulin constant regions, including native IgG and IgE constant region sequences, are well known in the art and are readily available, for example, from Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md. (1991).

Suitable vectors are prepared using standard techniques of recombinant DNA technology, and are, for example, described in "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology", 4 h edition (D. M. Weir & C. C. Blackwell, eds., Blackwell Science Inc., 1987); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991). Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors. After ligation, the vector containing the gene to be expressed is transformed into a suitable host cell.

Host cells can be any eukaryotic or prokaryotic hosts known for expression of heterologous proteins. Accordingly, the polypeptides of the present invention can be expressed in eukaryotic hosts, such as eukaryotic microbes (yeast) or cells isolated from multicellular organisms (mammalian cell cultures), plants and insect cells. Examples of mammalian cell lines suitable for the expression of heterologous polypeptides include monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line 293S (Graham et al, J. Gen. Virol. 36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary (CHO) cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216 [1980]; monkey kidney cells (CV1-76, ATCC CCL 70); African green monkey cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); human lung cells (W138, ATCC CCL 75); and human liver cells (Hep G2, HB 8065). In general myeloma cells, in particular those not producing any endogenous antibody, e.g. the non-immunoglobulin producing myelome cell line SP2/0, may be used for the production of the fusion molecules herein.

Eukaryotic expression systems employing insect cell hosts may rely on either plasmid or baculoviral expression systems. The typical insect host cells are derived from the fall army worm (Spodoptera frugiperda). For expression of a foreign protein these cells are infected with a recombinant form of the baculovirus Autographa californica nuclear polyhedrosis virus which has the gene of interest expressed under the control of the viral polyhedrin promoter. Other insects infected by this virus include a cell line known commercially as "High 5" (Invitrogen) which is derived from the cabbage looper (Trichoplusia ni). Another baculovirus sometimes used is the Bombyx mori nuclear polyhedorsis virus which infect the silk worm (Bombyx mori). Numerous baculovirus expression systems are commercially available, for example, from Invitrogen (Bac-N-Blue.TM.), Clontech (BacPAK.TM. Baculovirus Expression System), Life Technologies (BAC-TO-BAC.TM.), Novagen (Bac Vector System.TM.), Pharmingen and Quantum Biotechnologies). Another insect cell host is common fruit fly, Drosophila melanogaster, for which a transient or stable plasmid based transfection kit is offered commercially by Invitrogen (The DES.TM. System).

Saccharomyces cerevisiae is the most commonly used among lower eukaryotic hosts. However, a number of other genera, species, and strains are also available and useful herein, such as Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol. 28:165-278 (1988)). Yeast expression systems are commercially available, and can be purchased, for example, from Invitrogen (San Diego, Calif.). Other yeasts suitable for bi-functional protein expression include, without limitation, Kluyveromyces hosts (U.S. Pat. No. 4,943,529), e.g. Kluyveromyces lactis; Schizosaccharomyces pombe (Beach and Nurse, Nature 290:140 (1981); Aspergillus hosts, e.g. A. niger (Kelly and Hynes, EMBO J. 4:475-479 (1985])) and A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun. 112:284-289 (1983)), and Hansenula hosts, e.g. Hansenula polymorpha. Yeasts rapidly grow on inexpensive (minimal) media, the recombinant can be easily selected by complementation, expressed proteins can be specifically engineered for cytoplasmic localization or for extracellular export, and they are well suited for large-scale fermentation.

Prokaryotes may be hosts for the initial cloning steps, and are useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated. E. coli strains suitable for the production of the peptides of the present invention include, for example, BL21 carrying an inducible T7 RNA polymerase gene (Studier et al., Methods Enzymol. 185:60-98 (1990)); AD494 (DE3); EB105; and CB (E. coli B) and their derivatives; K12 strain 214 (ATCC 31,446); W3110 (ATCC 27,325); X1776 (ATCC 31,537); HB101 (ATCC 33,694); JM101 (ATCC 33,876); NM522 (ATCC 47,000); NM538 (ATCC 35,638); NM539 (ATCC 35,639), etc. Many other species and genera of prokaryotes may be used as well. Indeed, the peptides of the present invention can be readily produced in large amounts by utilizing recombinant protein expression in bacteria, where the peptide is fused to a cleavable ligand used for affinity purification.

Suitable promoters, vectors and other components for expression in various host cells are well known in the art and are disclosed, for example, in the textbooks listed above.

