US20030138440A1 - Multimeric proteins and methods of making and using same - Google Patents

Multimeric proteins and methods of making and using same Download PDF

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US20030138440A1
US20030138440A1 US10/199,957 US19995702A US2003138440A1 US 20030138440 A1 US20030138440 A1 US 20030138440A1 US 19995702 A US19995702 A US 19995702A US 2003138440 A1 US2003138440 A1 US 2003138440A1
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polypeptide
multimerization
seq
sequence
chimeric
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Fang Fang
Guang-Xiang Luo
Lori Kohlstaedt
Catherine Charles
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Perlan Therapeutics Inc
PERLAN THERAPEUTICS
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Perlan Therapeutics Inc
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Assigned to PERLAN THERAPEUTICS reassignment PERLAN THERAPEUTICS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KOHLSTAEDT, LORI ALLISON, CHARLES, CATHERINE HELEN, LUO, GUANG-XIANG
Assigned to PERLAN THERAPEUTICS, INC. reassignment PERLAN THERAPEUTICS, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: CFY BIOMEDICALS, INC.
Assigned to CFY BIOMEDICALS, INC. reassignment CFY BIOMEDICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FANG, FANG
Priority to US11/866,960 priority patent/US8394771B1/en
Priority to US13/794,457 priority patent/US20130243768A1/en
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

Definitions

  • the invention relates to engineered polypeptide sequences that mediate formation of oligomers (e.g., dimers, trimers, tetramers, hexamers, pentamers, and higher order olgomeric forms) between molecules attached thereto.
  • oligomers e.g., dimers, trimers, tetramers, hexamers, pentamers, and higher order olgomeric forms
  • Multimeric antibodies made by fusion with protein A or streptavidin may not be suitable for human use since protein A and streptavidin are highly immunogenic in humans.
  • Linking antibody fragments by a disulfide bond can lead to a dimeric recombinant antibody. It has been reported that single chain antibody scFv-based molecules can sometimes be made in multimeric forms by changing the length of linker between the VH and VL domains in scFv.
  • scFv When the linker is longer than 3 but shorter than 12 amino acid residues, scFv can form dimers, called “diabodies;” when the linker is less than 2 residues, or when no linker residue is used, scFv can form either trimers or tetramers, called “triabodies” and “tetrabodies.” Since these multimeric scFv molecules are structurally constrained, little affinity improvement resulted from higher valency. Triabodies have identical or lower affinities than the diabodies, and tetrabodies have less than one fold higher affinity than the diabodies (Kortt et al., Protein Engineering 10:423 (1997); and Gall et al., FEBS Lett. 453:164 (1999)).
  • the invention includes polypeptide sequences capable of conferring multimer formation.
  • a multimerization polypeptide is selected from the amino acid sequences set forth in SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163.
  • Multimerization polypeptides also include sequences having various amounts of sequence identity to the sequences disclosed herein, so long as the polypeptide is capable of multimerization.
  • a polypeptide has 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater identity to a multimerization polypeptide, for example, as set forth in any of SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163.
  • Subsequences modified forms, e.g., sequences having amino acid substitutions, additions or deletions of the multimerization polypeptides capable of multimerization are also included.
  • a modified form has one or more amino acid substitutions, provided that all of positions a or d of a seven residue repeat sequence (a.b.c.d.e.f.g), are either leucine, isoleucine or valine, said substituted polypeptide capable of conferring multimerization.
  • a modified form has one or more amino acid substitutions, provided that one or more positions a or d of a seven residue repeat sequence (a.b.c.d.e.f.g), are either leucine, isoleucine or valine, said substituted polypeptide capable of conferring multimerization.
  • at least one of positions a or d of a seven residue repeat sequence (a.b.c.d.e.f.g) are an amino acid other than leucine, isoleucine or valine.
  • the polypeptide has 1 to 5 amino acid substitutions.
  • a modified form has one or more amino acid substitutions in positions b, C, e, f or g, provided that said substituted polypeptide is capable of conferring multimerization.
  • the polypeptide has 1 to 5 amino acid substitutions.
  • Multimerization polypeptides are of various lengths.
  • the polypeptide has a sequence at least 11 amino acids in length, at least 15 amino acids in length, at least 18 amino acids in length, at least 22 amino acids in length, at least 27 amino acids in length, at least 31 amino acids in length.
  • the polypeptide has a sequence less than about 125 amino acids in length, less than about 100 amino acids in length, less than about 75 amino acids in length, less than about 50 amino acids in length.
  • Multimerization polypeptides including subsequences and modified forms thereof may be fused to any molecule thereby conferring multimer formation.
  • the molecule comprises a polypeptide sequence, e.g., a heterologous polypeptide.
  • the multimerization polypeptide is fused to the amino or carboxy terminus of the heterologous polypeptide, to form a chimeric polyeptide.
  • Chimeric polypeptides have a sequence length typically from about 18-30, 30-50, 50-75, 75-100, 100-150, 150-200, 200-250, 250-500 or 500-1000 amino acids, but maybe less or greater.
  • heterologous polypeptides are selected, for example, from: a binding protein (e.g., an antigen binding polypeptide), enzyme, receptor, ligand, nucleic acid binding protein, growth regulatory factor, differentiative factor, and chemotactic factor.
  • the antigen binding polypeptide comprises at least one antibody variable domain, such as a human or humanized variable domain.
  • the antigen binding polypeptide includes a single chain antibody, Fab, Fab′, (Fab′) 2 , or Fv antibody subsequence.
  • the antigen binding polypeptide comprises a multispecific or multifunctional antibody.
  • the antigen binding polypeptide binds to ICAM-1 or an epitope thereof, e.g., the antigen binding polypeptide inhibits human rhinovirus infection of a cell that expresses ICAM-1.
  • Multimers form hetero- or homo-dimer, -trimer, -tetramer, -pentamer or higher order oligomer. Binding between the monomeric substituents of the multimer may be measured by dissociation, or K D of the monomers.
  • K D dissociation
  • Various exemplary K D are 1 ⁇ 10 ⁇ 7 or less, 1 ⁇ 10 ⁇ 8 or less, or 1 ⁇ 10 ⁇ 9 .
  • linkers are included between the multimeric polypeptides and the molecule to which it is attached.
  • a linker comprises a polypeptide sequence, such as a human or humanized amino acid sequence.
  • Linkers can be of any size.
  • a polypeptide linker can be an amino acid sequence from about 5 to 20 amino acids, from about 10 to 30 amino acids, from about 25 to 50 amino acids, from about 30 to 60 amino acids, from about 50 to 75 amino acids, or greater or less in length.
  • a linker includes an amino acid sequence set forth in any of SEQ ID NO:43 (D30), SEQ ID NO:44 (D35), SEQ ID NO:45 (ED), SEQ ID NO:46 (EDC) or SEQ ID NO:47 (D63), or a subsequence thereof.
  • a pharmaceutical formulation includes a multimer fused to a molecule.
  • the molecule comprises a heterologus polypeptide sequence.
  • the invention further provides nucleic acids encoding the multimeric polypeptides alone, and in combination with heterologous polypeptides, as well as linked sequences.
  • Expression cassettes, vectors and cells e.g., bacterial, fungal, animal, plant, and insect, including the nucleic acids are included.
  • the invention additionally provides methods of producing a multimerization polypeptide having one or more seven residue repeat sequence, (a.b.c.d.e.f.g), that confers formation of a multimer.
  • a method includes modifying a polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein one or more of positions a or d are replaced with either leucine or isoleucine, thereby producing a multimerization polypeptide that confers multimer formation.
  • a method in another embodiment, includes modifying a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein positions a or d are replaced with valine and either of leucine or isoleucine, thereby producing a multimerization polypeptide that confers dimer formation.
  • the modified polypeptide forms a trimer, tetramer or pentamer, whereas the unmodified polypeptide forms a dimer; the modified polypeptide forms a tetramer or pentamer, whereas the unmodified polypeptide forms a dimer or trimer; the unmodified polypeptide forms a trimer or tetramer or pentamer.
  • the modified polypeptide has increased or decreased multimer stability in comparison to unmodified polypeptide.
  • the invention further provides methods of producing a chimeric polypeptide that forms a multimer.
  • a method includes producing a chimeric polypeptide comprising a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein one or more of positions a and d are either leucine or isoleucine, fused to a heterologous polypeptide thereby producing chimeric polypeptide that forms a trimer; a tetramer; or a pentamer.
  • a method includes producing a molecule including a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein positions a or d are valine and either of leucine or isoleucine, fused to the molecule thereby producing a molecule that forms a multimer; e.g., a trimer or tetramer or pentamer.
  • a method includes: incubating a polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g), wherein one or more of positions a and d are either leucine, isoleucine or valine, under conditions allowing formation of homo- or hetero-multimers; and assaying for the presence of homo- or hetero-multimers of the polypeptide, wherein formation of a homo- or hetero-multimer identifies a multimerization polypeptide comprising a seven residue repeat sequence, (a.b.c.d.e.f.g)
  • a method includes contacting RSV or a cell susceptible to RSV infection with an amount of a hetero- or homo-dimer, -trimer, -tetramer, -pentamer or higher order oligomer with a multimer effective to inhibit RSV infection of the cell. Additionally provided are methods of inhibiting RSV infection, inhibiting RSV progression or treating RSV infection of a subject.
  • a method in another embodiment, includes administering to a subject having or at risk of having RSV infection an amount of a multimer antibody, such as a hetero- or homo-dimer, -trimer, -tetramer or higher order oligomer of an antibody, effective to inhibit, inhibit progression or treat RSV infection of the subject.
