WO1991013984A2 - Proteines de liaison d'interleukine-4 - Google Patents

Proteines de liaison d'interleukine-4 Download PDF

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WO1991013984A2
WO1991013984A2 PCT/US1991/001612 US9101612W WO9113984A2 WO 1991013984 A2 WO1991013984 A2 WO 1991013984A2 US 9101612 W US9101612 W US 9101612W WO 9113984 A2 WO9113984 A2 WO 9113984A2
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cells
protein
human
binding
sequence
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WO1991013984A3 (fr
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William C. Fanslow
Richard J. Armitage
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Immunex Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to proteins which bind Interleukin-4 and, more specifically, to homogeneous preparations of a novel Interleukin-4 binding protein.
  • Interleukin-4 (IL-4) is one of many cytokines which regulate the function of immune cells. IL-4 acts upon immune cells by contacting and binding to a specific receptor (the IL-4 receptor) located on the surface of the immune cell. The binding of IL-4 to its specific receptor triggers a complex series of events within the responsive cell which sends a specific IL-4 signal to the cell. This signal results, among other things, in the proliferation of B cells, T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes.
  • IL-4 stimulates the proliferation of several E -2- and IL-3-dependent cell lines, induces the expression of class II major histocompatibility complex molecules on resting B cells, and enhances the secretion of IgE and IgGl isotypes by stimulated B cells.
  • Human IL-4 has been definitively characterized by recombinant DNA technology and by purification to homogeneity of the natural protein.
  • the immune system can turn against the body with harmful results.
  • autoimmune diseases such as arthritis and diabetes result when a misdirected immune response destroys joint tissues or pancreatic cells.
  • Transplant patients frequently suffer organ rejection as a result of the immune system attacking the transplanted organ as a foreign body.
  • the immune system overreacts to normal encounters with foreign substances, resulting in allergic conditions or asthma.
  • IL-4 plays a specific role in the activation of immune cells to destroy transplanted tissue and in promoting the production of specific types of antibodies, including the IgE antibody involved in allergic and asthmatic reactions.
  • Antagonists to IL-4 activity may therefore be of clinical use in regulating IL-4 mediated immune responses, for example in alleviating or preventing IgE-induced allergic responses or T cell mediated rejection of transplanted tissue.
  • the present invention relates to human IL-4bp- ⁇ . Specifically, the present invention provides substantially homogeneous DL-4bp- ⁇ .
  • the IL-4bp- ⁇ of the present invention binds to IL-4 and is therefore capable of inhibiting IL-4 binding to IL-4 receptor molecules.
  • the IL-4bp- ⁇ of the present invention is a glycoprotein which is isolable from human urine by purification on an IL-4 affinity column and has an apparent molecular weight of about 48,000 M r by SDS-PAGE and a partial N-terminal amino acid sequence of KVKPDMEVGXiYGEAETIL, wherein Xi is any amino acid.
  • the present invention also provides compositions for use in therapy, diagnosis, assay of IL-4bp- ⁇ , or in raising antibodies to IL-4bp- ⁇ , comprising effective quantities of IL-4bp- ⁇ prepared according to the foregoing processes.
  • IL-4bp- ⁇ The identification and purification of IL-4bp- ⁇ enables study of its structure and biological characteristics and the role it plays in the responses of various cell populations to IL-4 or other cytokine stimulation.
  • the figures are histograms showing the results of flow cytometry analysis of target cells which express IL-4 receptors.
  • Samples containing IL-4 binding proteins obtained from urine or cell supernatants were used to inhibit binding of biotinylated IL- 4 to the target cells.
  • the target cells are incubated with the fluorescing agent streptavidin-phycoerythrin (PE).
  • PE streptavidin-phycoerythrin
  • the histograms plot the log fluorescence intensity against the number of cells. A shift in the curve to the right thus indicates an increase in fluorescence intensity as a result of biotinylated IL-4 (not bound to IL-4 bp) binding to the target cells.
  • FIG. 1 shows the fluorescence profile of a PE-negative control.
  • Figure 2 shows an increase in fluorescence intensity as a result of biotinylated IL-4 binding to JMl cells relative to negative controls (Figure 1), while Figure 3 shows fluorescence intensity restored to control levels as a result of the addition of IL-4 affinity column eluate from urine containing IL-4bp- ⁇ inhibiting binding of biotinylated human IL-4 to JMl cells.