Whether a particular cell or cell line is suitable for the production of the polypeptides herein in a functionally active form, can be determined by empirical analysis. For example, an expression construct comprising the coding sequence of the desired molecule may be used to transfect a candidate cell line. The transfected cells are then grown in culture, the medium collected, and assayed for the presence of secreted polypeptide. The product can then be quantitated by methods known in the art, such as by ELISA with an antibody specifically binding the IgG, IgE portion of the molecule.

In certain instances, especially if the two polypeptide sequences making up the bifunctional molecule of the present invention are connected with a non-polypeptide linker, it may be advantageous to individually synthesize the IgE and IgG1 polypeptide sequences, e.g. by any of the recombinant approaches discussed above, followed by functionally linking the two sequences.

Alternatively, the two polypeptide sequences, or the entire molecule, may be prepared by chemical synthesis, such as solid phase peptide synthesis. Such methods are well known to those skilled in the art. In general, these methods employ either solid or solution phase synthesis methods, described in basic textbooks, such as, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984) and G. Barany and R. B. Merrifield, The Peptide: Analysis Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution synthesis.

The fusion molecules of the present invention may include amino acid sequence variants of native immunoglobulin (e.g. IgG and/or IgE). Such amino acid sequence variants can be produced by expressing the underlying DNA sequence in a suitable recombinant host cell, or by in vitro synthesis of the desired polypeptide, as discussed above. The nucleic acid sequence encoding a polypeptide variant may be prepared by site-directed mutagenesis of the nucleic acid sequence encoding the corresponding native (e.g. human) polypeptide. Site-directed mutagenesis using polymerase chain reaction (PCR) amplification may be used.(see, for example, U.S. Pat. No. 4,683,195 issued Jul. 28, 1987; and Current Protocols In Molecular Biology, Chapter 15 (Ausubel et al., ed., 1991). Other site-directed mutagenesis techniques are also well known in the art and are described, for example, in the following publications: Current Protocols In Molecular Biology, supra, Chapter 8; Molecular Cloning: A Laboratory Manual., 2nd edition (Sambrook et al., 1989); Zoller et al., Methods Enzymol. 100:468-500 (1983); Zoller & Smith, DNA 3:479-488 (1984); Zoller et al., Nucl. Acids Res., 10:6487 (1987); Brake et al., Proc. Natl. Acad. Sci. USA 81:4642-4646 (1984); Botstein et al., Science 229:1193 (1985); Kunkel et al., Methods Enzymol. 154:367-82 (1987), Adelman et al., DNA 2:183 (1983); and Carter et al., Nucl. Acids Res., 13:4331 (1986). Cassette mutagenesis (Wells et al., Gene 34:315 [1985]), and restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 [1986]) may also be used.

Amino acid sequence variants with more than one amino acid substitution may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously, using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from one another (e.g. separated by more than ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. The alternative method involves two or more rounds of mutagenesis to produce the desired mutant.

The polypeptides of the invention can also be prepared by the combinatorial peptide library method disclosed, for example, in International Patent Publication PCT WO 92/09300. This method is particularly suitable for preparing and analyzing a plurality of molecules, that are variants of given predetermined sequences, and is, therefore, particularly useful in identifying polypeptides with improved biological properties, which can then be produced by any technique known in the art, including recombinant DNA technology and/or chemical synthesis.

3. Therapeutic Uses of the Fusion Molecules of the Invention

The present invention provides a new therapeutic strategy for treating immediate hypersensitivity diseases mediated through the high-affinity IgE receptor. In particular, the invention provides compounds for use in the treatment of both allergic diseases where IgE bridging of Fc.epsilon.R receptors occurs and autoimmune disorders where autoantibodies bind to the Fc.epsilon.R.

4. Nature of the Diseases Targeted

Following the Gell and Coombs Classification, allergic reactions are classified depending on the type of immune response induced and the resulting tissue damage that develops as a result of reactivity to an antigen. A Type I reaction (immediate hypersensitivity) occurs when an antigen (called an allergen in this case) entering the body encounters mast cells or basophils which are sensitized as a result of IgE to that antigen being attached to its high-affinity receptor, Fc.epsilon.RI. Upon reaching the sensitized mast cell, the allergen cross-links IgE bound to Fc.epsilon.RI, causing an increase in intracellular calcium (Ca.sup.2+) that triggers the release of pre-formed mediators, such as histamine and proteases, and newly synthesized, lipid-derived mediators such as leukotrienes and prostaglandins. These autocoids produce the acute clinical symptoms of allergy. The stimulated basophils and mast cells will also produce and release proinflammatory mediators, which participate in the acute and delayed phase of allergic reactions.

