EP1237900A1 - Proteine de fusion optimisee par des sous-unites - Google Patents

Proteine de fusion optimisee par des sous-unites

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Publication number
EP1237900A1
EP1237900A1 EP00963585A EP00963585A EP1237900A1 EP 1237900 A1 EP1237900 A1 EP 1237900A1 EP 00963585 A EP00963585 A EP 00963585A EP 00963585 A EP00963585 A EP 00963585A EP 1237900 A1 EP1237900 A1 EP 1237900A1
Authority
EP
European Patent Office
Prior art keywords
fusion protein
protein
sequence
milk
transgenic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00963585A
Other languages
German (de)
English (en)
Other versions
EP1237900A4 (fr
Inventor
Dan Pollock
Harry M. Meade
Klaus Bosslet
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
rEVO Biologics Inc
Original Assignee
Genzyme Transgenics Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genzyme Transgenics Corp filed Critical Genzyme Transgenics Corp
Publication of EP1237900A1 publication Critical patent/EP1237900A1/fr
Publication of EP1237900A4 publication Critical patent/EP1237900A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01031—Beta-glucuronidase (3.2.1.31)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the invention relates to a fusion protein having a first and a second member, wherein the second member of the fusion protein assembles into a multimer and the other member is chosen, or modified, such that it promotes assembly of the second member into a preselected or an optimal number of subunits.
  • the first member, or the fusion protein assembles into a form having the same number of subunits as are present in an active, e.g., native, form of the second member. In preferred embodiments the first member, or the fusion protein, assembles into a form having fewer subunits than are present in an active, e.g., native, form of the second member. In preferred embodiments, the fusion protein assembles into a complex, e.g., a di-, tri-, tetra-, or higher multi-meric complex. Preferably, the fusion protein assembles into a dimer or a tetramer.
  • the fusion protein assembles into a complex having enzymatic activity.
  • the first member is a monomer.
  • it is a species which is normally monomeric, or which has been modified, e.g., by mutation of a site which modulates formation or maintenance of a multimer of subunits.
  • the monomeric form is useful because it does not prevent formation of a multimer by the second member.
  • the first member is a forms a dimmer, e.g., a heterodimer or homodimer.
  • a dimmer e.g., a heterodimer or homodimer.
  • it is a species which is normally dimeric, or which has been modified, e.g., by mutation of a site which modulates formation or maintenance of a multimerof subunits, to be dimeric.
  • the dimeric form is useful because it does not prevent formation of a multimer by the second member.
  • the fusion protein has the formula: R1-L-R2; R2-L-R1 ; R2-R1; or R1-R2, wherein Rl is a first member, e.g., an immunoglobulin subunit, L is a peptide linker and R2 is a second member, e.g., an enzyme subunit.
  • Rl and R2 are covalently linked, e.g., directly fused or linked via a peptide linker.
  • the first or the second member of the fusion protein, or both are modified by, e.g., substituting or deleting, a portion of the amino acid sequence.
  • the fusion protein includes a first member which is an Ig superfamily member, preferably an Ig subunit, which has been modified to inhibit formation of a multimeric form, e.g., a tetrameric form.
  • the modification which can be a change, insertion, or deletion of one or more amino acid residues, results in a subunit which does not form a multimer or which forms a lower order multimer that it normally would form, e.g., it forms a dimer rather than a tetramer.
  • a region which mediates formation or maintenance of a multimeric structure is modified and thereby wholly or partly inactivated.
  • a portion of an immunoglobulin subunit e.g., a heavy chain, e.g., the hinge region, is modified, e.g., deleted.
  • the modified immunoglobulin is monovalent.
  • the first member is a targeting agent, e.g., a polypeptide having a high affinity for a target, e.g., an antibody, a ligand, or an enzyme.
  • the first member is an immunoglobulin or a fragment thereof, e.g., an antigen binding fragment thereof.
  • the immunoglobulin is a monoclonal antibody, e.g., a human, murine (e.g., mouse) monoclonal antibody; or a recombinant monoclonal antibody.
  • the monoclonal antibody is a human antibody.
  • the monoclonal antibody is a recombinant antibody, e.g., a chimeric or a humanized antibody (e.g., it has a variable region, or at least a complementarity determining region (CDR), derived from a non-human antibody (e.g., murine) with the remaining portion(s) are human in origin); or a transgenically produced human antibody (e.g., an antibody produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell).
  • a recombinant antibody e.g., a chimeric or a humanized antibody (e.g., it has a variable region, or at least a complementarity determining region (CDR), derived from a non-human antibody (e.g., murine) with the remaining portion(s
  • the first member is a full-length antibody (e.g., an IgGl or IgG4 antibody) or includes only an antigen-binding portion (e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment).
  • the first member is an immunoglobulin subunit selected from the group consisting of a subunit of : IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgM, IgAl, IgA2, IgA.sub.sec, IgD, of IgE.
  • the immunoglobulin subunit is an IgG isotype, e.g., IgG3.
  • the first member is a monomer, e.g., a single chain antibody; or forms a dimer, e.g., a dimer of an immunoglobulin heavy chain and a light chain.
  • the first member is a monovalent antibody (e.g., it includes one pair of heavy and light chains, or antigen binding portions thereof). In other embodiments, the first member is divalent antibody (e.g., it includes two pairs of heavy and light chains, or antigen binding portions thereof).
  • the first member is an immunoglobulin that interacts with (e.g., binds to) a cell surface antigen on a target cell, e.g., a cancer cell.
  • a tumor cell antigen e.g., carcinoembryonic antigen (CEA), TAG- 72, her-2/neu, epidermal growth factor receptor, transferrin receptor, among others.
  • the first member localizes, e.g., increases the concentration of, a fusion protein in proximity to a target cell, e.g., a cancer cell.
  • the second member is capable of converting a precursor drug, e.g., a prodrug, to a toxic drug.
  • the first member is an immunoglobulin G (IgG) heavy and light chains
  • the second member is human beta-glucuronidase fusion protein.
  • the light chain of the first member has an amino acid sequence as shown in Figure IB (SEQ ID NO:2); the light chain of the first member has an amino acid sequence at least 60%, 70%, 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, most preferably at least 98%, 99% sequence identity or homology with an amino acid sequence from Figure IB (SEQ ID NO:2).
  • the light chain of the first member has an amino acid sequence that is encoded by a nucleotide sequence as shown in Figure IB (SEQ ID NO: 1), or Figure 2 (SEQ ID NO:37); the light chain of the first member has an amino acid sequence that is encoded by a nucleotide sequence at least 60%, 70%, 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, most preferably at least 98%, 99% sequence identity or homology with a nucleotide sequence shown in Figure IB (SEQ ID NOs:2, 3, or 4), or Figure 2 (SEQ ID NO:37); the light chain of the first member has an amino acid sequence that is encoded by a nucleotide sequence that is capable of hybridizing under stringent conditions to the nucleotide sequence shown in Figure IB.