  • Figure 4 also shows fluorescence intensity at control levels as a result of the addition of concentrated JMl cell supernatants inhibiting binding of biotinylated human IL-4 to JMl cells, suggesting that the JMl cell line also contains an IL-4 binding protein (referred to herein as IL-4bp- ⁇ ) similar to that purified from urine.
  • IL-4bp- ⁇ an IL-4 binding protein
  • Figures 5-7 show that IL-4bp- ⁇ has no effect on the binding of unrelated proteins.
  • Figure 6 shows an increase in fluorescence intensity (relative to PE negative controls of Figure 5) as a result of biotinylated IL-7 binding to 70Z3 cells.
  • Figure 7 also shows an increase in fluorescence intensity, indicating that addition of IL- 4bp- ⁇ (isolated from urine on the IL-4 affinity column) does not inhibit IL-7 binding to 70Z3 cells.
  • Figures 8-10 show that IL-4bp- ⁇ isolated from urine on the IL-4 affinity column inhibits binding of IL-4 to CTU cells transfected with the human IL-4 receptor gene, as shown by a decrease in fluorescence intensity (Figure 10) relative to positive controls ( Figure 9).
  • Figures 11-13 show no decrease in fluorescence intensity (relative positive controls using biotinylated murine H-4 to bind cells), indicating that IL-4bp- ⁇ (isolated from urine on the IL-4 affinity column) does not inhibit binding of murine EL-4 to CTLL cells transfected with the human IL-4 receptor gene, as shown by the failure of IL-4bp- ⁇ (Figure 13) to restore fluorescence intensity to the level of the negative control ( Figure 11).
  • IL-4bp- ⁇ refers to proteins which are capable of binding Interleukin-4 and have a partial N-terminal amino acid sequence substantially similar to the sequence of amino acids KVKPDMEVGXiYGEAETIL, wherein X ⁇ , is any amino acid.
  • Human IL-4bp- ⁇ has an apparent molecular weight by SDS-PAGE ranging from about 47 to about 50 kilodaltons (kDa), depending on the extent of glycosylation.
  • IL-4bp- ⁇ includes, but is not limited to, equivalent analogs or subunits of EL-4bp- ⁇ which are substantially similar to human IL-4bp- ⁇ and which exhibit at least some biological activity in common with IL-4bp- ⁇ .
  • IL-4bp- ⁇ derived from human urine
  • IL-4bp- ⁇ derived from JM- 1 cells
  • IL-4bp- ⁇ derived from JM- 1 cells
  • substantially similar when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of IL-4bp- ⁇ .
  • Substantially similar analog proteins will be greater than about 30 percent similar to the corresponding sequence of the human IL-4bp- ⁇ . Sequences having lesser degrees of similarity but comparable biological activity are considered to be equivalents.
  • the analog proteins will be greater than about 80 percent similar to the corresponding sequence of human EL-4bp, in which case they are defined as being "substantially identical.”
  • all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered substantially similar to a reference nucleic acid sequence. Percent similarity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482, 1981).
  • the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the sho ⁇ er of the two sequences.
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6145, 1986, as described by Schwartz and Dayhoff, ed., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • Recombinant means that a protein is derived from recombinant (e.g., microbial or mammalian) expression systems.
  • Microbial refers to recombinant proteins made in bacterial or fungal (e.g., yeast) expression systems.
  • recombinant microbial defines a protein produced in a microbial expression system which is essentially free of native endogenous substances. Protein expressed in most bacterial cultures, e.g., E. coli, will be free of glycan. Protein expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells.
  • Bioly active as used throughout the specification as a characteristic of IL-4bp- ⁇ , means that a particular molecule is capable of binding detectable quantities of IL-4 or inhibiting IL-4 from binding to membrane bound IL-4 receptor molecules and transducing an IL-4 signal.
  • Homogeneous refers to isolated and purified human IL-4bp- ⁇ which is substantially free of other human proteins of natural or endogenous origin and contains less than about 1% by mass of protein contaminants residual of production processes. Such compositions, however, can contain other proteins added as stabilizers, carriers, excipients or co-therapeutics. Homogeneous IL-4bp- ⁇ is detectable as a single protein band in a polyacrylamide gel by silver staining.