As discussed before, a large variety of allergens has been identified so far, and new allergens are identified, cloned and sequenced practically every day.

Ingestion of an allergen results in gastrointestinal and systemic allergic reactions. The most common food allergens involved are peanuts, shellfish, milk, fish, soy, wheat, egg and tree nuts such as walnuts. In susceptible people, these foods can trigger a variety of allergic symptoms, such as nausea, vomiting, diarrhea, urticaria, angioedema, asthma and full-blown anaphylaxis. Inhalation of airborne allergens results in allergic rhinitis and allergic asthma, which can be acute or chronic depending on the nature of the exposure(s). Exposure to airborne allergens in the eye results in allergic conjunctivitis. Common airborne allergens includes pollens, mold spores, dust mites and other insect proteins that are the most frequent cause of seasonal hay fever and allergic asthma.

Cutaneous exposure to an allergen, e.g. natural rubber latex proteins as found in latex gloves, may result in local allergic reactions manifest as hives (urticaria) at the places of contact with the allergen.

Systemic exposure to an allergen such as occurs with a bee sting, the injection of penicillin, or the use of natural rubber latex (NRL) gloves inside a patient during surgery may result in, cutaneous, gastrointestinal and respiratory reactions up to and including airway obstruction and full blown anaphylaxis. Hymenoptera stings are insects that commonly cause allergic reactions, often leading the anaphylactic shock. Examples include various bees including honeybees, yellow jackets, yellow hornets, wasps and white-faced hornets. Certain ants known as fire ants (Solenopsis invicta) are an increasing cause of allergy in the US as they expand their range in this country. Proteins in NRL gloves have become an increasing concern to health care workers and patients and at present, there is no successful form of therapy for this problem except avoidance.

5. Uses of Compounds for Targeted Diseases

The compounds disclosed herein can be used to acutely or chronically inhibit IgE mediated reaction to major environmental and occupational allergens, can be used to provide protection for allergy vaccination (immunotherapy) to induce a state of non-allergic reactivity during treatment for specific allergens and can also have a prophylactic effect against allergic disease by preventing allergic sensitization to environmental and occupational allergens when administered to at-risk individuals (e.g., those at genetic risk of asthma and those exposed to occupational allergens in the workplace).

The bifunctional epsilon-gamma compounds described can be used to prevent allergic reactions to any specific allergen or group of allergens. By occupying a critical number of Fc.epsilon.RI receptors, these molecules will inhibit the ability of basophils and mast cells to react to any allergen so as to prevent including, without limitation, asthma, allergic rhinitis, atopic dermatitis, food allergies, urticaria, angioedema, up to and including anaphylactic shock. Thus these compounds could be used acutely to desensitize a patient so that the administration of a therapeutic agent (e.g. penicillin) can be given safely. Similarly, they can be used to desensitize a patient so that standard allergen vaccination may be given with greater safety, e.g. peanut or latex treatment. They can also be used as chronic therapy to prevent clinical reactivity to prevent environmental allergens such as foods or inhalant allergens.

In addition, the chimeric epsilon-gamma compounds herein hold great promise for the treatment of chronic urticaria and angioedema. Urticaria is a skin symptom that may accompany allergies but often is idiopathic. It is a relatively common disorder caused by localized cutaneous mast cell degranulation, with resultant increased dermal vascular permeability culminating in pruritic wheals. Angioedema is a vascular reaction involving the deep dermis or subcutaneous or submucosal tissues caused by localized mast cell degranulation. This results in tissue swelling that is pruritic or painful. Chronic urticaria and angioedema often occur together although they occur individually as well. These conditions are common and once present for more than six months, they often last a decade or more. Although not fatal, they are very troubling to patients as the frequent recurring attaching disrupt daily activities and thereby result in significant morbidity. Standard therapy is often unsuccessful in these conditions and they are distressing to the point that chemotherapy with cyclosporine and other potent immunosuppressive drugs has recently been advocated. Increasing evidence suggests that as many as 60% of patients with these conditions actually have an autoimmune disease, in which they make functional antibodies against the Fc.epsilon.RI receptor. For further details, see Hide et al., N. Engl. J. Med. 328:1599-1604 (1993); Fiebiger et al., J. Clin. Invest. 96:2606-12 (1995); Fiebiger et a., J. Clin. Invest. 101:243-51 (1998); Kaplan, A. P., Urticaria and Angioedema, In: Inflammation: Basic Principles and Clinical Correlates (Galliin and Snyderman eds.), 3rd Edition, Lippincott & Wilkins, Philadelphia, 1999, pp. 915-928. The fusion molecules of the present invention are believed to form the basis for a novel and effective treatment of these diseases by safely blocking access to the Fc.epsilon.RI.