  • a multimer antibody such as a hetero- or homo-dimer, -trimer, -tetramer or higher order oligomer of an antibody, effective to inhibit, inhibit progression or treat RSV infection of the subject.
  • the subject has or is at risk of having asthma; a new born or between the ages of 1 to 5, 5 to 10 or 10 to 18; the cell is an epithelial cell; the multimer comprises a chimeric polypeptide, for example, an antibody such as a humanized antibody.
  • the antibody is administered locally; via inhalation or intranasaly.
  • a method includes administering to a subject having or at risk of having a common cold an amount of an antibody, or a hetero- or homo-dimer, -trimer, -tetramer or higher order oligomer of the antibody, effective to treat the common cold in the subject.
  • the treatment comprises inhibiting infection by HRV, progression of HRV infection or a symptom of HRV infection; the antibody is humanized; the antibody is administered locally; the antibody is administered via inhalation or intranasaly; the subject has or is at risk of having asthma; and the subject is a new born or between the ages of 1 to 5, 5 to 10 or 10 to 18.
  • FIG. 1 shows an expression vector (“Fab vector”) for expressing exemplary chimeric polypeptide comprising a multimerization domain (ATF1 ⁇ ), a linker (ED) and an antibody sequence (V L , V H , C L and C H ), as described in Examples 1-3.
  • Fab vector for expressing exemplary chimeric polypeptide comprising a multimerization domain (ATF1 ⁇ ), a linker (ED) and an antibody sequence (V L , V H , C L and C H ), as described in Examples 1-3.
  • FIGS. 2 A- 2 B show characterization of purified CFY196.
  • A) Purified CFY196 runs as a single peak on the HPLC size exclusion column;
  • FIGS. 3 A- 3 C show processed sedimentation velocity data from A) CFY 195 (peak is at 5.5 S); B) CFY192B; and C)CFY196 (peaks at 6.55). Note the narrower peak for CFY196.
  • FIG. 4 shows protection of HeLa cells from infection by HRV14 with monovalent Fab and multivalent Fab-ATF ⁇ domain chimeric proteins.
  • Chimeric proteins are denoted as follows: CFY193B, Fab-ED-ATF ⁇ LL; CFY196, Fab-ED-ATF ⁇ (1)LI+S; CFY192B, Fab-ED-ATF ⁇ (2)LI; and CFY195, Fab-ED-ATF ⁇ II.
  • FIG. 5 shows protection of HeLa cells from HRV15 infection with monovalent Fab19, bivalent monoclonal antibody RR/1, bivalent CFY202, trimeric CFY193B, and tetrameric CFY 196.
  • FIGS. 6 A- 6 B show a bispecific multimeric protein.
  • A) Fab moities illustrated with different hatching have different specificities or functions.
  • a linker sequence is illustrated as a hexagon.
  • B) This polypeptide is produced from one tricistronic RNA molecule.
  • the coding sequence for the first polypeptide chain translated from this message is illustrated by the hatched box, representing the anti-CD-3 light chain (LC-1).
  • the second DNA fragment encodes the central chimeric polypeptide consisting of the anti-CD-3 heavy chain (HC-1) linked to a hinge derived from IgD, followed by a dimerization domain, a second hinge, and the anti-CD19 light chain (LC-2).
  • the third RNA empty box
  • the restriction sites are as indicated.
  • FIGS. 7 A- 7 B show improvement in tetramerization of ATF ⁇ IL by substituting amino acids at solvent exposed positions: A) CFY1971; B) CFY1972: and C)CFY197.
  • FIG. 8 shows the results of a competition ELISA. The data is presented as a percent of inhibition of tracer antibody binding to ICAM-1.
  • the invention is based, at least in part, on the identification of peptide sequences that confer multimerization, referred to herein as “multimerization polypeptides,” “multimerization domains” or “multimerization devices.”
  • Invention multimerization polypeptides confer oligomer formation.
  • a multimerization polypeptide when fused to a second molecule, such as a heterologous polypeptide sequence, facilitates the formation of dimers, trimers, tetramers, pentamers or higher order oligomers or mixtures thereof among the polypeptides.
  • Such invention multimerization polypeptides are useful in a variety of diagnostic or therapeutic applications.
  • fusing a multimerization polypeptide to a binding protein such as an antigen binding polypeptide (e.g., antibody) can be used to increase the number of antigen binding sites via oligomer formation.
  • a binding protein such as an antigen binding polypeptide (e.g., antibody)
  • a binding protein such as an antigen binding polypeptide (e.g., antibody)
  • a binding protein such as an antigen binding polypeptide (e.g., antibody)
  • a binding protein such as an antigen binding polypeptide (e.g., antibody)
  • an antigen binding polypeptide e.g., antibody
  • a fully human or humanized multimerized antibody has decreased or absent immunogenicity in humans in comparison to non-humanized antibody.
  • the oligomer may comprise, for example, two or more molecules of the same protein (e.g., a homo-dimer, -trimer, -tetramer or higher oligomer) or a mixture of two or more different (i.e., non-identical) proteins (e.g. a hetero-dimer, -trimer,-tetramer or higher oligomer).
  • oligomeric antibodies may comprise the same antibody or two or more different antibodies, each of which have two or more functions or activities (e.g., bind to two or more epitopes).
  • Such hetero-oligomers also referred to as multifunctional oligomers (e.g., bifunctional, trifunctional, tetrafunctional, etc., as appropriate) due to the multiple functions of the proteins that comprise the oligomer, are useful in a variety of diagnostic and therapeutic applications.
  • a multifunctional oligomer comprising two or more different antibodies is useful in applications in which it is desired to utilize a multifunctional antibody.
  • a mutlifunctional antibody may include an antibody that binds to a cell surface receptor and an antibody that mediates the complement cascade so that cells bearing the receptor are targeted for killing.
  • the specific functionality of the oligomer can be determined by the skilled artisan depending upon the application.
  • a multimerization polypeptide that confer multimer formation.
  • a multimerization polypeptide is selected from any of the amino acid sequences set forth in SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163.
  • a multimerization polypeptide has 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequences set forth in SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163, provided that the multimerization polypeptide is capable of conferring multimerization.
  • a multimerization polypeptide confers formation of hetero- or homo-dimers, trimers, tetramers, pentamers, hexamers, higher order oligomers and mixtures thereof.
  • a multimerization polypeptide is a sequence that is made fully human or humanized based on the sequence of a human peptide that can form homo- or hetero-dimers, trimers, tetramers, or higher order oligomers.
  • Multimerization polypeptides and nucleic acids encoding multimerization polypeptides when not fused to heterologous polypeptides are distinct from known wild type leucine zipper sequences.
  • the multimerization polypeptides of the invention do not include wild type leucine zipper domain sequences known in the art, such as those present in naturally occurring GCN4, ATFa, ATF-7 (100% identical to the coiled-coil domain in ATF ⁇ ), ATF-2, cyclic AMP response element binding protein Pa (CREB-Pa), JUN-D and C-JUN.
  • the multimerization polypeptides of the invention are also distinct from the multimerization device described in WO 96/37621, wild type sequences derived from human p53, PF4, TSP-4, COMP, thrombospondin, dTAF II 42, dTAF II 31, dTAF II 62, dTAF II 80, histone 3 and histone 4.
  • invention multimerization polypeptides may be of any length provided that they are capable of conferring mutimerization.
  • a multimerization polypeptide has a sequence at least 11 amino acids in length, at least 15 amino acids in length, at least 18 amino acids in length; at least 22 amino acids in length; at least 25 amino acids in length; at least 29 amino acids in length; and at least 31 amino acids in length.
  • a multimerization polypeptide has a sequence less than about 100 amino acids in length; less than about 75 amino acids in length; and less than about 50 amino acids in length.
  • the invention also provides chimeric polypeptides that include a multimerization polypeptide fused to a heterologous polypeptide. Such chimeric polypeptides form oligomers (multimers) via the multimerization polypeptide.
  • a chimeric polypeptide includes a multimerization polypeptide selected from any of the amino acid sequences set forth in SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163.
  • a chimeric polypeptide includes a multimerization polypeptide having 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity to an amino acid sequence set forth in SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163, provided that the chimeric polypeptide forms multimers.
  • a chimeric polypeptide forms hetero- or homo-dimers, trimers, tetramers, pentamers, hexamers, higher order oligomers and mixtures thereof.
  • multimer and grammatical variations thereof refers to formation of an oligomeric complex between two or more distinct molecules.
  • polypeptide multimer this means that the polypeptide forms a higher order oligomer with itself (homo-multimer) or with other molecules (hetero-multimer).
  • a polypeptide that confers multimerization means an amino acid sequence that can confer the formation of a dimer, trimer, tetramer, pentamer, hexamers or any higher order oligomer with itself (homo-oligomer) or with one or more different proteins (hetero-oligomer) under appropriate conditions.
  • a chimeric polypeptide comprising a multimerization polypeptide fused to a heterologous polypeptide is able to form dimers, trimers, tetramers, pentamers, hexamers, or higher order oligomers with itself or with different proteins having domains capable of interacting with the multimerization polypeptide portion.
  • Multimers therefore additionally include monovalent or oligomeric (e.g., dimer, trimer, tetramer, etc.) chimeric polypeptides either joined directly or indirectly through covalent or non-covalent binding.
  • the multimerization polypeptide may be directly linked to the heterologous polypeptide via a covalent bond, such as a chemical cross linking agent.