  • the heavy chain of the first member has an amino acid sequence that is encoded by a nucleotide sequence as shown in Figure 4B (SEQ ID NO: 5), or Figure 5 (SEQ ID NO: 12); the heavy chain of the first member has an amino acid sequence that is encoded by a nucleotide sequence at least 60%, 70%, 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, most preferably at least 98%, 99% sequence identity or homology with a nucleotide sequence shown in Figure 4B (SEQ ID NO:5), or Figure 5 (SEQ ID NO: 12); the heavy chain of the first member has an amino acid sequence that is encoded by a nucleotide sequence that is capable of hybridizing under stringent conditions to the nucleotide sequence shown in Figure 4B, or 5.
  • the fusion protein includes a peptide linker and the peptide linker has one or more of the following characteristics: a) it allows for the rotation of the first and the second member relative to each other; b) it is resistant to digestion by proteases; c) it does not interact with the first or the second; d) it allows the fusion protein to form a complex (e.g., a di-, tri-, terra-, or multi-meric complex) that retains enzymatic activity; and e) it promotes folding and/or assembly of the fusion protein into an active complex.
  • a complex e.g., a di-, tri-, terra-, or multi-meric complex
  • the fusion protein includes a peptide linker and the peptide linker is 5 to 60, more preferably, 10 to 30, amino acids in length; the peptide linker is 20 amino acids in length; the peptide linker is 17 amino acids in length; each of the amino acids in the peptide linker is selected from the group consisting of Gly, Ser, Asn, Thr and Ala; the peptide linker includes a Gly-Ser element.
  • the fusion protein includes a peptide linker and the peptide linker includes a sequence having the formula (Ser-Gly-Gly-Gly-Gly)y wherein y is
  • the peptide linker includes a sequence having the formula (Ser-Gly-Gly-Gly-Gly)3.
  • the peptide linker includes a sequence having the formula ((Ser-Gly-Gly-Gly)3-Ser-Pro).
  • the fusion protein is produced recombinantly, e.g., produced in a host cell (e.g., a cultured cell), or in a transgenic animal, e.g., a transgenic mammal (e.g., a goat, a cow, or a rodent (e.g., a mouse).
  • a transgenic mammal e.g., a goat, a cow, or a rodent (e.g., a mouse).
  • the fusion protein is produced in a transgenic mammal
  • the method further includes: providing a transgenic animal, which includes a transgene which provides for the expression of a fusion protein described herein; allowing the transgene to be expressed; and, preferably, recovering fusion protein, from the milk of the transgenic mammal.
  • the fusion protein can further include: a signal sequence which directs the secretion of the fusion protein, e.g., a signal from a secreted protein (e.g., a signal from a protein secreted into milk; or an immunoglobulin secretory signal); and
  • a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein e.g., a protein secreted into milk, or an immunoglobulin, to promote secretion, e.g., in the milk of a transgenic mammal, of the fusion protein.
  • the fusion protein is made in a mammary gland of the transgenic mammal, e.g., a ruminant, e.g., a goat or a cow.
  • the fusion protein is secreted into the milk of the transgenic mammal, e.g., a ruminant, e.g., a dairy animal, e.g., a goat or a cow.
  • a ruminant e.g., a dairy animal, e.g., a goat or a cow.
  • the fusion protein is secreted into the milk of a transgenic mammal at concentrations of at least about 0.1 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 5 mg/ml or higher.
  • the fusion protein is made under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter.
  • a milk specific promoter e.g., a milk serum protein or casein promoter.
  • the milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter.
  • the promoter is a goat ⁇ casein promoter.
  • the transgene encoding the fusion protein is a nucleic acid construct which includes:
  • a promoter e.g., a mammary epithelial specific promoter, e.g., a milk protein promoter
  • nucleotide sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g. a protein secreted into milk, or an immunoglobulin, to allow secretion, e.g., in the milk of a transgenic mammal, of the fusion protein; (e) one or more nucleotide sequences which encode a fusion protein, e.g., an immunoglobulin-enzyme fusion protein as described herein; and
  • a 3' untranslated region from a mammalian gene e.g., a mammary epithelial specific gene, (e.g., a milk protein gene).
  • elements a (if present), b, c, d (if present), and f of the transgene are from the same gene; the elements a (if present), b, c, d (if present), and f of the transgene are from two or more genes.
  • the signal sequence, the promoter sequence and the 3' untranslated sequence can be from a mammary epithelial specific gene, e.g., a milk serum protein or casein gene (e.g., a ⁇ casein gene).
  • the signal sequence, the promoter sequence and the 3' untranslated sequence are from a goat ⁇ casein gene.
  • the promoter of the transgene is a mammary epithelial specific promoter, e.g., a milk serum protein or casein promoter (e.g., a ⁇ casein promoter).
  • the milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter.
  • the promoter is a goat ⁇ casein promoter.
  • the signal sequence encoded by the transgene is an amino terminal sequence which directs the expression of the protein to the exterior of a cell, or into the cell membrane.
  • the signal sequence can be obtained from an immunoglobulin protein.
  • the signal sequence is from a protein which is secreted into the milk, e.g., the milk of the transgenic animal.
  • the one or more nucleotide sequences encoding a fusion protein include one or more of: a nucleotide sequence encoding a first member, e.g., an immunoglobulin heavy chain (or an antigen binding portion thereof) operably linked to a second member, e.g., an enzyme; (optionally) a nucleotide sequence encoding an immunoglobulin light chain (or an antigen binding portion thereof), or both.
  • the nucleotide sequences encoding the heavy chain fusion and the light chain are operatively linked in a single construct, e.g., a single cosmid.
  • the nucleotide sequences encoding the heavy chain fusion and the light chain are introduced into a transgenic animal in separate constructs. Preferably, when linked, the nucleotide sequences are arranged in the following order:
  • Nl is a first member, e.g., an immunoglobulin heavy chain (or an antigen binding portion thereof) operably linked to a second member, e.g., an enzyme; and N2 is an immunoglobulin light chain (or an antigen binding portion thereof).
  • the nucleotide sequences can be in any orientation with respect to each other, e.g., sense/sense; reverse/reverse; sense/reverse; or reverse/sense.
  • the 3' untranslated region of the transgene includes a polyadenylation site, and is obtained from a mammalian gene, e.g., a mammary epithelial specific gene, e.g., a milk serum protein gene or casein gene.
  • the 3' untranslated region can be obtained from a casein gene (e.g., a ⁇ casein gene), a beta lactoglobulin gene, whey acid protein gene, or lactalbumin gene.
  • the 3' untranslated region is from a goat ⁇ casein gene.
  • the transgene e.g., the transgene as described herein, integrates into a germ cell and/or a somatic cell of the transgenic animal.
  • the invention features, a method for providing a transgenically produced fusion protein, e.g., a fusion protein as described herein, in the milk, of a transgenic mammal.