  • IL-4bp- ⁇ can be purified from human urine as follows. Normal human urine is collected aseptically and stored. The urine is then concentrated using a commercially available protein concentration filter, for example, an Amicon Centriprep-10 ® or Millipore Pellicon ultrafiltration unit. Cellular debris is preferably removed from the retentate by filtering on 0.22 ⁇ filters. The concentrate is then applied to a suitable purification matrix that is capable of purifying protein from a complex solution.
  • the purification matrix comprises EL-4 molecules bound to a suitable support.
  • the purification matrix comprises an anti-IL-4bp- ⁇ antibody molecule bound to a suitable support.
  • the matrix to which the IL-4 or antibody is bound can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
  • one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify an IL-4bp- ⁇ composition.
  • RP-HPLC reversed-phase high performance liquid chromatography
  • the present invention provides substantially homogeneous mammalian IL-4bp- ⁇ proteins. Such proteins are substantially free of contaminating endogenous materials and, optionally, without associated native-pattern glycosylation.
  • Human IL-4bp- ⁇ is isolated from human urine as a glycoprotein having an apparent molecular weight by SDS-PAGE of about 48 kilodaltons (kDa).
  • Derivatives of EL-4bp- ⁇ within the scope of the invention also include various structural forms of the primary protein which retain biological activity. Due to the presence of ionizable amino and carboxyl groups, for example, an IL-4bp- ⁇ protein may be in the form of acidic or basic salts, or in neutral form. Individual amino acid residues may also be modified by oxidation or reduction.
  • the primary amino acid structure may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants.
  • Covalent derivatives are prepared by linking particular functional groups to IL-4bp- ⁇ amino acid side chains or at the N- or C-termini.
  • Other derivatives of IL-4bp- ⁇ within the scope of this invention include covalent or aggregative conjugates of IL-4bp- ⁇ or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions.
  • the conjugated peptide may be a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co-translationally or post- ranslationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast ⁇ -factor leader).
  • IL-4bp- ⁇ protein fusions can comprise peptides added to facilitate purification or identification of IL-4bp- ⁇ (e.g., poly-His).
  • the amino acid sequence of IL-4bp- ⁇ can also be linked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp- Asp-Lys (DYKDDDDK) (Hopp et al., Bio/Technology 6:1204, 1988.)
  • the latter sequence is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein.
  • This sequence is also specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing. Fusion proteins capped with this peptide may also be resistant to intracellular degradation in E. coli.
  • IL-4bp- ⁇ derivatives may also be used as immunogens, reagents in immunoassays, or as binding agents for affinity purification procedures of IL-4 or other binding ligands.
  • IL-4bp- ⁇ derivatives may also be obtained by cross-linking agents, such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at cysteine and lysine residues.
  • IL-4bp- ⁇ proteins may also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen bromide- activated, bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehvde cross-linking).
  • substrates such as cyanogen bromide- activated, bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehvde cross-linking).
  • IL-4bp- ⁇ may be used to selectively bind (for purposes of assay or purification) anti-IL-4bp- ⁇ antibodies or IL-4.
  • the present invention also includes IL-4bp- ⁇ with or without associated native- pattern glycosylation.
  • IL-4bp- ⁇ expressed in yeast or mammalian expression systems, e.g., COS-7 cells, may be similar or significantly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system.
  • Expression of IL-4bp- ⁇ DNAs in bacteria such as E. coli provides non-glycosylated molecules.
  • Functional mutant analogs of mammalian IL-4bp- ⁇ having inactivated N- glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site- specific muta genesis techniques.
  • N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-Ai-Z, where Ai is any amino acid except Pro, and Z is Ser or Thr.
  • asparagine provides a side chain amino group for covalent attachment of carbohydrate.
  • Such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between Ai and Z, or an amino acid other than Asn between Asn and A i .
  • IL-4bp- ⁇ derivatives may also be obtained by mutations of IL-4bp- ⁇ .
  • An IL- 4bp- ⁇ mutant as referred to herein, is a polypeptide homologous to IL-4bp- ⁇ but which has an amino acid sequence different from native IL-4bp- ⁇ because of a deletion, insertion or substitution.
  • mammalian IL-4bp- ⁇ is presumably encoded by multi-exon genes.
  • Alternative mRNA constructs which can be attributed to different mRNA splicing events following transcription, and which share large regions of identity or similarity with the cDNAs claimed herein, are considered to be within the scope of the present invention.