  • multimerization polypeptide can be connected to the heterologous polypeptide via a linker sequence (e.g., a peptide sequence such as the antibody hinge sequence).
  • a linker sequence e.g., a peptide sequence such as the antibody hinge sequence.
  • inclusion of a linker ensures that the multimerization polypeptide does not block function of the heterologous polypeptide and that the heterologous polypeptide does not block multimerization function.
  • a linker thus allows each antigen-binding domain in the multimer enough flexibility to bind antigen.
  • linker amino acid sequences may be fully human, humanized or non-human amino acid sequences, unmodified or modified as set forth herein.
  • a fully human or humanized linker sequence may be particularly useful for therapeutic purposes.
  • compositions comprising a chimeric polypeptide that include a multimerization polypeptide fused to a heterologous polypeptide are distinct from the known native (i.e., naturally occurring) and recombinant proteins that contain a multimerization polypeptide sequence.
  • scFv fused to yeast based protein GCN4-LI domain which forms tetravalent scFv (Pack et al., J. Mol. Biol. 246:28 (1995)) and scFv fused to wild type leucine zipper domains from jun and fos (U.S. Pat. No.
  • 5,910,573 are distinct from chimeric polypeptide that includes a multimerization polypeptide fused to a heterologous polypeptide.
  • Invention chimeric polypeptides may include multimerization sequences as known in the art, provided that the chimeric polypeptides form multimers and are distinct from known proteins that contain the multimerization sequences known in the art.
  • heterologous when used in reference to a polypeptide, means that the polypeptide is not normally contiguous with the other polypeptide in its natural environment.
  • an invention chimeric polypeptide including a multimerization polypeptide fused to a heterologous polypeptide means that the multimerization polypeptide does not exist fused with the heterologous polypeptide in normal cells.
  • a chimeric polypeptide including a multimerization polypeptide fused to a heterologous polypeptide is a molecule that does not normally exist in nature, i.e., such a molecule is produced by the hand of man, e.g., artificially produced through recombinant DNA technology.
  • fusion when used in reference to two or more molecules (e.g., polypeptides) means that the molecules are covalently attached or linked. Any method of attachment of the molecules is contemplated. Thus, the chemical nature of the linkage between the multimer and the molecule to which it is attached is not limited. A particular example for the linkage of two protein sequences is an amide bond or equivalent.
  • chimera when used in reference to a protein, means that the protein is comprised of one or more amino acid residues from two or more different proteins (i.e., non-identical proteins).
  • a particular example of a chimera containing amino acid residues from two or more different proteins is a polypeptide comprising a multimerization polypeptide and a heterologous polypeptide.
  • a particular example of a chimera containing amino acid residues from three or more different proteins is a polypeptide comprising a multimerization polypeptide, a linker (e.g. hinge) and a heterologous polypeptide.
  • amino acid sequences that confer multimerization mediate protein-protein binding via Van der Waals' forces, hydrophobic interactions, hydrogen bonding or charge-charge bonds.
  • Molecules may also form an oligomer if they are covalently linked to each other.
  • two distinct proteins, chemically synthesized, in vitro translated or isolated or purified from a cell or a sample may be chemically cross-linked together via a non-amide bond to form an oligomer.
  • two molecules that exist as separate entities and that do not form oligomers through non-covalent interaction but are joined together via covalent bonds are also considered to be a multimer.
  • an oligomer or a multimer of the invention may be formed through covalent bonding, non-covalent bonding or mixtures thereof.
  • the coiled-coil domain is composed of interacting, amphipathic ⁇ helices characterized by a seven-residue repeat sequence (a heptad repeat), a.b.c.d.e.f.g, with hydrophobic residues predominant at positions a and d (positions one and four), and polar residues generally elsewhere (Harbury et al., Science 262:1401 (1993)).
  • the leucine zipper domains are coiled-coil domains that typically have leucine at the d position of the heptad repeats.
  • Naturally occurring coiled-coils are typically made up of multiple heptad repeats, for example, three or more sequences, (a.b.c.d.e.f.g) 1 -(a.b.c.d.e.f.g) 2 -(a.b.c.d.e.f.g) 3 , etc.
  • the designation “(a.b.c.d.e.f.g) n ” merely refers to two or more additional half (3-4 amino acids) or full length (7 amino acids) heptad repeat sequences, where each half or full (a.b.c.d.e.f.g.) repeat need not have the identical amino acid sequence (see, e.g., Tables 3A and 3B).
  • tetramers are likely to form from coiled-coil sequences that have leucine at position a and isoleucine at position d, as shown by ATF ⁇ -LI, ATF1-LI, ATF2-LI, CJUN-LI, JUND-LI and CREB-LI domains.
  • Coiled-coil sequences with isoleucine at position a and leucine at position d may form dimers, such as CREB-IL, CJUN-IL and ATF2-IL, tetramers such as ATF ⁇ -IL, or dimers such as ATF1-IL.
  • leucine at both positions a and d either trimers or tetramers form.
  • ATF ⁇ -LL, ATF1-LL, CJUN-LL form trimer, and ATF2-LL and CREB-LL both form tetramers.
  • Coiled-coils with isoleucine at both positions a and d have a tendency to form trimers; for example, ATF ⁇ -II and ATF1-II form trimer.
  • mutliple forms exist because amino acids outside the hydrophobic core, positions b, c, e, f, and g, can modulate the multimerization state of the coiled coil by creating a network of inter- and intra-helical hydrogen bonds and salt bridges.
  • This network of hydrophilic bonds changes the relative orientation of the helices as it tends toward optimal geometry, resulting in the observed effect on multimerization state. Accordingly, it is possible in a context dependent manner to change one or a few residues at individual b, c, e, f and g positions to modulate the multimerization state.
  • the resulting multimerization polypeptides tend to be unstable, but can confer mixtures of dimer and tetramer formation.
  • the coiled-coil domain of JUN-D, amino acids 249-280 which has 48% identity to ATF ⁇ domain (Table 3B) was modified in accordance with the invention.
  • JUND-LI forms trimer and JUND-LL forms a dimer (Table 8).
  • the coiled-coil domain of C-JUN, amino acids 277-308 has 39% identity to ATF ⁇ domain.
  • C-JUN-LI forms tetramer
  • C-JUN-LL forms trimer
  • C-JUN-IL forms dimer (Table 8).
  • multimerization domains with two or more (a.b.c.d.e.f.g.) heptad repeat sequences will tolerate amino acids other than leucine, isoleucine or valine at the a and d positions.
  • one or more of the a and d positions may be amino acids other than leucine, isoleucine or valine.
  • a single hydrophilic residue may be inserted at an a or d position in a sequence including at least three heptad repeats to modulate the multimerization state.
  • the invention includes multimerization polypeptides in which the a and d positions in a given heptad repeat sequence may be amino acids other than leucine, isoleucine or valine.
  • the strength of the binding between the monomers that comprise the multimer also referred to herein as stability or tightness, may be altered by modifying (a.b.c.d.e.f.g.) heptad repeat sequences.
  • the invention includes multimerization polypeptides in which the a, b, c, d, e, f and g positions may be modified to alter the multimerization state as well as increase or decrease the strength of binding between the multimers.
  • Each of the multimeric domains derived from coiled-coil sequences may exist as mixtures of monomer, dimer, trimer, tetramer or higher oligomer.
  • the major multimeric form, which constitutes over 50% of the mass, is the designated multimer form. For example, if a multimer is designated a tetramer, at least 50% of the mass is in the tetrameric form.
  • multimerization polypeptides of the invention include, for example, SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163. Additional multimerization polypeptides may be identified by assaying putative coiled-coil sequences, composed of one or more heptad repeats (a.b.c.d.e.f.g) for oligomer formation using routine detection assays as set forth herein or known in the art (see, e.g., Example 6).
  • a sequence including a heptad repeat, (a.b.c.d.e.f.g), wherein one or more positions a and d are either leucine, isoleucine or valine can be incubated under conditions allowing formation of a hetero- or homo-oligomer.
  • the leucine zipper domain of cAMP response element binding protein-Pa (CREB-Pa, amino acids 393-424) is 74% identical to ATF ⁇ leucine zipper domain sequence (Table 3C).
  • Variants of CREB-Pa lecine zipper domain, CREB-LL and CREB-LI, formed tetramers and CREB-IL formed dimer (Table 8).
  • ATF-I leucine zipper domain (amino acids 238-269) is 29% identical to ATF ⁇ leucine zipper domain sequence.
  • Variants of ATF-1 leucine zipper domain, ATF1-LI form tetramers; ATF1-IL, ATF1-LL and ATF1-II all form trimers (Table 8).
  • Additional multimerization domains can be produced by first identifying a wild type coiled-coil domain. For example, five leucine zipper domains were identified by searching the public database (Table 3B). These domains show 32% to 77% identity to the ATF ⁇ leucine zipper domain, and less than 50% identity to GCN4 leucine zipper domain (Table 3C). The wild type residues at positions a and d were replaced with leucine or isoleucine (Table 3D).
  • Additional coiled-coil domains that may be modifed in accordance with the invention, include, for example, three and four helix bundles such as lung surfactant D protein, tetranectin, and mannose binding protein; and ROP, cytochrome B562, and tetrabrachion stalk, respectively.
  • multimerization domains can therefore be identified by comparison to sequence databases, and mutating the sequence as set forth herein.
  • one or more coiled-coil forming sequences may be selected from the protein database (e.g., GEN-BANK, SWISS-PROT) and isoleucine, leucine or valine may be introduced into one or more a or d positions of the heptad repeats to alter the multimerization status of the sequence.