  • the method includes obtaining milk from a transgenic mammal, which includes a fusion protein encoding transgene, e.g., one which has been introduced into its germline, e.g., a nucleic acid construct as described herein, that result in the expression of the protein-coding sequence of fusion protein in mammary gland epithelial cells, thereby secreting the fusion protein in the milk of the mammal.
  • the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats.
  • the preferred transgenic mammal is a goat.
  • the fusion protein is secreted into the milk of a transgenic mammal at concentrations of at least about 0.1 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 5 mg/ml or higher.
  • the transgene encoding the immunoglobulin-enzyme fusion protein is a nucleic acid construct which includes:
  • a promoter e.g., a mammary epithelial specific promoter, e.g., a milk protein promoter
  • a nucleotide sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g., a protein secreted into milk, or an immunoglobulin, to allow secretion, e.g., in the milk of a transgenic mammal, of the non- secreted protein;
  • fusion protein e.g., a fusion protein as described herein;
  • elements a (if present), b, c, d (if present), and f of the transgene are from the same gene; the elements a (if present), b, c, d (if present), and f of the transgene are from two or more genes.
  • the signal sequence, the promoter sequence and the 3' untranslated sequence can be from a mammalian gene, e.g., a mammary epithelial specific gene, e.g., a milk serum protein or casein gene (e.g., a ⁇ casein gene).
  • a mammary epithelial specific gene e.g., a milk serum protein or casein gene (e.g., a ⁇ casein gene).
  • the signal sequence, the promoter sequence and the 3' untranslated sequence are from a goat ⁇ casein gene.
  • the promoter of the transgene is a mammary epithelial specific promoter, e.g., a milk serum protein or casein promoter (e.g., a ⁇ casein promoter).
  • the milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter.
  • the promoter is a goat ⁇ casein promoter.
  • the signal sequence encoded by the transgene is an amino terminal sequence which directs the expression of the protein to the exterior of a cell, or into the cell membrane.
  • the signal sequence is from a protein which is secreted into the milk, e.g., the milk of the transgenic animal.
  • the transgene e.g., the transgene as described herein, integrates into a germ cell and/or a somatic cell of the transgenic animal.
  • the invention features, a transgene, e.g., a nucleic acid construct, preferably, an isolated nucleic acid construct, which includes:
  • a promoter e.g., a mammary epithelial specific promoter, e.g., a milk protein promoter
  • a nucleotide sequence which encodes a signal sequence which can direct the secretion of the fusion protein e.g. a signal sequence from a milk specific protein, or an immunoglobulin
  • a nucleotide sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein e.g. a protein secreted into milk, or an immunoglobulin, to allow secretion, e.g., in the milk of a transgenic mammal, of the fusion protein protein;
  • fusion protein e.g., a fusion protein as described herein;
  • the signal sequence encoded by the transgene is an amino terminal sequence which directs the expression of the protein to the exterior of a cell, or into the cell membrane.
  • the signal sequence is from a milk specific protein, or an immunoglobulin.
  • the signal sequence directs secretion of the encoded fusion protein into the milk of a transgenic animal, e.g., a transgenic mammal.
  • the composition includes milk.
  • the transgenic mammals has germ cells and somatic cells containing a transgene that encodes a fusion protein, e.g., a transgene which encodes a fusion protein described herein.
  • the fusion protein expressed in the transgenic animal is under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter.
  • a milk specific promoter e.g., a milk serum protein or casein promoter.
  • the milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter.
  • the promoter is a goat ⁇ casein promoter.
  • the transgenic organism is a transgenic plant or animal.
  • Preferred transgenic animals include: mammals; birds; reptiles; marsupials; and amphibians.
  • Suitable mammals include: ruminants; ungulates; domesticated mammals; and dairy animals.
  • Particularly preferred animals include: mice, goats, sheep, camels, rabbits, cows, pigs, horses, oxen, and llamas.
  • Suitable birds include chickens, geese, and turkeys.
  • the transgenic protein is secreted into the milk of a transgenic animal, the animal should be able to produce at least 1, and more preferably at least 10, or 100, liters of milk per year.
  • the fusion protein is under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter.
  • a milk specific promoter e.g., a milk serum protein or casein promoter.
  • the milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter.
  • the promoter is a goat ⁇ casein promoter.
  • the invention features, a method of selectively killing an aberrant or diseased cell which expresses on its surface a target antigen, e.g., a cancer cell expressing a cell suface antigen.
  • the method includes: contacting said aberrant or diseased cell with an effective amount of a fusion protein, e.g., a fusion protein described herein, wherein either the first or the second member of the fusion protein recognizes said target antigen, such that selective killing of the cell occurs.
  • the subject method can be used on cells in culture, e.g. in vitro or ex vivo (e.g., cultures comprising cancer cells).
  • the invention features, a method of selectively killing an aberrant or diseased cell which expresses on its surface a target antigen, e.g., a cancer cell expressing a cell suface antigen.
  • the method includes: introducing into said aberrant or diseased cell a nucleic acid encoding a fusion protein, e.g., a fusion protein described herein, wherein either the first or the second member of the fusion protein recognizes said target antigen, such that selective killing of the cell occurs.
  • the invention provides, a method of treating in a subject, a disorder characterized by aberrant growth or activity of a cell which expresses on its surface a target antigen, e.g., a cancer cell expressing a target antigen.
  • the method includes administering to the subject an effective amount of a fusion protein, or a nucleic acid encoding a fusion protein (e.g., a fusion protein described herein), wherein either the first or the second member of the fusion protein recognizes said target antigen.
  • the term includes, for example, a recombinant DNA which is inco ⁇ orated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences.
  • Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional fusion protein sequence.
  • transgenic organism refers to a transgenic animal or plant.
  • a transgenic animal is a non-human animal in which one or more, and preferably essentially all, of the cells of the animal contain a transgene introduced by way of human intervention, such as by transgenic techniques known in the art.
  • the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • non-human animals include vertebrates, e.g., mammals and non-mammals, such as non-human primates, ruminants, birds, amphibians, reptiles and rodents, e.g., mice and rats.
  • the term also includes rabbits.
  • Figure 1A is a schematic diagram of a construct containing the genomic sequence of the light chain (LC) of humanized anti-carcinoembryonic antigen antibody 431.
  • the location of the signal peptide sequence (s) and the light chain variable (Vk) and the Ck regions are also indicated.
  • the location of the restriction enzyme sites is also indicated.
  • Figure IB depicts the nucleotide and amino acid sequence for the light chain of humanized anti-carcinoembryonic antigen antibody 431. The location of the restriction enzyme sites is indicated.
  • Figure 2 depicts the nucleotide sequence for a Sal I insert containing the coding sequences for light chain of humanized anti-carcinoembryonic antigen antibody 431.