  • Bioequivalent analogs of IL-4bp- ⁇ proteins may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity.
  • cysteine residues can be deleted or replaced with other amino acids to prevent formation of incorrect intramolecular disulfide bridges upon renaturation.
  • Other approaches to mutagenesis involve modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present.
  • substitutions should be made conservatively; i.e., the most preferred substitute amino acids are those having physicochemical characteristics resembling those of the residue to be replaced.
  • Subunits of IL-4bp- ⁇ may be constructed by deleting terminal or internal residues or sequences.
  • the resulting protein is an IL-4bp- ⁇ molecule which retains its ability to bind IL-4.
  • Mutations in nucleotide sequences constructed for expression of analog IL-4bp- ⁇ must, of course, preserve the reading frame phase of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which would adversely affect translation of the EL-4bp- ⁇ mRNA.
  • a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed IL-4bp- ⁇ mutants screened for the desired activity.
  • nucleotide sequence which encodes IL-4bp- ⁇ will be expressed in the final product, for example, nucleotide substitutions may be made to enhance expression, primarily to avoid secondary structure loops in the transcribed mRNA (see EPA 75,444A, incorporated herein by reference), or to provide codons that are more readily translated by the selected host, e.g., the well-known E. coli preference codons for E. coli expression.
  • Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:13, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462, which are incorporated by reference herein.
  • Homogeneous human E -4bp- ⁇ may also be produced by recombinant DNA techniques. Briefly, a recombinant DNA clone encoding IL-4bp- ⁇ is isolated and inserted into an appropriate expression vector, which is then integrated into a host microorganism. The host microorganism expresses the recombinant IL-4bp- ⁇ protein and the protein is purified from cell extracts or cell supernatants.
  • Recombinant production of genes first requires the isolation of a recombinant DNA clone encoding a particular gene or mRNA sequence.
  • recombinant DNA clones are isolated from primary cells or cell lines which constitutively express IL-4bp- ⁇ .
  • the isolated DNA is preferably in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns. Genomic DNA containing the relevant sequences can also be used.
  • Such isolated DNA fragments are inse ⁇ ed into a recombinant expression vector to amplify or express the DNA which encodes the desired protein.
  • Recombinant expression vectors include synthetic or cDNA-derived DNA fragments encoding human IL-4bp- ⁇ or bioequivalent analogs operably linked to suitable transcriptional or translational regulatory or structural elements derived from mammalian, microbial, viral or insect genes.
  • suitable transcriptional or translational regulatory or structural elements include a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation initiation and termination, as described in detail below.
  • the ability to replicate in a host usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated.
  • Structural elements intended for use in yeast expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein may include an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • DNA regions are operably linked when they are functionally related to each other.
  • DNA for a signal peptide is operably linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
  • operably linked means contiguous and, in the case of secretory leaders, contiguous and in reading frame.
  • DNA sequences encoding human IL-4bp- ⁇ which are to be expressed in a microorganism will preferably contain no introns that could prematurely terminate transcription of DNA into mRNA; however, premature termination of transcription may be desirable, for example, where it would result in mutants having advantageous C- terminal truncations, for example, deletion of nucleotides which encode amino acids not necessary for biological activity or binding activity. Due to code degeneracy, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence.
  • the recombinant expression vectors carrying the recombinant IL-4bp- ⁇ insert are then transformed into a substantially homogeneous monoculture of suitable host microorganisms, for example, bacteria such as E. coli, yeast such as S. cerevisiae, or a mammalian cell line such as Chinese Hamster Ovary (CHO) cells, which stably integrate the vector/insert into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid.
  • suitable host microorganisms for example, bacteria such as E. coli, yeast such as S. cerevisiae, or a mammalian cell line such as Chinese Hamster Ovary (CHO) cells, which stably integrate the vector/insert into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid.
  • suitable host microorganisms for example, bacteria such as E. coli, yeast such as S.
  • Transformed host cells are cells which have been transformed or transfected with IL-4bp- ⁇ vectors constructed using recombinant DNA techniques.
  • Transformed host cells ordinarily express IL-4bp, but host cells transformed for purposes of cloning or amplifying IL-4bp- ⁇ DNA do not need to express IL-4bp- ⁇ .