  • the multimerization state may be determined using the assays described herein (Example 6).
  • the heptad repeat may be substituted with one or more point mutations, additions, or deletions at positions b, c, e, f and g where the additional mutations are selected to modulate or stabilize the multimeric state that is formed, that is, to alter the type of multimer formed, e.g., trimer vs. tetramer, or to modulate the binding strength between the monomers that form the multimer.
  • the additional mutations are selected to modulate or stabilize the multimeric state that is formed, that is, to alter the type of multimer formed, e.g., trimer vs. tetramer, or to modulate the binding strength between the monomers that form the multimer.
  • one or more positions b, c, e, f and g may be modified alone or in combination with modifications at one or more a or d positions.
  • mutations include those that make additional interhelical hydrogen bonds or salt bridges, for example, between e and g positions of a four helix bundle.
  • the mutations can also be selected by considering the effect of change on the network of hydrophilic bonds on the surface of the domain. Inspection of the hydrophilic bonding network is generally predictive but multimer status can be confirmed using the multimerization assays described herein (Example 6).
  • the modified sequence forms a trimer or tetramer or higher order oligomer instead of a dimer
  • one or more residues may be added at the helix's N or C terminus, or at the N or C terminus of the series of heptad repeat sequences to improve stability or tightness of the multimer.
  • the phrase to increase or improve “tightness” or “stability” means that the multimer formed is less likely to dissociate into its constituent monomers.
  • a general approximation of multimer tighteness or stability can be assessed by the narrowness or broadness of the peak formed in sedimentation velocity studies. Tighteness or stability can also be assayed, for example, by denaturing the multimer formed while simultaneously monitoring circular dichroism.
  • a specific example of such a modification is the addition of a terminal serine (Example 11).
  • the invention provides methods of producing a multimerization polypeptide comprising one or more seven residue coiled-coil, or leucine zipper repeat sequence, (a.b.c.d.e.f.g).
  • a method includes producing a polypeptide comprising a coiled-coil sequence, (a.b.c.d.e.f.g), wherein positions a and d are replaced with either leucine or isoleucine, thereby producing a multimerization polypeptide that confers dimer, trimer, tetramer or pentamer formation.
  • a method in another embodiment, includes producing a coiled-coil sequence, (a.b.c.d.e.f.g), wherein positions a or d are replaced with valine and either of leucine or isoleucine or valine, thereby producing a multimerization polypeptide that confers dimer and tetramer formation.
  • a method includes synthesizing a coiled-coil sequence, (a.b.c.d.e.f.g), wherein positions a and d are either leucine or isoleucine or valine to produce a multimerization polypeptide that confers dimer, trimer or tetramer formation.
  • a method includes synthesizing a coiled-coil sequence, (a.b.c.d.e.f.g), wherein positions a or d are valine and either of leucine or isoleucine to produce a multimerization polypeptide that confers dimer and tetramer formation.
  • positions a or d are replaced such that they are predominantly (e.g., greater than 50%) either leucine, isoleucine or valine.
  • one or more positions b, c, e, f and g are replaced, e.g., one or more hydrophobic residues are substituted with hydrophilic residues.
  • one or more amino acids are added to the N- or C-terminus of the multimerization domain or flanking a heptad repeat within the domain.
  • the invention also provides methods of identifying multimerization polypeptides comprising a coiled-coil sequence, (a.b.c.d.e.f.g), wherein positions a or d are either leucine, isoleucine or valine.
  • a method includes, incubating a polypeptide comprising a coiled-coil sequence, (a.b.c.d.e.f.g), wherein positions a or d are either leucine, isoleucine or valine, under conditions allowing formation of homo- or hetero-multimers, and assaying for the presence of homo- or hetero-multimers of the polypeptide.
  • the polypeptide comprises a heterologous polypeptide fused to a polypeptide comprising the coiled-coil sequence, (a.b.c.d.e.f.g).
  • the polypeptide contains a linker between the heterologous polypeptide and the polypeptide comprising the coiled-coil sequence, (a.b.c.d.e.f.g).
  • the homo- or hetero-multimer that is detected is a dimer, trimer, tetramer, pentamer, hexamer, or higher order oligomer.
  • proteins proteins, polypeptide and “peptide” are used interchangeably herein to refer to two or more contiguous amino acids, also referred to as “residues,” covalently linked through an amide bond or equivalent. Proteins are of unlimited length and may be comprised of L- or D-amino acids as well as mixtures thereof. Amino acids may be linked by non-natural and non-amide chemical bonds including, for example, those formed with glutaraldehyde, N-hydoxysuccinimide esters, bifunctional maleimides, or N,N′-dicyclohexylcarbodiimide (DCC).
  • DCC N,N′-dicyclohexylcarbodiimide
  • Non-amide bonds include, for example, ketomethylene, aminomethylene, olefin, ether, thioether and the like (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins , Vol. 7, pp 267-357, “Peptide and Backbone Modifications,” Marcel Decker, NY).
  • Polypeptides may have one or more cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond.
  • Polypeptides may be modified in vitro or in vivo, e.g., post-translationally modified to include, for example, sugar residues, phosphate groups, ubiquitin, fatty acids or lipids.
  • Polypeptides further include amino acid structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues.
  • antibody refers to a protein that binds to other molecules (antigens) via heavy and light chain variable domains, V H and V L , respectively.
  • Antibodies include IgG, IgD, IgA, IgM and IgE.
  • the antibodies may be intact immunoglobulin molecules, two full length heavy chains linked by disulfide bonds to two full length light chains, as well as subsequences (i.e. fragments) of immunoglobulin molecules, with our without constant region, that bind to an epitope of an antigen, or subsequences thereof (i.e. fragments) of immunoglobulin molecules, with or without constant region, that bind to an epitope of an antigen.
  • Antibodies may comprise full length heavy and light chain variable domains, V H and V L , individually or in any combination.
  • Polypeptide sequences can be made using recombinant DNA technology of polypeptide-encoding nucleic acids via cell expression or in vitro translation, or chemical synthesis of polypeptide chains using methods known in the art.
  • Antibodies and subsequences can be expressed from recombinantly produced antibody-encoding nucleic acid, such as a polynucleotide isolated from hybridoma cells or selected from a library of naturally occurring or synthetic antibody genes (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1989; Harlow and Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1999; Fitzgerald et al., J.A.C.S.
  • Polypeptide sequences can also be produced by a chemical synthesizer (see, e.g., Applied Biosystems, Foster City, Calif.).
  • multifunctional means that the composition referred to has two or more activities or functions (e.g., bifunctional, trifunctional, tetrafunctional, etc.).
  • a multifunctional polypeptide has two or more of antigen binding, enzyme activity, ligand or receptor binding, toxin, etc.).
  • a multifunctional oligomer comprises a mixture of two or more polypeptides each having at least one function or activity, such as antigen binding and enzyme activity, ligand or receptor binding, toxin, etc.
  • An antibody that binds to a particular antigen, and which also has an attached polypeptide with enzyme activity is one particular example of a bifunctional antibody.
  • Candidate functions for multifunctional olgiomers other than antigen binding and in addition to enzyme activity include, for example, detectable domains such as immunoglobulin, T7 and polyhistidine amino acid sequences, toxins (e.g., ricin, cholera, pertussis), cell surface proteins such as receptors, ligands (substrates, agonists and antagonists), adhesion proteins (e.g., streptavidin, avidin, lectins), growth factors, differentiative factors, chemotactic factors and proenzymes.
  • detectable domains such as immunoglobulin, T7 and polyhistidine amino acid sequences, toxins (e.g., ricin, cholera, pertussis), cell surface proteins such as receptors, ligands (substrates, agonists and antagonists), adhesion proteins (e.g., streptavidin, avidin, lectins), growth factors, differentiative factors, chemotactic factors and proenzymes.
  • Multifunctional multimers further include multispecific (e.g., bispecific, trispecific, tetraspecific, etc.) compositions.
  • multispecific means an antigen binding polypeptide (e.g., an antibody) that binds to different antigenic epitopes.
  • the different epitopes may be present on the same antigen or on different antigens.
  • a multispecific antibody oligomer comprises a mixture of two or more antibodies each having different epitope binding specificity.
  • Multispecific oligomers may be comprised of individual antigen binding polypeptides each of which have distinct variable domains.
  • one of the antigen binding polypeptides of the oligomer may have two variable domains each of which recognize a different epitope.
  • Multifunctional polypeptides can be produced through chemical crosslinking of the selected molecules (which have been produced by synthetic means or by expression of nucleic acid that encodes the polypeptides) or through recombinant DNA technology combined with in vitro, or cellular expression of the polypeptide, and subsequent oligomerization.
  • Multispecific antibodies can be similarly produced through recombinant technology and expression, fusion of hybridomas that produce antibodies with different epitopic specificities, or expression of multiple nucleic acid encoding antibody variable chains with different epitopic specificities in a single cell.
  • FIGS. 6A and 6B A specific example of a bispecific antibody is illustrated in FIGS. 6A and 6B.
  • the CREB-IL multimerization polypeptide is positioned at the center of a molecule linking Fab from two different antibodies producing a tetravalent binding antibody.
  • Use of multimeric polypeptides conferring trimer or tetramer formation would produce hexavalent and octavalent molecules, respectively.
  • the Fab moities in the bispecific protein can have different specificities.
  • a flexible linker (e.g., hinge) sequence can be used to join the Fab portion of each protein.