In addition the chimeric epsilon-gamma compounds herein may be used for the treatment of inflammatory arthritis, e.g. rheumatoid arthritis or other autoimmune conditions depending on the role of mast cells and basophils in those diseases. Mast cells have been historically thought of primarily as a critical component of IgE-mediated allergic diseases through degranulation and cytokine production triggered by allergen driven aggregation of IgE bound to the high affinity IgE receptor (Fc.epsilon.RI). More recent studies have provided evidence that mast cells also may play a key role in autoimmune disease and especially those with autoantibody-dependent immune pathology. Activation and subsequent degranulation of mast cells via Fc.epsilon.RI cross-linking and anaphylatoxins generated through complement pathways are thought to be important for these processes. Thus prevention and/or inhibition of mast cell activation, degranulation and cytokine production provide a potential therapeutic target in autoimmune diseases such as inflammatory arthritis (Benoist and Mathis Arthritis Res. 2000 vol. 2:90-94). In addition to inflammatory arthritis, mast cells appear to be very important in experimental allergic encephalomyelitis (EAE), multiple sclerosis, and certain type of autoimmune skin disease such as bullous pemphigoid.

6. Compositions and Formulations of the Invention

For therapeutic uses, including prevention, the compounds of the invention can be formulated as pharmaceutical compositions in admixture with pharmaceutically acceptable carriers or diluents. Methods for making pharmaceutical formulations are well known in the art.

Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, Pa. 1990. See, also, Wang and Hanson "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers", Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42-2S (1988). A suitable administration format can best be determined by a medical practitioner for each patient individually.

Pharmaceutical compositions of the present invention can comprise a fusion molecule of the present invention along with conventional carriers and optionally other ingredients.

Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, inhalation, or by injection. Such forms should allow the agent or composition to reach a target cell whether the target cell is present in a multicellular host or in culture. For example, pharmacological agents or compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the agent or composition from exerting its effect.

Carriers or excipients can also be used to facilitate administration of the compound. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. The compositions or pharmaceutical compositions can be administered by different routes including, but not limited to, oral, intravenous, intra-arterial, intraperitoneal, subcutaneous, intranasal or intrapulmonary routes. The desired isotonicity of the compositions can be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes.

For systemic administration, injection may be used e.g., intramuscular, intravenous, intra-arterial, etc. For injection, the compounds of the invention are formulated in liquid solutions, such as in physiologically compatible buffers such as Hank's solution or Ringer's solution. Alternatively, the compounds of the invention are formulated in one or more excipients (e.g., propylene glycol) that are generally accepted as safe as defined by USP standards. They can, for example, be suspended in an inert oil, suitably a vegetable oil such as sesame, peanut, olive oil, or other acceptable carrier.

They are suspended in an aqueous carrier, for example, in an isotonic buffer solution at pH of about 5.6 to 7.4. These compositions can be sterilized by conventional sterilization techniques, or can be sterile filtered. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents. Useful buffers include for example, sodium acetate/acetic acid buffers. A form of repository or "depot" slow release preparation can be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many, hours or days following transdermal injection or delivery. In addition, the compounds can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

Alternatively, certain molecules identified in accordance with the present invention can be administered orally. For oral administration, the compounds are formulated into conventional oral dosage forms such as capsules, tablets and tonics.

Systemic administration can also be by transmucosal or transdermal. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be, for example, through nasal sprays or using suppositories.