  • Expressed IL-4bp- ⁇ will be deposited in the cell membrane or secreted into the culture supernatant, depending on the IL-4bp- ⁇ DNA selected.
  • Suitable host cells for expression of mammalian IL-4bp- ⁇ include prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E.
  • coli or bacilli Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems could also be employed to produce mammalian IL-4bp- ⁇ using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985), the relevant disclosure of which is hereby incorporated by reference.
  • Prokaryotic expression hosts may be used for expression of IL-4bp- ⁇ that do not require extensive proteolytic and disulfide processing.
  • Prokaryotic expression vectors generally comprise one or more phenotypic selectable markers, for example a gene encoding proteins conferring antibiotic resistance or supplying an autorrophic requirement, and an origin of replication recognized by the host to ensure amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene 2:95, 1977).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.
  • Promoters commonly used in recombinant microbial expression vectors include the ⁇ -lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281 :544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4051, 1980; and EPA 36,776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982).
  • Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the ⁇ PL promoter include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in £. coli RR1 (ATCC 53082).
  • Recombinant IL-4bp- ⁇ proteins may also be expressed in yeast hosts, preferably from the Saccharomyces genus, such as S. cerevisiae. Yeast of other genera, such as Pichia or Kluyveromyces may also be employed. Yeast vectors will generally contain an origin of replication from the 2 ⁇ yeast plasmid or an autonomously replicating sequence (ARS), promoter, DNA encoding IL-4bp- ⁇ , sequences for polyadenylation and transcription termination and a selection gene. Preferably, yeast vectors will include an origin of replication and selectable marker permitting transformation of both yeast and E. coli, e.g., the ampicillin resistance gene of E. coli and S.
  • ARS autonomously replicating sequence
  • cerevisiae trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, and a promoter derived from a highly expressed yeast gene to induce transcription of a structural sequence downstream.
  • the presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem.255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem.
  • enolase such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • Suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA 73,657.
  • Preferred yeast vectors can be assembled using DNA sequences from pBR322 for selection and replication in £. coli (Amp r gene and origin of replication) and yeast DNA sequences including a glucose-repressible ADH2 promoter and ⁇ -factor secretion leader.
  • the ADH2 promoter has been described by Russell et al. (/. Biol. Chem. 258:2614, 1982) and Beier et al. (Nature 300:124, 1982).
  • the yeast ⁇ -factor leader which directs secretion of heterologous proteins, can be inserted between the promoter and the structural gene to be expressed. See, e.g., Kurjan et al., Cell 30:933, 1982; and Bitter et al., Proc. Natl.
  • the leader sequence may be modified to contain, near its 3' end, one or more useful restriction sites to facilitate fusion of the leader sequence to foreign genes.
  • Suitable yeast transformation protocols are known to those of skill in the an; an exemplary technique is described by Hinnen et al., Proc. Natl. Acad. Sci.
  • Trp + transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ⁇ g/ml adenine and 20 ⁇ g/ml uraciL
  • Host strains transformed by vectors comprising the ADH2 promoter may be grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ⁇ g/ml adenine and 80 ⁇ g/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and held at 4"C prior to further purification.
  • Suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:115, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines.
  • Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • the transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources.
  • viral sources for example, commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence.
  • the early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature 273:113, 1978).
  • Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind HI site toward the Bgl I site located in the viral origin of replication is included.
  • mammalian genomic IL-4bp- ⁇ promoter, control and/or signal sequences may be utilized, provided such control sequences are compatible with the host cell chosen.
  • Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol.3:280, 1983).
  • a useful system for stable high level expression of mammalian receptor cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986).
  • pDC302 A particularly preferred eukaryotic vector for expression of IL-4bp DNA is pDC302 and is disclosed below in Example 4D.
  • pDC302 was derived from the mammalian high expression vector pDC201 and contains regulatory sequences from SV40, adenovirus-2, and human cytomegalovirus.
  • a sample of pDC302 has been deposited with the American Type Culture Collection (ATCC) under the name ⁇ CAV/NOT-IL-7R, deposit accession number 68014.
  • ATCC American Type Culture Collection
  • Purified mammalian IL-4 receptors or analogs are prepared by culturing suitable host vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts as described above.
  • supernatants from systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
  • a suitable affinity matrix can comprise an IL-4 or lectin or antibody molecule bound to a suitable support.