  • the exemplary bispecific antibody polypeptide may be produced from one tricistronic RNA molecule (FIG. 6B).
  • subsequence or “fragment” means a portion of the full length molecule.
  • a subsequence of a multimerization polypeptide is one or more amino acids less in length than full length polypeptide (e.g. one or more internal or terminal amino acid deletions from either amino or carboxy-termini). Subsequences therefore can be any length up to the full length molecule.
  • antibody subsequences include, for example, chimeric polypeptides in which a heterologous domain comprises an Fab, Fab′, (Fab′) 2 , Fv, or single chain antibody (SCA) fragment (e.g., scFv).
  • Subsequences include portions, which retain at least part of the function or activity of full length sequence. For example, an antibody subsequence will retain the ability to selectively bind to an antigen even though the binding affinity of the antibody subsequence may be greater or less than the binding affinity of the full length antibody.
  • an Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.
  • An (Fab′) 2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • An Fab′ fragment of an antibody molecule can be obtained from (Fab′) 2 by reduction with a thiol reducing agent, which yields a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab′ fragments are obtained per antibody molecule treated in this manner.
  • An Fv fragment is a fragment containing the variable region of a light chain V L and the variable region of a heavy chain V H expressed as two chains.
  • the association may be non-covalent or may be covalent, such as a chemical cross-linking agent or an intermolecular disulfide bond (Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659 (1972); Sandhu, Crit. Rev. Biotech. 12:437 (1992)).
  • a single chain antibody (“SCA”) is a genetically engineered or enzymatically digested antibody containing the variable region of a light chain V L and the variable region of a heavy chain, linked by a flexible linker, such as a polypeptide sequence, in either V L -linker-V H orientation or in V H -linker-V L orientation.
  • a single chain Fv fragment can be produced by linking two variable domains via a disulfide linkage between two cysteine residues.
  • binding means that the compositions referred to have affinity for each other. “Specific binding” is where the binding is selective between two molecules. A particular example of specific binding is that which occurs between an antibody and an antigen. Typically, specific binding can be distinguished from non-specific when the dissociation constant (K D ) is less than about 1 ⁇ 10 ⁇ 5 M or less than about 1 ⁇ 10 ⁇ 6 M or 1 ⁇ 10 ⁇ 7 M. Specific binding can be detected, for example, by ELISA, immunoprecipitation, coprecipitation, with or without chemical crosslinking, two-hybrid assays and the like. Appropriate controls can be used to distinguish between “specific” and “non-specific” binding.
  • K D dissociation constant
  • Full length antibodies, subsequences (e.g., single chain forms) or modified forms, fully human, humanized or non-human may be present as heteromeric or homomeric dimers, trimers, tetramers, pentamers, hexamers or any higher order oligomer that retains at least a part of the antigen binding activity of the monomer.
  • Antibody multimers include oligomeric (e.g., dimer, trimer, tetramer, etc.) combinations of different antibodies that are multispecific (e.g., bispecific, trispecific, tetraspecific, etc.) or multifunctional (e.g., bifunctional, trifunctional, tetrafunctional, etc.).
  • the invention further provides linker polypeptide sequences.
  • the invention linker sequences can be fused to a heterologous polypeptide to form a chimeric polypeptide.
  • a linker comprises a portion of a hinge region from an immunoglobulin.
  • a linker is derived from or modeled after a human or humanized immunoglobulin.
  • a linker sequence comprises a portion of an immunoglobulin hinge region selected from an amino acid sequence set forth in any of (SEQ ID NO:43 (D30), SEQ ID NO:44 (D35), SEQ ID NO:45 (ED), SEQ ID NO:46 (EDC) or SEQ ID NO:47 (D63)).
  • a linker sequence comprises an amino acid sequence from about 2 to 20 amino acids, from about 5 to 10 amino acids, from about 10 to 30 amino acids, from about 25 to 50 amino acids, from about 30 to 60 amino acids, from about 50 to 75 amino acids.
  • a linker sequence comprises an amino acid sequence from about 2 to 20 amino acids, from about 5 to 10 amino acids, from about 10 to 30 amino acids, from about 25 to 50 amino acids, from about 30 to 60 amino acids, from about 50 to 75 amino acids, from about 75 to 100 amino acids and which includes an amino acid sequence set forth in any of (SEQ ID NO:43 (D30), SEQ ID NO:44 (D35), SEQ ID NO:45 (ED), SEQ ID NO:46 (EDC) or SEQ ID NO:47 (D63)).
  • linker refers to a molecule or group of molecules that connects two or more molecules to each other.
  • a flexible linker between two molecules joined to each other allows enough free rotation of one or more of the molecules so that the molecules do not block each others' function.
  • a linker such as an amino acid sequence located between a multimerization polypeptide and a heterologous polypeptide of a chimeric polypeptide, e.g., a humanized antibody, allows the antibody to bind to antigen without significant steric interference from other multimers within the oligomer.
  • the invention also provides chimeric polypeptides including a multimerization polypeptide fused to a heterologous polypeptide that further include a linker polypeptide between the multimerization polypeptide and heterologous polypeptide. Also provided are chimeric polypeptides comprising linker sequences fused to a heterologous sequence.
  • Polypeptides of the invention including multimerization polypeptides, chimeric polypeptides oligomers, and linkers, include modified forms such as sequences having one or more amino acid substitutions, additions or deletions (i.e., subsequences), provided the modification does not destroy function.
  • the term “modification” therefore denotes an alteration of the molecule that does not destroy an activity or function of the modified molecule.
  • a modified multimerization polypeptide will therefore retain, for example, at least in part, the ability to form oligomers, even if the oligomers formed are less stable due to decreased affinity, e.g., form dimers instead of trimers, form trimers instead of tetramers, etc.
  • a modified heterologous polypeptide will retain at least a part of one or more functions or activities associated with the polypeptide (e.g., protein binding activity, enzyme activity, ligand activity, nucleic acid binding activity, growth regulatory activity, cell differentiative activity, or chemotactic activity).
  • a chimeric polypeptide that includes a modified antibody sequence such as an antibody subsequence or antibody having one or more amino acid additions or insertions will retain, at least in part, antigen binding capability.
  • Modifications therefore include amino acid additions, insertions, deletions and substitutions, for example.
  • An example of an addition is where one or more amino acids are added to the N- or C-terminal end multimerization polypeptide.
  • An example of an insertion is where an amino acid is inserted into the sequence.
  • An example of a deletion is where one or more amino acids are deleted from the N- or C-terminal end, or internally within the sequence.
  • Modifications may improve an activity or function of the modified molecule or provide a distinct functionality.
  • affinity of a multimerization polypeptide may be increased by addition of all or a portion of a heptad repeat sequence (e.g., a half turn).
  • Addition of a heptad repeat sequence, or a portion of a heptad repeat sequence may also change the oligomer from forming dimer to trimer or tetramer formation.
  • Exemplary multimerization polypeptides are set forth, for example, in SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163, each contain 4 and one-half heptad repeat sequences (see Table 3A). These and other heptad containing sequences may therefore be modified. For example, full length (7 amino acids) or half (3 to 4 amino acids of the motif) heptad repeat sequences may be added.
  • multimerization polypeptides of the invention including exemplary multimerization polypeptides set forth in SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; and SEQ ID NOs:154 to 163, may be modified to delete half (3 to 4 amino acids) or full (7 amino acids) length heptad repeat, for example.
  • Exemplary amino acid substitutions include conservative amino acid substitutions.
  • conservative amino acid substitution means the replacement of one amino acid by a biologically or chemically similar residue.
  • Biologically similar means that the substitution is compatible with biological activity, e.g., for a multimerization polypeptide, oligomer formation.
  • Particular examples of conservative substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, and the like.
  • a multimerization polypeptide has 1-3,3-5 or 5-10 amino acid substitutions, provided that positions a (one) or d (four) of a coiled-coil heptad repeat sequence, (a.b.c.d.e.f.g), are either leucine, isoleucine or valine, and that the substituted polypeptide be capable of multimerization.
  • positions a and d are predominantly either leucine, isoleucine or valine.
  • a multimerization polypeptide has 1-3,3-5 or 5-10 amino acid substitutions at positions b, C, e, f or g, e.g., to modulate the type of multimer that forms or to modulate the stability or tightness of the multimer formed.
  • one or more of the amino acid substitutions are conservative amino acid substitutions.
  • the substitution is with a human amino acid.
  • Modifications also include derivatized sequences, for example, amino acids in which free amino groups form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups; the free carboxy groups from salts, methyl and ethyl esters; free hydroxl groups that form O-acyl or O-alkyl derivatives, as well as naturally occurring amino acid derivatives, for example, 4-hydroxyproline, for proline, 5-hydroxylysine for lysine, homoserine for serine, omithine for lysine, etc. Also included are modifications that confer covalent bonding, for example, a disulfide linkage between two cysteine residues thereby producing a cyclic polypeptide. Modifications can be produced using any of a variety of methods well known in the art (e.g., PCR based sited-directed, deletion and insertion mutagenesis, chemical modification and mutagenesis, chemical cross-linking, etc.).
  • Modifications also include addition of functional entities such as tags (e.g., polyhistidine, T7, immunoglobulin, etc.), gold particles, covalently or non-covalently attached to the multimerization polypeptide, chimeric polypeptide or oligomers.
  • tags e.g., polyhistidine, T7, immunoglobulin, etc.
  • gold particles covalently or non-covalently attached to the multimerization polypeptide, chimeric polypeptide or oligomers.