  • Figure 3 is a schematic diagram of a construct (Be 458) which includes the Sal I insert containing the coding sequences for light chain of humanized anti-carcinoembryonic antigen antibody 431. Also indicated is the location of the silencer, 5' ⁇ -casein untranslated region, the light chain coding region, and the 3' ⁇ -casein untranslated region.
  • Figure 4 A is a schematic diagram of a construct containing the genomic sequence of the heavy chain (HC) of humanized anti-carcinoembryonic antigen antibody 431 linked to the ⁇ -glucuronidase sequence.
  • HC heavy chain
  • FIG. 4B depicts the nucleotide and amino acid sequence for the heavy chain of humanized anti-carcinoembryonic antigen antibody 431. The location of the restriction enzyme sites is indicated.
  • Figure 6 is a schematic diagram of a construct (Be 454) containing the mutant heavy chain of humanized anti-carcinoembryonic antigen antibody 431 linked to the ⁇ - glucuronidase sequence. The location of the silencer, 5' ⁇ -casein untranslated region, the heavy chain mutant/ ⁇ -glucuronidase fusion coding region, and the 3' ⁇ -casein untranslated region. The location of the restriction enzyme sites is also indicated.
  • Figure 8 is an enlarged view of the mutations to ⁇ -glucuronidase
  • the present invention provides, at least in part, transgenically produced fusion proteins wherein one member of the fusion protein assembles into a multimer and the other member is chosen, or modified, to promote assembly into the optimal number of subunits.
  • the fusion protein includes an immunoglobulin subunit (e.g., an immunoglobulin heavy or light chain) fused to a toxin (e.g., a subunit of an enzyme).
  • the immunogloblulin-enzyme fusion proteins described herein serve to target a cytotoxic agent (e.g. the enzyme) to an undesirable cell, e.g., a tumor cell.
  • the fusion proteins described in the Examples below can be used to target, to a tumor cell.
  • a non-toxic prodrug can be administered. This prodrug is converted to a highly cytotoxic drug by the action of the targeted enzyme localized at the tumor site, permitting to achieve therapeutic levels of the drug without unacceptable toxicity for the patients.
  • a monoclonal antibody against a target antigen, e.g., a cell surface protein (e.g., receptor) on a cell can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.
  • the preferred animal system for preparing hybridomas is the murine system.
  • Hybridoma production in the mouse is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Human monoclonal antibodies (mAbs) directed against human proteins can be generated using transgenic mice carrying the complete human immune system rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al.
  • Monoclonal antibodies can also be generated by other methods known to those skilled in the art of recombinant DNA technology.
  • An alternative method referred to as the "combinatorial antibody display” method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies (for descriptions of combinatorial antibody display see e.g., Sastry et al. 1989 PNAS 86:5728; Huse et al. 1989 Science 246:1275; and Orlandi et al. 1989 PNAS 86:3833). After immunizing an animal with an immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned.
  • Methods are generally known for obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR.
  • mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies (Larrick et al., 1991 , Biotechniques 11 :152- 156).
  • a similar strategy can also been used to amplify human heavy and light chain variable regions from human antibodies (Larrick et al., 1991, Methods: Companion to Methods in Enzymology 2:106-110).
  • RNA is isolated from B lymphocytes, for example, peripheral blood cells, bone marrow, or spleen preparations, using standard protocols (e.g., U.S. Patent No. 4,683,202; Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et al., PNAS (1989) 86:5728-5732; and Huse et al. (1989) Science 246:1275-1281.) First-strand cDNA is synthesized using primers specific for the constant region of the heavy chain(s) and each of the K and ⁇ light chains, as well as primers for the signal sequence.
  • variable region PCR primers the variable regions of both heavy and light chains are amplified, each alone or in combinantion, and ligated into appropriate vectors for further manipulation in generating the display packages.
  • Oligonucleotide primers useful in amplification protocols may be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences may also be incorporated into the primers to allow for the cloning of the amplified fragment into a vector in a predetermined reading frame for expression.
  • the V-gene library cloned from the immunization-derived antibody repertoire can be expressed by a population of display packages, preferably derived from filamentous phage, to form an antibody display library.
  • the display package comprises a system that allows the sampling of very large variegated antibody display libraries, rapid sorting after each affinity separation round, and easy isolation of the antibody gene from purified display packages.
  • kits for generating phage display libraries e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene Swr Z4RTM phage display kit, catalog no. 240612
  • methods and reagents particularly amenable for use in generating a variegated antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No.
  • the V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene subsequently cloned into the desired expression vector or phage genome.
  • a flexible linker As generally described in McCafferty et al., Nature (1990) 348:552-554, complete Vjr and VL domains of an antibody, joined by a flexible (Gly4-Ser)3 linker can be used to produce a single chain antibody which can render the display package separable based on antigen affinity. Isolated scFV antibodies immunoreactive with the antigen can subsequently be formulated into a pharmaceutical preparation for use in the subject method.
  • the antibody library is screened with the target antigen, or peptide fragment thereof, to identify and isolate packages that express an antibody having specificity for the target antigen.
  • Nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques.
  • Specific antibody molecules with high affinities for a surface protein can be made according to methods known to those in the art, e.g, methods involving screening of libraries (Ladner, R.C., et al, U.S. Patent 5,233,409; Ladner, R.C., et al, U.S. Patent 5,403,484). Further, the methods of these libraries can be used in screens to obtain binding determinants that are mimetics of the structural determinants of antibodies.
  • the Fv binding surface of a particular antibody molecule interacts with its target ligand according to principles of protein-protein interactions, hence sequence data for VJJ and VL (the latter of which may be of the K or ⁇ chain type) is the basis for protein engineering techniques known to those with skill in the art.
  • sequence data for VJJ and VL is the basis for protein engineering techniques known to those with skill in the art.
  • Details of the protein surface that comprises the binding determinants can be obtained from antibody sequence information, by a modeling procedure using previously determined three-dimensional structures from other antibodies obtained from NMR studies or crytallographic data. See for example Bajorath, J. and S. Sheriff, 1996, Proteins: Struct., Fund., and Genet. 24 (2), 152-157; Webster, D.M. and A. R. Rees, 1995, "Molecular modeling of antibody- combining sites,"in S.
  • a variegated peptide library is expressed by a population of display packages to form a peptide display library.
  • the display package comprises a system that allows the sampling of very large variegated peptide display libraries, rapid sorting after each affinity separation round, and easy isolation of the peptide-encoding gene from purified display packages.
  • Peptide display libraries can be in, e.g., prokaryotic organisms and viruses, which can be amplified quickly, are relatively easy to manipulate, and which allows the creation of large number of clones.
  • Preferred display packages include, for example, vegetative bacterial cells, bacterial spores, and most preferably, bacterial viruses (especially DNA viruses).
  • the present invention also contemplates the use of eukaryotic cells, including yeast and their spores, as potential display packages. Phage display libraries are described above.