  • an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
  • a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.
  • RP-HPLC steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify an IL-4bp- ⁇ composition.
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.
  • Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • Microbial cells employed in expression of recombinant mammalian IL-4bp- ⁇ can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • Fermentation of yeast which express mammalian IL-4bp- ⁇ as a secreted protein greatly simplifies purification.
  • Secreted recombinant protein resulting from a large- scale fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog.296:111, 1984).
  • This reference describes two sequential, reversed- phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.
  • Human IL-4bp- ⁇ synthesized in recombinant culture is characterized by the presence of non-human cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover human IL-4bp- ⁇ from the culture. These components ordinarily will be of yeast, prokaryotic or non- human higher eukaryotic origin and preferably are present in innocuous contaminant quantities, on the order of less than about 1 percent by weight. Further, recombinant cell culture enables the production of IL-4bp- ⁇ free of proteins which may be normally associated with IL-4bp- ⁇ as it is found in nature in its species of origin, e.g. in cells, cell exudates or body fluids.
  • IL-4bp-B Compositions comprising an effective amount of IL-4bp- ⁇ proteins and a suitable diluent and carrier, and methods for suppressing DL-4-dependent immune responses in humans comprising administering an effective amount of IL-4bp- ⁇ .
  • soluble cytokine receptors e.g., IL-4 receptor, IL-1 receptor or TNF receptor.
  • purified IL-4bp- ⁇ is administered to a patient, preferably a human, for treatment in a manner appropriate to the indication.
  • IL- 4bp compositions administered to suppress immune function can be given by bolus injection, continuous infusion, sustained release from implants, or other suitable technique.
  • an IL-4bp- ⁇ therapeutic agent will be administered in the form of a composition comprising purified protein in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to recipients at the dosages and concentrations employed.
  • compositions entails combining IL-4bp- ⁇ with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients.
  • antioxidants such as ascorbic acid
  • chelating agents such as EDTA, glutathione and other stabilizers
  • excipients e.g., EDTA, glutathione and other stabilizers
  • product is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.
  • IL-4bp- ⁇ dosages can be determined in trials; generally, IL-4bp- ⁇ dosages of from about 1 ng/kg/day to about 10 mg/kg day are expected to induce a biological effect.
  • the amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth.
  • IL-4bp- ⁇ is administered for the purpose of inhibiting IL-4-mediated immune responses in a human.
  • IL-4bp- ⁇ may be useful to inhibit Ig-E antibody formation in the treatment of IL-4-mediated IgE-induced immediate hypersensitivity reactions, such as allergic rhinitis (common hay fever), bronchial asthma, atopic dermatitis and gastrointestinal food allergy.
  • IL-4bp- ⁇ compositions may also be used to regulate IL-4-mediated T-cell functions.
  • T cell dependent functions were formerly thought to be mediated principally by IL-2, recent studies have shown that under some circumstances T-cell growth and proliferation can be mediated by growth factors such as IL-4.
  • IL-4bp- ⁇ may be useful in suppressing or inhibiting T-cell dependent responses to alloantigen.
  • diseases or conditions are caused by an immune response to alloantigen, including allograft rejection and graft- versus-host reaction.
  • IL-4bp- ⁇ may suppresses lymphoproliferation and inflammation which result upon activation of T cells.
  • IL-4bp- ⁇ may therefore be potentially effective in the clinical treatment of, for example, rejection of allografts
  • IL-4bp- ⁇ may also be useful in clinical treatment of autoimmune dysfunctions, such as rheumatoid arthritis, diabetes, which are dependent upon the activation of T- cells against antigens not recognized as being indigenous to the host.
  • IL-2 plays in the proliferation and differentiation of T-cells
  • combination therapy using IL-4bp- ⁇ , IL-4 receptor and IL-2 receptors may be preferred in the treatment of T-cell dependent dysfunctions.
  • the following examples are offered by way of illustration, and not by way of limitation.
  • Human IL-4bp- ⁇ was purified to homogeneity from human urine as follows.
  • a human IL-4 affinity column was prepared by coupling 360 ug of recombinant human IL-4 to 1 ml AminoLinkTM gel (Pierce) according to the manufacturer's suggestions.
  • Normal human urine was collected aseptically and stored at 4 * C.