  • the invention provides modified polypeptides having one or more activities (e.g., retain at least part of multimer activity, antigen binding activity, etc.) of unmodified polypeptide.
  • Modifications include radioactive and non-radioactive detectable labels attached to or incorporated into the molecule.
  • identity means that two or more referenced entities are the same. Thus, where two polypeptide sequences are identical, they have the same amino acid sequence. “Areas of identity” means that a portion of two or more referenced entities are the same. Thus, where two polypeptide sequences are identical over one or more parts of their sequence, they share identity in these areas.
  • substantially identity means that the identity is structurally or functionally significant. That is, the identity is such that the molecules are structurally identical or perform the same function (e.g., biological function) even though the molecules differ. Due to variation in the amount of sequence conservation between structurally and functionally related proteins, the amount of sequence identity for molecules having substantial identity will depend upon the type of region/domain and its function.
  • sequence homology For nucleic acid sequences, 50% sequence homology and above may constitute substantial identity. Substantial homology for proteins can be significantly less, for example, as little as 30% sequence identity, but typically is more, e.g., 50%, 60%, 75%, 85% or more.
  • BLAST e.g., BLAST 2.0
  • Altschul et al. J. Mol. Biol. 215:403 (1990), publicly available through NCBI at http:/www.ncbi.nlm.nih.gov
  • exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2.
  • a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 and the like.
  • compositions when used as a modifier of invention compositions (e.g., multimerization polypeptides, chimeras, linkers, antibodies, subsequences, modified forms, nucleic acids encoding same, cells, vectors, etc.), means that the compositions are made by the hand of man and are separated from their naturally occurring in vivo environment. Generally, compositions so separated are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane.
  • An “isolated” polypeptide can also be “substantially pure” when free of most or all of the materials with which it normally is associated in nature.
  • an isolated polypeptide that also is substantially pure does not include polypeptides or polynucleotides present among millions of other sequences, such as antibodies of an antibody library or nucleic acids in a genomic or cDNA library, for example.
  • Purity can be at least about 60% or more by mass. The purity can also be about 70% or 80% or more, and can be greater, for example, 90% or more. Purity can be determined by any appropriate method, including, for example, UV spectroscopy, mass spectroscopy, chromatography (e.g., HPLC, gas phase), gel electrophoresis (e.g., silver or coomassie staining) and sequence analysis (nucleic acid and peptide).
  • the invention also provides nucleic acids encoding invention polypeptides, including multimerization polypeptides, chimeras, linkers, subsequences, modified forms and multimers thereof.
  • a nucleic acid encodes a polypeptide set forth in SEQ ID NOs:1 to 7; SEQ ID NOs:9 to 37; or SEQ ID NOs:154 to 163.
  • a nucleic acid encodes a chimeric polypeptide comprising a sequence set forth in any of (SEQ ID NOs:1 to 36 and SEQ ID NOs:154 to 163), fused to a heterologous domain.
  • a nucleic acid sequence encodes (SEQ ID NO:43 (D30), SEQ ID NO:44 (D35), SEQ ID NO:45 (ED), SEQ ID NO:46 (EDC) or SEQ ID NO:47 (D63)).
  • nucleic acid refers to at least two or more ribo- or deoxyribonucleic acid base pairs that are linked through a phosphodiester bond or equivalent.
  • Nucleic acids include polynucleotides and polynculeosides.
  • Nucleic acids include single, double or triple, circular or linear molecules.
  • a nucleic acid molecule may belong exclusively or in a mixture to any group of nucleotide-containing molecules, as exemplified by, but not limited to, the following groups of nucleic acid molecules: RNA, DNA, cDNA, genomic nucleic acids, non-genomic nucleic acids, naturally occurring and non naturally occurring nucleic acids and synthetic nucleic acids. This includes, by way of example, nucleic acids associated with any organelle, such as the mitochondria, ribosomal RNA, and nucleic acid molecules comprised chimerically of one or more components that are not naturally occurring along with naturally occurring components.
  • nucleic acid molecule may contain in part one or more non-nucleotide-based components as exemplified by, but not limited to, amino acids and sugars.
  • a ribozyme that is in part nucleotide-based and in part protein-based is considered a “nucleic acid molecule.”
  • Nucleic acids can be of any length. Nucleic acid lengths typically range from about 20 to 10 Kb, 10 to 5 Kb, 1 to 5 Kb or less, 1000 to about 500 base pairs or less in length. Nucleic acids can also be shorter, for example, 100 to about 500 base pairs, or from about 12 to 25, 25 to 50, 50 to 100, 100 to 250, or about 250 to 500 base pairs in length.
  • nucleic acids include sequences and subsequences degenerate with respect to nucleic acids that encode (SEQ ID NO: 1 to 7, SEQ ID NOs:9 to 37, SEQ ID NOs:154 to 163, SEQ ID NO:43 (D30), SEQ ID NO:44 (D35), SEQ ID NO:45 (ED), SEQ ID NO:46 (EDC) or SEQ ID NO:47 (D63)).
  • Nucleic acids also include sequences set forth in Tables 1, 2 and 4, sequences complementary thereto and subsequences thereof. Such nucleic acids are useful for hybridization to detect the presence or an amount of chimeric polypeptide in a sample (in vitro, cell, culture medium, tissue or organ, serum, in a subject, etc.).
  • Nucleic acids of the invention can be produced using various standard cloning and chemical synthesis techniques. Such techniques include, but are not limited to: 1) nucleic acid amplification, e.g., polymerase chain reaction (PCR), with genomic DNA or cDNA targets, using primers (e.g., a degenerate primer mixture) capable of annealing to antibody sequence; 2) chemical synthesis of nucleic acid sequences which can then be cloned into a plasmid, propagated amplified and purified and; 3) computer searches of databases for related sequences. Purity of nucleic acids can be determined through sequencing, gel electrophoresis and the like.
  • PCR polymerase chain reaction
  • primers e.g., a degenerate primer mixture
  • the invention further provides expression cassettes comprising a nucleic acid encoding a chimeric polypeptide operably linked to an expression control element.
  • an expression control element “operably linked” to a nucleic acid means that the control element modulates transcription and as appropriate, translation of the transcript.
  • nucleic acid there need not be physical linkage to nucleic acid in order to control expression.
  • physical linkage is not required for the elements to be operably linked.
  • a minimal element can be linked to a nucleic acid encoding a chimeric polypeptide.
  • a second element that controls expression of an operably linked nucleic acid encoding a protein that functions “in trans” to bind to the minimal element can influence expression of the chimeric polypeptide. Because the second element regulates expression of chimeric polypeptide, the second element is operably linked to the nucleic acid encoding the chimeric polypeptide.
  • expression control element refers to nucleic acid that influences expression of an operably linked nucleic acid. Promoters and enhancers are particular non-limiting examples of expression control elements.
  • a “promotor sequence” is a DNA regulatory region capable of initiating transcription of a downstream (3′ direction) coding sequence. The promoter sequence includes a minimum number of bases necessary to initiate transcription. Enhancers also regulate gene expression but can function a distance from the transcription start site of the gene to which it is operably linked. Enhancers also function at either 5′ or 3′ ends of the gene, as well as within the gene (e.g., in introns or coding sequences).
  • An expression control element can confer expression in a manner that is “constitutive,” such that transcription of the operably linked nucleic acid occurs without the presence of a signal or stimuli.
  • Expression control elements can confer expression in a manner that is “regulatable,” that is, a signal or stimuli increases or decreases expression of the operably linked nucleic acid.
  • a regulatable element that increases expression of the operably linked nucleic acid in response to a signal or stimuli is also referred to as an “inducible element.”
  • a regulatable element that decreases expression of the operably linked nucleic acid in response to a signal or stimuli is referred to as a “repressible element” (i.e., the signal decreases expression such that when the signal, is removed or absent, expression is increased).
  • Expression control elements include elements active in a particular tissue or cell type, referred to herein as a “tissue-specific expression control elements.” Tissue-specific expression control elements are typically active in specific cell or tissue because they are recognized by transcriptional activator proteins, or other regulators of transcription, that are unique to a specific cell or tissue type.
  • Expression control elements additionally include elements that confer expression at a particular stage of the cell cycle or differentiation. Accordingly, the invention further includes expression control elements that confer constitutive, regulatable, tissue-specific, cell cycle specific, and differentiation stage specific expression.
  • Expression control elements include full-length nucleic acid sequences, such as native promoter and enhancer elements, as well as subsequences or nucleotide variants thereof (e.g., substituted/mutated or other forms that differ from native sequences) which retain all or part of full-length or non-variant control element function (confer regulation, e.g., retain some amount of inducibility in response to a signal or stimuli).
  • constitutive promoters such as T7 and the like, as well as inducible promoters such as ⁇ L of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) may be used.
  • inducible promoters e.g., ecdysone
  • yeast constitutive or inducible promoters may be used (see, e.g., Ausubel et al., In: Current Protocols in Molecular Biology, Vol. 2, Ch. 13, ed., Greene Publish. Assoc.
  • a constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (R. Rothstein In: DNA Cloning, A Practical Approach , Vol.11, Ch. 3, ed. D. M. Glover, IRL Press, Wash., D.C. (1986)).
  • constitutive promoters of viral or other origins may be used.
  • SV40, or viral long terminal repeats (LTRs) and the like, or inducible promoters derived from the genome of mammalian cells (e.g., metallothionein IIA promoter; heat shock promoter, steroid/thyroid hormone/retinoic acid response elements) or from mammalian viruses (e.g., the adenovirus late promoter; the inducible mouse mammary tumor virus LTR) can be used for expression.