  • soluble receptor e.g., a target antigen
  • affinity chromatography with an appropriate "receptor”, e.g., a target antigen
  • identification of the isolated binding agents or ligands by conventional techniques (e.g., mass spectrometry and NMR).
  • the soluble receptor is conjugated to a label (e.g., fluorophores, colorimetric enzymes, radioisotopes, or luminescent compounds) that can be detected to indicate ligand binding.
  • a label e.g., fluorophores, colorimetric enzymes, radioisotopes, or luminescent compounds
  • immobilized compounds can be selectively released and allowed to diffuse through a membrane to interact with a receptor.
  • Combinatorial libraries of compounds can also be synthesized with "tags" to encode the identity of each member of the library (see, e.g., W.C. Still et al, International Application WO 94/08051 ).
  • this method features the use of inert but readily detectable tags, that are attached to the solid support or to the compounds. When an active compound is detected, the identity of the compound is determined by identification of the unique accompanying tag.
  • This tagging method permits the synthesis of large libraries of compounds which can be identified at very low levels among to total set of all compounds in the library.
  • modified antibody is also intended to include antibodies, such as monoclonal antibodies, chimeric antibodies, and humanized antibodies which have been modified by, e.g., deleting, adding, or substituting portions of the antibody.
  • an antibody can be modified by deleting the hinge region, thus generating a monovalent antibody. Any modification is within the scope of the invention so long as the antibody has at least one antigen binding region specific.
  • Chimeric mouse-human monoclonal antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted, (see Robinson et al, International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al.,
  • the chimeric antibody can be further humanized by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions.
  • General reviews of humanized chimeric antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207 and by Oi et al., 1986, BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from 7E3, an anti- GPIIbIII a antibody producing hybridoma. The recombinant DNA encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector. Suitable humanized antibodies can alternatively be produced by CDR substitution U.S.
  • Patent 5,225,539 Jones et al. 1986 Nature 321 :552-525; Verhoeyan et al. 1988 Science
  • All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to the Fc receptor.
  • An antibody can be humanized by any method, which is capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody.
  • chimeric and humanized antibodies in which specific amino acids have been substituted, deleted or added.
  • preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen.
  • amino acids located in the human framework region can be replaced with the amino acids located at the corresponding positions in the mouse antibody. Such substitutions are known to improve binding of humanized antibodies to the antigen in some instances.
  • Antibodies in which amino acids have been added, deleted, or subsituted are referred to herein as modified antibodies or altered antibodies.
  • the first member of the fusion proteins of the present invention is a targeting agent, e.g., a polypeptide having a high affinity for a target, e.g., an antibody, a ligand, or an enzyme.
  • a targeting agent e.g., a polypeptide having a high affinity for a target, e.g., an antibody, a ligand, or an enzyme.
  • the fusion proteins of the invention can be used to selectively direct (e.g., localize) the second member of the fusion protein to the vicinity of an undesirable cell.
  • the first member can be an immunoglobulin that interacts with (e.g., binds to a target antigen).
  • the target antigen is present on the surface of a cell, e.g., an aberrant cell such a hyperproliferative cell (e.g., a cancer cell).
  • exemplary target antigens include carcinoembryonic antigen (CEA), TAG-72, her-2/neu, epidermal growth factor receptor, transferrin receptor, among others.
  • target cell shall mean any undesirable cell in a subject (e.g., a human or animal) that can be targeted by a fusion protein of the invention.
  • exemplary target cells include tumor cells, such as carcinoma or adenocarcinoma-derived cells (e.g., colon, breast, prostate, ovarian and endometrial cancer cells) (Thor, A. et al. (1997) Cancer Res 46: 3118; Soisson A. P. et al. (1989) Am. J. Obstet. Gynecol: 1258-63).
  • the first and second members of the fusion protein can be linked to each other, preferably via a linker sequence.
  • the linker sequence should separate the first and second members of the fusion protein by a distance sufficient to ensure that each member properly folds into its secondary and tertiary structures.
  • Preferred linker sequences (1) should adopt a flexible extended conformation, (2) should not exhibit a propensity for developing an ordered secondary structure which could interact with the functional first and second members, and (3) should have minimal hydrophobic or charged character, which could promote interaction with the functional protein domains.
  • Typical surface amino acids in flexible protein regions include Gly, Asn and Ser. Permutations of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence.
  • Other near neutral amino acids, such as Thr and Ala can also be used in the linker sequence.
  • a linker sequence length of 20 amino acids can be used to provide a suitable separation of functional protein domains, although longer or shorter linker sequences may also be used.
  • the length of the linker sequence separating the first and second members can be from 5 to 500 amino acids in length, or more preferably from 5 to 100 amino acids in length.
  • the linker sequence is from about 5-30 amino acids in length.
  • the linker sequence is from about 5 to about 20 amino acids, and is advantageously from about 10 to about 20 amino acids.
  • Amino acid sequences useful as linkers of the first and second member include, but are not limited to, (SerGly4)y wherein y is greater than or equal to 8, or Gly4SerGly5Ser.
  • a preferred linker sequence has the formula (SerGly4)4-
  • Another preferred linker has the sequence ((Ser-Ser-Ser-Ser-Gly)3-
  • the first and second members can be directly fused without a linker sequence.
  • C-terminal amino acid regions which can be used to separate the functional domains and prevent steric interference.
  • the C-terminus of first member can be directly fused to the N-terminus of second, or viceversa.
  • a fusion protein of the invention can be prepared with standard recombinant DNA techniques using a nucleic acid molecule encoding the fusion protein.
  • a nucleotide sequence encoding a fusion protein can be synthesized by standard DNA synthesis methods.
  • a nucleic acid encoding a fusion protein can be introduced into a host cell, e.g., a cell of a primary or immortalized cell line. The recombinant cells can be used to produce the fusion protein.
  • a nucleic acid encoding a fusion protein can be introduced into a host cell, e.g., by homologous recombination. In most cases, a nucleic acid encoding the fusion protein is incorporated into a recombinant expression vector.
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, those that direct expression of the nucleotide sequence only in certain host cells (e.g. , tissue-specific regulatory sequences) and those that direct expression in a regulatable manner (e.g., only in the presence of an inducing agent). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of fusion protein desired, and the like.
  • the fusion protein expression vectors can be introduced into host cells to thereby produce fusion proteins encoded by nucleic acids.
  • Recombinant expression vectors can be designed for expression of fusion proteins in prokaryotic or eukaryotic cells.
  • fusion proteins can be expressed in bacterial cells such as E. coli, insect cells (e.g., in the baculovirus expression system), yeast cells or mammalian cells.
  • suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • yeast S. cerevisiae examples include pYepSecl (Baldari et al, (1987) EMBO J.
  • Baculovirus vectors available for expression of fusion proteins in cultured insect cells include the pAc series (Smith et al. , (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).