  • the collected urine was subjected to ultrafiltration using a Centriprep-10 15 ml concentrator device (Amicon).
  • the retentate was continually concentrated at 4 * C until a 150-330 fold concentration by volume was achieved.
  • the concentrated urine was sterile filtered on 0.22 ⁇ filters and stored in sterile polypropylene tubes at 4 * C.
  • Urine concentrate (0.5-1.0 ml) was loaded onto the human IL-4 affinity column, prepared as described above. After 1 hr of incubation at room temperature, unbound material (flow through) was collected and the column washed with 10 column volumes of PBS/0.5M NaCl to remove non-specifically bound protein. Urinary proteins specifically binding to the IL-4 matrix were eluted from the column with lOOmM citrate buffer, pH 2.8. Eight to 10 500 ⁇ l fractions were collected and were immediately neutralized with 1.0M Hepes, pH 8.5. Protein in each fraction was estimated by A28O using an extinction coefficient of 1.0. Protein was repeatedly detected in fractions 3-5 corresponding to 1.5 column volumes.
  • Fractions 3-5 were pooled and concentrated using an Centricon-10 microconcentrator (Amicon) and protein estimated in the unpooled fractions and the IL-4 binding protein (IL-4bp) concentrate using the Pierce Bicinchoninic acid (BCA) protein assay reagent with bovine serum albumin as the standard.
  • Samples of the IL-4bp- ⁇ concentrate were subjected to SDS- PAGE under reducing conditions on 8-25% and 10-15% gradient gels (Pharmacia) using M r standards ranging from 12-95,000 daltons. Protein bands were detected by standard silver staining methods, such as is described by Urdal et al., Proc. Natl. Acad. Sci. USA 81:64 ! (1984).
  • Amino terminal protein sequence of human IL-4bp- ⁇ from the IL-4 affinity column purification was obtained by excising bands from the gels and subjecting to automated Edman degradation on an Applied Biosystems Model 477A Protein Sequencer essentially as described by March et al. (Nature 315:641, 1985), except that PTH amino acids were automatically injected and analyzed on line with an Applied Biosystems Model 120 A HPLC using a gradient and detection system supplied by the manufacturer. The following amino terminal sequence was determined from the results of two separated sequencing runs:
  • Amino acid Xi was ambiguous.
  • the sequence of amino acids 1-8 was used to search the NBRF old and new protein sequence data base. No identical proteins were found by this search.
  • the yield of IL-4bp from 500 ml of concentrated urine was calculated as 40 ⁇ g by BCA determination and the sequence was obtained from 3 ⁇ g of this material.
  • the sequence data indicated that the amount of protein in this preparation was 20 picomoles or 940 ng.
  • the total yield of B -4bp- ⁇ from 500 ml of human urine was 12.5 ⁇ g.
  • Human IL-4bp- ⁇ was then characterized functionally in a variety of assays as set forth in Example 3.
  • the functional ability of IL-4bp- ⁇ to bind IL-4 was determined by examining the ability of IL-4bp- ⁇ to inhibit binding of IL-4 to various target cells which express IL-4 receptors.
  • the target cells were stained with biotinylated human and murine IL-4 and human IL-7, followed by streptavidin-phycoerythrin, and analyzed by flow cytometry, as follows.
  • the IL-4 affinity column eluate from urine (2ug) was incubated for 1 hour at 4"C with either 50 ng of biotinylated human or murine IL-4 in a total volume of 50 ⁇ l or with 250ng of biotinylated human IL-7 in a total volume of 25ul.
  • the IL-4 affinity column eluate from from JMl supernatants (4.5ug) was incubated for 1 hour at 4 * C with 250ng of biotinylated human IL-4. All dilutions were made in PBS +0.02% NaN3.
  • Controls consisted of PBS alone in the place of column eluate. 5xl0 5 target cells were then added and incubated 30 minutes at 4'C. JMl cells were added to JMl supernatant eluate/biotin-IL-4; 70Z3 cells (a murine pre-B cell) were added to urine eluate/biotin-IL-7; and CTLL cells (a murine IL-4bp+ T-cell line, available as ATCC TIB 214, which was transfected with the human IL-4bp gene) were added to urine eluate/biotin-huIL-4 and to urine eluate/biotin-muIL-4.