  • the invention also provides transformed cells and progeny thereof into which nucleic acids encoding invention polypeptides have been introduced by means of recombinant DNA techniques in vitro, ex vivo or in vivo.
  • the transformed cells and progeny thereof can be propagated and the introduced nucleic acid transcribed, or encoded protein expressed. It is understood that a progeny cell may not be identical to the parental cell, since there may be mutations that occur during replication.
  • Transformed cells include but are not limited to prokaryotic and eukaryotic cells such as bacteria, fungi, plant, insect, and animal (e.g., mammalian, including human) cells.
  • the cells may be present in culture, in a cell, tissue or organ ex vivo or present in a subject.
  • transformed means a genetic change in a cell following incorporation of nucleic acid (e.g., a transgene) exogenous to the cell.
  • a “transformed cell” is a cell into which, or a progeny of which a nucleic acid molecule has been introduced by means of recombinant DNA techniques. Cell transformation may be carried out as described herein or using techniques known in the art. Accordingly, methods of producing cells containing the nucleic acids and cells expressing the chimeric polypeptides of the invention are also provided.
  • vector refers to, e.g., a plasmid, virus, such as a viral vector, or other vehicle known in the art that can be manipulated by insertion or incorporation of a nucleic acid, for genetic manipulation (i.e., “cloning vectors”), or can be used to transcribe or translate the inserted nucleic acid (i.e., “expression vectors”).
  • cloning vectors can be used to transcribe or translate the inserted nucleic acid
  • expression vectors are useful for introducing nucleic acids, including a nucleic acid that encodes a chimeric polypeptide operably linked with an expression control element, and expressing the encoded protein in vitro (e.g., in solution or in solid phase), in cells or in vivo.
  • a vector generally contains at least an origin of replication for propagation in a cell.
  • Control elements including expression control elements as set forth herein, present within a vector, are included to facilitate transcription and translation.
  • expression control element is intended to include, at a minimum, one or more components whose presence can influence expression, and can include components other than or in addition to promoters or enhancers, for example, leader sequences and fusion partner sequences, internal ribosome binding sites (IRES) elements for the creation of multigene, or polycistronic, messages, splicing signal for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA, polyadenylation signal to provide proper polyadenylation of the transcript of a gene of interest, stop codons, etc.
  • IRS internal ribosome binding sites
  • Vectors can include a selection marker.
  • selection marker means a gene that allows for the selection of cells containing the gene.
  • “Positive selection” refers to a process whereby only cells that contain the selection marker will survive upon exposure to the positive selection.
  • Drug resistance is one example of a positive selection marker; cells containing the marker will survive in culture medium containing the selection drug whereas cells which do not contain the marker will die.
  • markers include drug resistance genes such as neo, which confers resistance to G418, hygr, which confers resistance to hygromycin, orpuro which confers resistance to puromycin, among others.
  • Other positive selection marker genes include genes that allow identification or screening of cells containing the marker. These genes include genes for fluorescent proteins (GFP), the lacZ gene, the alkaline phosphatase gene, and surface markers such as CD8, among others.
  • Vectors can contain negative selection markers.
  • Negative selection refers to a process whereby cells containing a negative selection marker are killed upon exposure to an appropriate negative selection agent.
  • HSV-tk herpes simplex virus-thymidine kinase
  • GANC drug gancyclovir
  • the gpt gene renders cells sensitive to 6-thioxanthine.
  • Additional selection systems may be used, including, but not limited to the hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al., Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and the adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes. Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman et al., Proc. Natl. Acad. Sci.
  • Vectors included are those based on viral vectors, such as retroviral, adeno-associated virus, adenovirus, reovirus, lentivirus, rotavirus genomes, simian virus 40 (SV40) or bovine papilloma virus, etc., modified for introducing and expressing a nucleic acid in a cell (Cone et al., Proc. Natl. Acad. Sci. USA 81:6349 (1984); Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982; Sarver et al, Mol. Cell. Biol. 1:486 (1981)).
  • viral vectors such as retroviral, adeno-associated virus, adenovirus, reovirus, lentivirus, rotavirus genomes, simian virus 40 (SV40) or bovine papilloma virus, etc.
  • Additional viral vectors useful for expression include parvovirus, rotavirus, Norwalk virus, coronaviruses, paramyxo and rhabdoviruses, togavirus (e.g., Sindbis virus and semliki forest virus) and vesicular stomatitis virus.
  • parvovirus rotavirus
  • Norwalk virus coronaviruses
  • paramyxo and rhabdoviruses e.g., Sindbis virus and semliki forest virus
  • togavirus e.g., Sindbis virus and semliki forest virus
  • vesicular stomatitis virus e.g., Sindbis virus and semliki forest virus
  • Mammalian expression systems further include vectors specifically designed for in vivo and ex vivo expression.
  • Such systems include adeno-associated virus (AAV) vectors (U.S. Pat. No. 5,604,090) which have been shown to provide expression of Factor IX in humans and in mice at levels sufficient for therapeutic benefit (Kay et al., Nat. Genet. 24:257 (2000); Nakai et al., Blood 91:4600 (1998)).
  • Adenoviral vectors U.S. Pat. Nos. 5,700,470, 5,731,172 and 5,928,944
  • herpes simplex virus vectors U.S. Pat. No.
  • vectors U.S. Pat. Nos. 5,624,820, 5,693,508, 5,665,577, 6,013,516 and 5,674,703 and WIPO publications WO92/05266 and WO92/14829
  • papilloma virus vectors e.g., human and bovine papilloma virus
  • Vectors also include cytomegalovirus (CMV) based vectors (U.S. Pat. No. 5,561,063).
  • CMV cytomegalovirus
  • Vectors that efficiently deliver genes to cells of the intestinal tract have been developed and also maybe used (see, e.g., U.S. Pat. Nos. 5,821,235, 5,786,340 and 6,110,456).
  • yeast vectors that facilitate integration of foreign nucleic acid sequences into a chromosome, via homologous recombination, for example, are known in the art and can be used.
  • Yeast artificial chromosomes YAC are typically used when the inserted nucleic acids are too large for more conventional vectors (e.g., greater than about 12 kb).
  • nucleic acid encoding humanized antibody and humanized antibody into target cells can also be carried out by conventional methods known in the art such as osmotic shock (e.g., calcium phosphate), electroporation, microinjection, cell fusion, etc. Introduction of nucleic acid and polypeptide in vitro, ex vivo and in vivo can also be accomplished using other techniques.
  • osmotic shock e.g., calcium phosphate
  • electroporation e.g., calcium phosphate
  • microinjection e.g., cell fusion
  • Introduction of nucleic acid and polypeptide in vitro, ex vivo and in vivo can also be accomplished using other techniques.
  • a polymeric substance such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers.
  • a nucleic acid can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules, or poly (methylmethacrolate) microcapsules, respectively, or in a colloid drug delivery system.
  • Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • liposomes for introducing various compositions into cells, including nucleic acids, is known to those skilled in the art (see, e.g., U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282).
  • a carrier comprising a natural polymer, or a derivative or a hydrolysate of a natural polymer, described in WO 94/20078 and U.S. Pat. No. 6,096,291, is suitable for mucosal delivery of molecules, such as polypeptides and polynucleotides.
  • Piperazine based amphilic cationic lipids useful for gene therapy also are known (see, e.g., U.S. Pat. No.
  • Cationic lipid systems also are known (see, e.g., U.S. Pat. No. 5,459,127). Accordingly, viral and non-viral vector means of delivery into cells or tissue, in vitro, in vivo and ex vivo are included.
  • kits comprising one or more compositions of the invention, including pharmaceutical formulations, packaged into suitable packaging material.
  • a kit includes a chimeric polypeptide or hetero- or homo-oligomer thereof.
  • a kit includes a nucleic acid encoding a chimeric polypeptide.
  • the nucleic acids further include an expression control element conferring expression in a cell; an expression vector; a viral expression vector; an adeno-associated virus expression vector; an adenoviral expression vector; and a retroviral expression vector.
  • a kit includes a label or packaging insert including instructions for expressing a chimeric polypeptide (e.g., humanized antibody) or a nucleic acid encoding a chimeric polypeptide in cells in vitro, in vivo, or ex vivo.
  • a kit includes a label or packaging insert including instructions for treating a subject (e.g., a subject having or at risk of having asthma) with a chimeric polypeptide (e.g., humanized antibody) or a nucleic acid encoding a chimeric polypeptide in vivo, or ex vivo.
  • the term “packaging material” refers to a physical structure housing the components of the kit.
  • the packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, etc.).
  • the label or packaging insert can include appropriate written instructions, for example, practicing a method of the invention, e.g., treating HRV or RSV infection or the common cold. Kits of the invention therefore can additionally include instructions for using the kit components in a method of the invention.
  • Instructions can include instructions for practicing any of the methods of the invention described herein.
  • invention pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration to a subject. Instructions may additionally include indications of a satisfactory clinical endpoint or any adverse symptoms that may occur, or additional information required by the Food and Drug Administration for use on a human subject.
  • the instructions may be on “printed matter,” e.g., on paper or cardboard within the kit, on a label affixed to the kit or packaging material, or attached to a vial or tube containing a component of the kit.
  • Instructions may comprise voice recording or video tape and additionally be included on a computer readable medium, such as a disk (floppy diskette or hard disk), optical CD such as CD- or DVD-ROM/RAM, magnetic tape, electrical storage media such as RAM and ROM and hybrids of these such as magnetic/optical storage media.