  • mammalian expression vectors examples include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al (1987), EMBO J. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the recombinant expression vector can contain additional nucleotide sequences.
  • the recombinant expression vector may encode a selectable marker gene to identify host cells that have inco ⁇ orated the vector.
  • the recombinant expression vector can encode a signal sequence operatively linked to sequences encoding the amino- terminus of the fusion protein such that upon expression, the fusion protein is synthesized with the signal sequence fused to its amino terminus.
  • This signal sequence directs the fusion protein into the secretory pathway of the cell and is then cleaved, allowing for release of the mature fusion protein (i.e., the fusion protein without the signal sequence) from the host cell.
  • a signal sequence to facilitate secretion of proteins or peptides from mammalian host cells is known in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals.
  • a gene that encodes a selectable marker can be introduced into the host cells along with the gene encoding the fusion protein.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the fusion protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • a recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • DNA constructs can be introduced into the germ line of a mammal to make a transgenic mammal.
  • one or several copies of the construct can be inco ⁇ orated into the genome of a mammalian embryo by standard transgenic techniques. It is often desirable to express the transgenic protein in the milk of a transgenic mammal. Mammals that produce large volumes of milk and have long lactating periods are preferred. Preferred mammals are ruminants, e.g., cows, sheep, camels or goats, e.g., goats of Swiss origin, e.g., the Alpine, Saanen and Toggenburg breed goats. Other preferred animals include oxen, rabbits and pigs.
  • ruminants e.g., cows, sheep, camels or goats, e.g., goats of Swiss origin, e.g., the Alpine, Saanen and Toggenburg breed goats.
  • Other preferred animals include oxen, rabbits and pigs.
  • a transgenic non-human animal is produced by introducing a transgene into the germline of the non-human animal.
  • Transgenes can be introduced into embryonal target cells at various developmental stages. Different methods are used depending on the stage of development of the embryonal target cell. The specific line(s) of any animal used should, if possible, be selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
  • fusion protein transgene can be introduced into a mammal by microinjection of the construct into the pronuclei of the fertilized mammalian egg(s) to cause one or more copies of the construct to be retained in the cells of the developing mammal(s).
  • the egg can be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both.
  • One common method is to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.
  • Milk Specific Promoters Useful transcriptional promoters are those promoters that are preferentially activated in mammary epithelial cells, including promoters that control the genes encoding milk proteins such as caseins, beta lactoglobulin (Clark et al., (1989) Bio/Technology 1_: 487- 492), whey acid protein (Gorton et al. (1987) Bio/Technology 5: 1183-1187), and lactalbumin (Soulier et al., (1992) FEBS Letts. 297: B).
  • caseins such as caseins, beta lactoglobulin (Clark et al., (1989) Bio/Technology 1_: 487- 492), whey acid protein (Gorton et al. (1987) Bio/Technology 5: 1183-1187), and lactalbumin (Soulier et al., (1992) FEBS Letts. 297: B).
  • the alpha, beta, gamma or kappa casein gene promoter of any mammalian species can be used to provide mammary expression; a preferred promoter is the goat beta casein gene promoter (DiTullio, (1992) Bio/Technology K):74-77).
  • Milk-specific protein promoter or the promoters that are specifically activated in mammary tissue can be isolated from cDNA or genomic sequences. Preferably, they are genomic in origin. DNA sequence information is available for mammary gland specific genes listed above, in at least one, and often in several organisms. See, e.g., Richards et al., J. Biol. Chem.
  • a preferred insulator is a DNA segment which encompasses the 5' end of the chicken ⁇ -globin locus and corresponds to the chicken 5' constitutive hypersensitive site as described in PCT Publication 94/23046, the contents of which is inco ⁇ orated herein by reference.
  • a construct can also include a 3' untranslated region downstream of the DNA sequence coding for the non-secreted protein. Such regions can stabilize the RNA transcript of the expression system and thus increases the yield of desired protein from the expression system.
  • the 3' untranslated regions useful in the constructs of this invention are sequences that provide a poly A signal. Such sequences may be derived, e.g., from the SV40 small t antigen, the casein 3' untranslated region or other 3' untranslated sequences well known in the art.
  • the 3' untranslated region is derived from a milk specific protein. The length of the 3' untranslated region is not critical but the stabilizing effect of its poly A transcript appears important in stabilizing the RNA of the expression sequence.
  • Prior art methods can include making a construct and testing it for the ability to produce a product in cultured cells prior to placing the construct in a transgenic animal.
  • the transgenic fusion protein can be produced in milk at relatively high concentrations and in large volumes, providing continuous high level output of normally processed peptide that is easily harvested from a renewable resource.
  • a fusion protein can be expressed in a transgenic organism, e.g., a transgenic plant, e.g., a transgenic plant in which the DNA transgene is inserted into the nuclear or plastidic genome. Plant transformation is known as the art. See, in general, Methods in Enzymology Vol. 153 ("Recombinant DNA Part D") 1987, Wu and Grossman Eds., Academic Press and
  • plant protoplasts are electroporated in the presence of plasmids or nucleic acids containing the relevant genetic construct. Electrical impulses of high field strength reversibly permeabihze biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form a plant callus. Selection of the transformed plant cells with the transformed gene can be accomplished using phenotypic markers.
  • Parts obtained from a transgenic plant such as flowers, seeds, leaves, branches, fruit, and the like are covered by the invention, provided that these parts include cells which have been so transformed. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention, provided that these parts comprise the introduced DNA sequences. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention.
  • the gapped region (through the termination codon and new Sal I site) was then sequenced to confirm that no changes were made in sequence.
  • the Example describes the generation of a heavy chain/ ⁇ -glucuronidase fusion construct using the heavy chain nucleotide sequence from a humanized monoclonal antibody against carcinoembryonic antigen (431) subcloned into a mammary specific expression vector (Bel 63) and a commercial mammalian expression vector (pcDNA3).
  • the Hind III -Xba I fragment containing the heavy chain/ ⁇ -glucuronidase fusion sequence was subcloned into pGEM3z to facilitate further manipulation.
  • the internal Sal I site had to be changed for the ptupose of subcloning the fragment into a beta casein expression vector.
  • This Example described the generation of a construct which includes the light chain and the heavy chain/ ⁇ -glucuronidase fusion, along with their corresponding upstream and downstream beta casein sequences ligated together into a single cosmid.
  • both chains along with their own corresponding upstream and downstream beta casein sequences were ligated together into a single cosmid.
  • this supercosl (Stiratagene) was modified by inserting the following oligonucleotides into the Bam HI site:
  • the manipulated DNA fragments were tested in tissue culture using the pcDNA3 constructs described above transfected into cos 7 cells using the standard protocol for Lipofectamine using Opti-MEM (Gibco-BRL). Conditioned media (DMEM +10%FBS) was removed after 48 hours and run on a 10 -20% SDS-PAGE gels for Western blotting.