  • IL-4bp- ⁇ isolated to homogeneity by IL-4 affinity purification from JMl cell supernatants, inhibits binding of biotinylated human EL-4 to JMl cells ( Figure 3);
  • Table A shows the percent inhibition of biotinylated cytokine binding compared to staining with biotinylated cytokine alone.
  • Preparations of purified recombinant IL-4bp- ⁇ is employed to generate monoclonal antibodies against IL-4bp- ⁇ using conventional techniques, such as those disclosed in U. S. Patent 4,411,993. Such antibodies are likely to be useful in interfering with IL-4 binding to IL-4 receptors, for example, in ameliorating toxic or other undesired effects of IL-4.
  • IL-4bp- ⁇ used as immunogen is emulsified in complete Freund's adjuvant and injected in amounts ranging from 10-100 ⁇ l subcutaneously into Lewis rats. Three weeks later, the immunized animals are boosted with additional immunogen emulsified in incomplete Freund's adjuvant and boosted every three weeks thereafter. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot-blot assay, ELISA (enzyme-linked immunosorbent assay), or inhibition of binding of 125 I-IL-4 to extracts of IL-4bp- ⁇ bearing cells. Other assay procedures are also suitable.
  • Hybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a HAT selective medium (hypoxanthine, aminopterin, and thymidine) to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT selective medium hyperxanthine, aminopterin, and thymidine
  • Hybridoma clones thus generated are screened for reactivity with IL-4bp- ⁇ .
  • Initial screening of hybridoma supernatants utilize an antibody capture and binding of partially purified 125 I-IL-4bp- ⁇ .
  • Positive hybridomas screened by this method are tested by a modified antibody capture to detect blocking antibody.
  • Positive antibodies are capable of immunoprecipitating human IL-4bp- ⁇ protein from appropriate cells.
  • the anti-IL-4bp- ⁇ hybridomas are then injected into the peritoneal cavities of nude mice to produce ascites containing high concentrations (>1 mg/ml) of anti-IL-4bp- ⁇ monoclonal antibody.
  • the resulting monoclonal antibody is purified by ammonium sulfate precipitation followed by gel exclusion chromatography, and/or affinity chromatography based on binding of antibody to Protein G.

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Abstract

Protéines homogènes de liaison d'interleukine-4 (IL-4bp-β), compositions comprenant IL-4bp-β, et procédé d'obtention de IL-4bp-β homogène et de IL-4bp-β homogène recombinée.
PCT/US1991/001612 1990-03-09 1991-03-08 Proteines de liaison d'interleukine-4 WO1991013984A2 (fr)

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US5714146A (en) * 1992-08-26 1998-02-03 Board Of Regents Of The University Of Washington IL-4 bone therapy

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WO1989009621A2 (fr) * 1988-04-06 1989-10-19 Mary Alice Ritter Composes pour le diagnostic et/ou le traitement de tumeurs

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WO1989009621A2 (fr) * 1988-04-06 1989-10-19 Mary Alice Ritter Composes pour le diagnostic et/ou le traitement de tumeurs

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Title
Cell, volume 59, 20 October 1989, Cell Press, B. Mosley et al: "The murine inter-leukin-4 receptor: Molecular cloning and characterization of secreted and membrane bound forms", pages 335-348 *
Proceedings of the National Academy of Sciences of the USA, volume 87, June 1990, (Washington, DC, US), R. Fernandez-Botran et al.: "A soluble high-affinity, interleukin-4-binding protein in present in the biological fluids of mice", pages 4202-4206 *
Proceedings of the National Academy of Sciences of the USA, volume 87, no. 3, February 1990, (Washington, DC, US), N. Harada et al.: "Expression cloning of a cDNA encoding the murine inter-leukin 4 receptor based on ligand binding", pages 857-861 *
The Journal of Experimental Medicine, volume 171, no. 3, 1 March 1990, The Rockefeller University Press, R.L. Idzerda et al.: "Human inter-leukin-4 receptor confers biological responsiveness and defines a novel receptor superfamily" pages 861-873 *
The Journal of Immunology, volume 144, no. 11, 1 June 1990, The American Assosiation of Immunologist, (US), M.P. Beckmann et al.: "Monoclonal antibodies block murine IL-4 receptor function", pages 4212-4217 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714146A (en) * 1992-08-26 1998-02-03 Board Of Regents Of The University Of Washington IL-4 bone therapy

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