One route for administration of the compounds of the invention may be inhalation for intranasal and/or intrapulmonary delivery. For administration by inhalation, usually inhalable dry powder compositions or aerosol compositions are used, where the size of the particles or droplets is selected to ensure deposition of the active ingredient in the desired part of the respiratory tract, e.g. throat, upper respiratory tract or lungs. Inhalable compositions and devices for their administration are well known in the art. For example, devices for the delivery of aerosol medications for inspiration are known. One such device is a metered dose inhaler that delivers the same dosage of medication to the patient upon each actuation of the device. Metered dose inhalers typically include a canister containing a reservoir of medication and propellant under pressure and a fixed volume metered dose chamber. The canister is inserted into a receptacle in a body or base having a mouthpiece or nosepiece for delivering medication to the patient. The patient uses the device by manually pressing the canister into the body to close a filling valve and capture a metered dose of medication inside the chamber and to open a release valve which releases the captured, fixed volume of medication in the dose chamber to the atmosphere as an aerosol mist. Simultaneously, the patient inhales through the mouthpiece to entrain the mist into the airway. The patient then releases the canister so that the release valve closes and the filling valve opens to refill the dose chamber for the next administration of medication. See, for example, U.S. Pat. No. 4,896,832 and a product available from 3M Healthcare known as Aerosol Sheathed Actuator and Cap.

Another device is the breath actuated metered dose inhaler that operates to provide automatically a metered dose in response to the patient's inspiratory effort. One style of breath actuated device releases a dose when the inspiratory effort moves a mechanical lever to trigger the release valve. Another style releases the dose when the detected flow rises above a preset threshold, as detected by a hot wire anemometer. See, for example, U.S. Pat. Nos. 3,187,748; 3,565,070; 3,814,297; 3,826,413; 4,592,348; 4,648,393; 4,803,978.

Devices also exist to deliver dry powdered drugs to the patient's airways (see, e.g. U.S. Pat. No. 4,527,769) and to deliver an aerosol by heating a solid aerosol precursor material (see, e.g. U.S. Pat. No. 4,922,901). These devices typically operate to deliver the drug during the early stages of the patient's inspiration by relying on the patient's inspiratory flow to draw the drug out of the reservoir into the airway or to actuate a heating element to vaporize the solid aerosol precursor.

Devices for controlling particle size of an aerosol are also known, see, for example, U.S. Pat. Nos. 4,790,305; 4,926,852; 4,677,975; and 3,658,059.

For topical administration, the compounds of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.

If desired, solutions of the above compositions can be thickened with a thickening agent such as methyl cellulose. They can be prepared in emulsified form, either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents can be employed including, for example, acacia powder, a non-ionic surfactant (such as a Tween), or an ionic surfactant (such as alkali polyether alcohol sulfates or sulfonates, e.g., a Triton).

Compositions useful in the invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components can be mixed simply in a blender or other standard device to produce a concentrated mixture which can then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.

The amounts of various compounds for use in the methods of the invention to be administered can be determined by standard procedures. Generally, a therapeutically effective amount is between about 100 mg/kg and 10.sup.-12 mg/kg depending on the age and size of the patient, and the disease or disorder associated with the patient. Generally, it is an amount between about 0.05 and 50 mg/kg, or between about 1.0 and 10 mg/kg for the individual to be treated. The determination of the actual dose is well within the skill of an ordinary physician.

The compounds of the present invention may be administered in combination with one or more further therapeutic agents for the treatment of IgE-mediated allergic diseases or conditions.

Such further therapeutic agents include, without limitation, corticosteroids, beta-antagonists, theophylline, leukotriene inhibitors, allergen vaccination, and biologic response modifiers such as soluble recombinant human soluble IL-4 receptors (Immunogen), and therapies that target Toll-like receptors. (see, e.g. Barnes, The New England Journal of Medicine 341:2006-2008 (1999)). Thus the compounds of the present invention can be used to supplement traditional allergy therapy, such as corticosteroid therapy performed with inhaled or oral corticosteroids.

7. Articles of Manufacture

The invention also provides articles of manufacture comprising the single-chain fusion compounds herein The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also be an inhalation device such as those discussed above. At least one active agent in the composition is a fusion compound of the invention. The label or package insert indicates that the composition is used for treating the condition of choice, such as an allergic condition, e.g. asthma or any of the IgE-mediated allergies discussed above. The article of manufacture may further comprise a further container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
 

Claim 1 of 1 Claim

1. A fusion molecule comprising the polypeptide sequence CH.epsilon.2-CH.epsilon.3-CH.epsilon.4-.gamma.hinge-CH.gamma.2-CH.gamma.3- , wherein the sequence comprises the sequence of SEQ ID NO: 19.

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