  • Detection was with the ECL kit from Amersham. Mab 2149/80 was the only antibody that showed a signal on the western blot. For the light chain, samples were again run under reducing conditions and electroblotted onto nitrocellulose. The nitrocellulose was then incubated overnight with horse radish peroxidase conjugated goat anti-human Kappa chain antibody (Cappel no. 55233). Detection was with the ECL kit from Amersham.
  • the first mutation was to remove the hinge region of the construct.
  • the second mutation removes the hinge and linker sequence (ala-ala-ala-ala- val) (SEQ ID NO:31) at the beginning of the ⁇ -glucuronidase coding sequence, fusing the CH2 portion to ⁇ glucuronidase.
  • gapped heteroduplex mutagenesis was again used.
  • the construct Behring HC5 (which contains the fusion protein in pGem3Z with both ends modified and an internal Sal I site removed) was linearized with (Xba I). A second aliquot was cut with BstE2 plus Not I. When boiled together and cooled some of each strand anneal forming the heteroduplex containing a single stranded gap, in this case between the BstE2 and Not I sites. Two new constructs were then made, sequencing over the gapped portion to make sure no other mutations were made inadvertently.
  • GTC #403 using the oligonucleotide "Behr hinge-alternate" (in bold below) removes the hinge region and part of the introns immediately preceding and after it.
  • CH2 coding sequence ⁇ -glucuronidase (SEQ ID NO: 33)
  • the mutated fusion protein coding sequence can then be excised using Sal I and subcloned into an appropriate expression vector.
  • Bc454 Bc450 with heavy chain mutant 403 (minus hinge)
  • Bc456 Bc450 with heavy chain mutant 406 (minus hinge/linker)
  • Figure 5 depicts the nucleotide and amino acid sequence for the mutant heavy chain of humanized anti-carcinoembryonic antigen antibody 431 lacking the hinge region.
  • Figure 6 is a schematic diagram of a construct (Be 454) containing the mutant heavy chain of humanized anti-carcinoembryonic antigen antibody 431 linked to the ⁇ - glucuronidase sequence. The location of the silencer, 5' ⁇ -casein untranslated region, the heavy chain mutant/ ⁇ -glucuronidase fusion coding region, and the 3 ' ⁇ -casein untranslated region.
  • the previous examples describe the testing of the original fusion protein and two heavy chain mutants in the milk expression system.
  • the original fusion proteins were tested both without the insulator and also co injected with a separate insulator fragments.
  • the heavy chain mutants on the other hand, were tested with the insulator integrated into the construct.
  • the concentration of the fusion protein produced in milk was estimated by comparing the signal of a sample to that of a standard on a Western blot. Later, experiments measured activity rather than concentration based on Western blots. The activity measurements were more accurate. Except for the first set of constructs, Bcl74 + Bcl75, estimates of protein concentration by Western blot are rough estimates. Generally, lines that express well appear to be in the 1-2 mg/ml range.
  • Swiss-origin goats e.g., the Alpine, Saanen, and Toggenburg breeds, are preferred in the production of transgenic goats.
  • estrus in the donors is synchronized on Day 0 by 6 mg subcutaneous norgestomet ear implants (Syncromate-B, CEVA Laboratories, Inc., Overland Park, KS).
  • Prostaglandin is administered after the first seven to nine days to shut down the endogenous synthesis of progesterone.
  • FSH - Schering Co ⁇ ., Kenilworth, NJ follicle-stimulating hormone
  • the implant is removed on Day 14. Twenty-four hours following implant removal the donor animals are mated several times to fertile males over a two-day period (Selgrath, et al., Theriogenology, 1990. pp. 1195-1205).
  • a 20 gauge needle is placed in the uterus approximately 0.5 cm from the uterotubal junction.
  • Ten to twenty ml of sterile phosphate buffered saline (PBS) is flushed through the cannulated oviduct and collected in a Petri dish. This procedure is repeated on the opposite side and then the reproductive tract is replaced in the abdomen.
  • PBS phosphate buffered saline
  • 10-20 ml of a sterile saline glycerol solution is poured into the abdominal cavity to prevent adhesions.
  • the linea alba is closed with simple interrupted sutures of 2.0 Polydioxanone or Supramid and the skin closed with sterile wound clips.
  • Fertilized goat eggs are collected from the PBS oviductal flushings on a stereomicroscope, and are then washed in Ham's F12 medium (Sigma, St. Louis, MO) containing 10% fetal bovine serum (FBS) purchased from Sigma. In cases where the pronuclei are visible, the embryos is immediately microinjected. If pronuclei are not visible, the embryos can be placed in Ham's F12 containing 10% FBS for short term culture at 37°C in a humidified gas chamber containing 5% CO2 in air until the pronuclei become visible (Selgrath, et al., Theriogenology, 1990. pp. 1195-1205).
  • One-cell goat embryos are placed in a microdrop of medium under oil on a glass depression slide. Fertilized eggs having two visible pronuclei are immobilized on a flame- polished holding micropipet on a Zeiss upright microscope with a fixed stage using Normarski optics.
  • a pronucleus is microinjected with the DNA construct of interest, e.g., a BC355 vector containing the fusion protein gene operably linked to the regulatory elements of the goat beta-casein gene, in injection buffer (Tris-EDTA) using a fine glass microneedle (Selgrath, et al., Theriogenology, 1990. pp. 1195-1205).
  • the surviving embryos are placed in a culture of Ham's F12 containing 10% FBS and then incubated in a humidified gas chamber containing 5% CO2 in air at 37°C until the recipient animals are prepared for embryo transfer (Selgrath, et al., Theriogenology, 1990. p. 1195-1205). Preparation of recipients:
  • Estrus synchronization in recipient animals is induced by 6 mg norgestomet ear implants (Syncromate-B).
  • the animals On Day 13 after insertion of the implant, the animals are given a single non-superovulatory injection (400 LU.) of pregnant mares serum gonadotropin (PMSG) obtained from Sigma. Recipient females are mated to vasectomized males to ensure estrus synchrony (Selgrath, et al., Theriogenology, 1990. pp. 1195-1205).
  • All embryos from one donor female are kept together and transferred to a single recipient when possible.
  • the surgical procedure is identical to that outlined for embryo collection outlined above, except that the oviduct is not cannulated, and the embryos are transferred in a minimal volume of Ham's F12 containing 10% FBS into the oviductal lumen via the fimbria using a glass micropipet. Animals having more than six to eight ovulation points on the ovary are deemed unsuitable as recipients. Incision closure and post-operative care are the same as for donor animals (see, e.g., Selgrath, et al., Theriogenology, 1990. pp. 1195-1205).
  • Pregnancy is determined by ultrasonography 45 days after the first day of standing estrus.
  • a second ultrasound exam is conducted to confirm pregnancy and assess fetal stress.
  • the pregnant recipient doe is vaccinated with tetanus toxoid and Clostridium C&D.
  • Selenium and vitamin E (Bo-Se) are given IM and Ivermectin was given SC.
  • the does are moved to a clean stall on Day 145 and allowed to acclimatize to this environment prior to inducing labor on about Day 147. Parturition is induced at Day 147 with 40 mg of PGF2a (Lutalyse®, Upjohn Company, Kalamazoo Michigan). This injection is given IM in two doses, one 20 mg dose followed by a 20 mg dose four hours later.
  • the doe is under periodic observation during the day and evening following the first injection of
  • Lutalyse® on Day 147. Observations are increased to every 30 minutes beginning on the morning of the second day. Parturition occurred between 30 and 40 hours after the first injection. Following delivery the doe is milked to collect the colostrum and passage of the placenta is confirmed.
  • genomic DNA is isolated from two different cell lines to avoid missing any mosaic transgenics.
  • a mosaic animal is defined as any goat that does not have at least one copy of the transgene in every cell. Therefore, an ear tissue sample (mesoderm) and blood sample are taken from a two day old F ⁇ animal for the isolation of genomic DNA (Lacy, et al., A Laboratory Manual, 1986, Cold Springs Harbor, NY; and Herrmann and Frischauf, Methods Enzymology, 1987. 152: pp. 180-183).
  • the DNA samples are analyzed by the polymerase chain reaction (Gould, et al., Proc. Natl. Acad. Sci, 1989. 86:pp.
  • transgenic founder (FQ) goats as well as other transgenic goats.
  • the transgenic FQ founder goats for example, are bred to produce milk, if female, or to produce a transgenic female offspring if it is a male founder.
  • This transgenic founder male can be bred to non-transgenic females, to produce transgenic female offspring.
  • Transmission of the transgene of interest, in the goat line is analyzed in ear tissue and blood by PCR and Southern blot analysis.
  • Southern blot analysis of the founder male and the three transgenic offspring shows no rearrangement or change in the copy number between generations.
  • the Southern blots are probed with immunoglobulin- enzyme fusion protein cDNA probe.
  • the blots are analyzed on a Betascope 603 and copy number determined by comparison of the transgene to the goat beta casein endogenous gene.
  • the expression level of the transgenic protein, in the milk of transgenic animals is determined using enzymatic assays or Western blots.

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Abstract

L'invention porte sur un procédé d'élaboration d'une protéine de fusion comportant un premier élément fusionné avec un deuxième élément, toux deux étant choisis pour que la protéine de fusion forme un complexe présentant plusieurs sous-unités qui optimisent l'activité de la forme multimère du deuxième élément.
EP00963585A 1999-09-17 2000-09-18 Proteine de fusion optimisee par des sous-unites Withdrawn EP1237900A4 (fr)

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US39907999A 1999-09-17 1999-09-17
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PCT/US2000/025558 WO2001019842A1 (fr) 1999-09-17 2000-09-18 Proteine de fusion optimisee par des sous-unites

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US20030035798A1 (en) 2000-08-16 2003-02-20 Fang Fang Humanized antibodies
JP2005522192A (ja) 2001-07-19 2005-07-28 パーラン セラピューティクス， インコーポレイテッド マルチマータンパク質およびマルチマータンパク質を作製および使用する方法
CA2929846C (fr) * 2013-11-19 2020-09-15 Regeneron Pharmaceuticals, Inc. Animaux non humains ayant un gene de facteur activant les cellules b humanise

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WO1995027782A1 (fr) * 1994-04-08 1995-10-19 Ppl Therapeutics (Scotland) Ltd Production de peptides utiles en tant que proteines de fusion dans du lait de mammifere transgenique
WO1998037224A1 (fr) * 1997-02-25 1998-08-27 Genzyme Transgenics Corporation Proteines non secretees produites de maniere transgenique
WO1998050563A1 (fr) * 1997-05-01 1998-11-12 Ppl Therapeutics (Scotland) Ltd. Methodes de production d'un peptide amide par utilisation d'une proteine de fusion
US5959171A (en) * 1994-08-17 1999-09-28 Pharming B.V. Method for the production of biologically active polypeptides in a mammal's
WO1999066054A2 (fr) * 1998-06-15 1999-12-23 Genzyme Transgenics Corp. Fusion analogue d'erythropoietine-albumine serique humaine

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IL111748A0 (en) * 1993-12-03 1995-01-24 Zeneca Ltd Proteins
US5635363A (en) * 1995-02-28 1997-06-03 The Board Of Trustees Of The Leland Stanford Junior University Compositions and methods for the detection, quantitation and purification of antigen-specific T cells

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Publication number Priority date Publication date Assignee Title
WO1995027782A1 (fr) * 1994-04-08 1995-10-19 Ppl Therapeutics (Scotland) Ltd Production de peptides utiles en tant que proteines de fusion dans du lait de mammifere transgenique
US5959171A (en) * 1994-08-17 1999-09-28 Pharming B.V. Method for the production of biologically active polypeptides in a mammal's
WO1998037224A1 (fr) * 1997-02-25 1998-08-27 Genzyme Transgenics Corporation Proteines non secretees produites de maniere transgenique
WO1998050563A1 (fr) * 1997-05-01 1998-11-12 Ppl Therapeutics (Scotland) Ltd. Methodes de production d'un peptide amide par utilisation d'une proteine de fusion
WO1999066054A2 (fr) * 1998-06-15 1999-12-23 Genzyme Transgenics Corp. Fusion analogue d'erythropoietine-albumine serique humaine

Non-Patent Citations (3)

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Title
LEE S H ET AL: "Production of biomedical proteins in the milk of transgenic dairy cows: the state of the art" JOURNAL OF CONTROLLED RELEASE, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 29, 1994, pages 213-221, XP002120416 ISSN: 0168-3659 *
NEWTON D L ET AL: "ANTITRANSFERRIN RECEPTOR ANTIBODY-RNASE FUSION PROTEIN EXPRESSED INTHE MAMMARY GLAND OF TRANSGENIC MICE" JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 231, 1999, pages 159-167, XP002934495 ISSN: 0022-1759 *
See also references of WO0119842A1 *

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WO2001019842A1 (fr) 2001-03-22
EP1237900A4 (fr) 2005-08-03
MXPA02002768A (es) 2002-08-30
RU2002110116A (ru) 2004-03-10
JP2003509038A (ja) 2003-03-11
BR0014524A (pt) 2002-06-11
CA2384766A1 (fr) 2001-03-22
CN1379782A (zh) 2002-11-13
NZ517774A (en) 2005-01-28
IL148549A0 (en) 2002-09-12
KR20020039346A (ko) 2002-05-25
AU3883101A (en) 2001-04-17
WO2001019842A9 (fr) 2002-11-14
HUP0202702A2 (hu) 2002-12-28
NO20021244L (no) 2002-05-13
AU781462B2 (en) 2005-05-26

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