  • kits can additionally include a buffering agent, a preservative, or a protein/nucleic acid stabilizing agent.
  • the kit can also include control components for assaying for activity, e.g., a control sample or a standard.
  • Each component of the kit can be enclosed within an individual container or in a mixture and all of the various containers can be within single or multiple packages.
  • an invention composition can be packaged into a hand pump container or pressurized (e.g., aerosol) container for spraying the composition into the throat or nasal or sinus passages of a subject.
  • the molecules of the invention can be incorporated into pharmaceutical compositions.
  • Such pharmaceutical compositions are useful for administration to a subject in vivo or ex vivo, and for providing therapy for a physiological disorder or condition treatable with a polypeptide of the invention.
  • compositions include “pharmaceutically acceptable” and “physiologically acceptable” carriers, diluents or excipients.
  • pharmaceutically acceptable and “physiologically acceptable” include solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration.
  • Such formulations can be contained in a liquid; emulsion, suspension, syrup or elixir, or solid form; tablet (coated or uncoated), capsule (hard or soft), powder, granule, crystal, or microbead.
  • Supplementary active compounds e.g., preservatives, antibacterial, antiviral and antifungal agents
  • compositions can be formulated to be compatible with a particular local or systemic route of administration.
  • pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by particular routes.
  • routes of administration for compositions of the invention are inhalation or intranasal delivery. Additional routes include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, transdermal (topical), transmucosal, and rectal administration.
  • parenteral e.g., intravenous, intradermal, subcutaneous, oral, transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as as
  • compositions for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal.
  • Isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride can be included in the composition.
  • Including an agent which delays absorption, for example, aluminum monostearate and gelatin can prolong absorption of injectable compositions.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of above ingredients followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients as above.
  • methods of preparation include, for example, vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays, inhalation devices (e.g., aspirators) or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • invention polypeptides and nucleic acids encoding them can be prepared with carriers that protect against rapid elimination from the body, such as a controlled release formulation or a time delay material such as glyceryl monostearate or glyceryl stearate.
  • the compositions can also be locally or systemically delivered using implants and microencapsulated delivery systems to achieve sustained delivery or for controlled release.
  • Biodegradable, biocompatable polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to cells or tissues using antibodies or viral coat proteins) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • compositions for administration in the methods of the invention are known in the art (see, e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993)).
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier or excipient.
  • Multimeric antibodies of the invention include antibodies that protect against virus infection of cells.
  • antibodies that bind ICAM-1 that have been fused with a multimerization polypeptide form multimers that can protect cells from HRV infection (FIGS. 6 and 7).
  • CFY196 Fab-ATF ⁇ (1)LI+S
  • CFY197 Fab-ATF ⁇ (3)IL
  • CFY193B Fab-ATF ⁇ (2)LL
  • RSV respiratory syncytial virus
  • ICAM-1 as a co-receptor for cell infection
  • antibody or ligand that binds ICAM-1 and that have been fused with a multimerization polypeptide to form multimers can also protect cells from RSV infection.
  • the invention provides antibody and ligand multimers that protect against RSV infection of cells.
  • protective efficacy is the amount of an antibody which can protect 50% of susceptible cells from infection (i.e. EC 50 ) under experimental conditions (see, e.g., Example 6).
  • protective efficacy in EC 50 is the amount of antibody that protects 50% of cells from RSV infection.
  • an antibody having a protective efficacy 5 times greater than another antibody e.g., non-humanized
  • Multimeric antibodies typically exhibit greater protective efficacy than a monomeric counterpart.
  • an antibody multimer has a protective efficacy at least 2 to 5 times greater than the antibody monomer.
  • an antibody multimer has a protective efficacy at least 5 to 10 times greater than antibody monomer.
  • an antibody multimer has a protective efficacy at least 10 to 20 times greater than the antibody monomer.
  • an antibody has a protective efficacy at least 20 to 30 times greater or more than the antibody monomer, for example, 30 to 50 times, 50 to 100 times, or 100 to 1000 times or more.
  • Chimeric polypeptides of the invention include multimerization polypeptide fused to antibody that binds to ICAM-1.
  • antibody binding to ICAM-1 inhibits viral binding or the ability to infect or penetrate the cell thereby inhibiting viral infection or proliferation.
  • pathogens such as respiratory syncytial and other viruses (e.g., HRV and coxackie A virus), bacteria, fungi and protozoa (e.g., malaria) that bind to ICAM-1.
  • pathogens such as respiratory syncytial and other viruses (e.g., HRV and coxackie A virus), bacteria, fungi and protozoa (e.g., malaria) that bind to ICAM-1.
  • the multimerized antibodies are useful for inhibiting HRV and RSV infection as well as for inhibiting any microorganism or other pathogen in which ICAM-1 receptor participates.
  • the invention provides multimerized antibodies (including fully or partially humanized forms) that inhibit pathogen infection of cells where infection is mediated, at least in part, by binding to ICAM-1, and methods for inhibiting pathogen infection of cells where infection is mediated, at least in part, by binding to ICAM-1.
  • a method includes contacting a virus or cell with an amount of multimerized antibody that binds to ICAM-1 sufficient to inhibit viral infection of the cell.
  • the multimerized antibody is humanized.
  • a method includes administering to a subject an amount of multimerized antibody that binds to ICAM-1 sufficient to inhibit viral infection of the subject.
  • the virus is RSV, coxackie A virus and HRV.
  • a method includes administering to a subject an amount of multimerized antibody that binds to ICAM-1 sufficient to inhibit infection of the subject by a pathogen.
  • a method includes administering to a subject an amount of multimerized antibody that binds to ICAM-1 sufficient to ameliorate a symptom of the infection, e.g., a symptom of the common cold.
  • the term “ameliorate,” when used in reference to a condition such as a symptom of a disease means to reduce one or more symptoms of the condition.
  • symptoms associated with the common cold include fever, headache, chills, sneezing, coughing, congestion, sore throat, runny nose, sore muscles, general malaise, etc.
  • a pathogen associated with the common cold e.g., HRV
  • to ameliorate the common cold or a pathogen associated with the common cold means to reduce one or more of fever, headache, chills, sneezing, coughing, congestion, sore throat, runny nose, sore muscles, general malaise, etc.
  • invention multimerized antibodies can be used to treat undesirable conditions, such as diseases or disorders in which ICAM-1 plays a role.
  • undesirable conditions such as diseases or disorders in which ICAM-1 plays a role.
  • LFA-1 interaction with ICAM-1 participates in inflammation.
  • An invention antibody may be used to inhibit this interaction thereby modulating (e.g., decrease) local or systemic inflammation.
  • a method includes administering to a subject enough multimerized antibody to reduce or prevent inflammation.
  • an invention antibody may be used to modulate immune response in order to reduce or prevent organ transplant rejection or autoimmune diseases or cancer or metastasis.
  • the invention provides multimerized antibodies that modulate immune responsiveness (e.g., inflammation) and other cellular processes in which ICAM-1 participates, as well as methods for modulating immune response pathways and other cellular processes in which ICAM-1 participates.
  • the invention also provides methods for inhibiting infection (e.g., prophylaxis), inhibiting progression or treating a pathogenic infection of a subject.
  • infection e.g., prophylaxis
  • ICAM-1 binding molecules may be delivered intra-nasally or intra-orally as sprays or drops to a subject.
  • a method includes administering to a subject having or at risk of having an RSV infection an amount of multimerized antibody sufficient to inhibit infection, inhibit progression or to treat RSV infection of the subject.
  • a method includes administering to a subject having or at risk of having an coxackie A virus or HRV infection an amount of multimerized antibody sufficient to inhibit infection, inhibit progression or to treat coxackie A virus or HRV infection of the subject.
  • a method includes administering to a subject having or at risk of having malaria an amount of multimerized antibody sufficient to inhibit infection, inhibit progression or to treat malaria of the subject.
  • the invention further provides methods of decreasing or inhibiting (i.e., ameliorating) one or more symptoms of a pathogen infection (e.g., caused by RSV, coxackie A virus, HRV or malaria).
  • a method includes administering to a subject having one or more symptoms associated with RSV, coxackie A virus, HRV or malaria an amount of a multimerized antibody sufficient to decrease or inhibit one or more symptoms associated with, RSV, coxackie A virus, HRV or malaria in the subject.
  • Symptoms decreased or inhibited include, for example, for RSV, one or more of pneumonia, fever, bronchitis, and upper respiratory tract infection; for coxackie A virus, one or more of fever, headache, chills, sneezing, coughing, congestion, sore throat, etc; for malaria, one or more of fever, chill, enlarged liver, anemia.
  • a method includes administering to a subject having pneumonia, fever, bronchitis, or upper respiratory tract infection an amount of a multimerized antibody sufficient to decrease or inhibit one or more symptoms of pneumonia, fever, bronchitis, or upper respiratory tract infection in the subject.
  • the humanized antibody is administered locally.
  • the multimerized antibody is administered via inhalation or intranasaly.
  • the subject has or is at risk of having asthma.
  • the methods of the invention may be practiced prior to infection (i.e. prophylaxis) or after infection, before or after acute or chronic symptoms of the infection or physiological condition or disorder develops (e.g., before organ transplantation).
  • Administering a composition prior to or immediately following development of symptoms may lessen the severity of the symptoms in the subject.
  • Administering a composition prior to development of symptoms in the subject may decrease contagiousness of the subject thereby decreasing the likelihood of other subjects becoming infected from the infected subject.
  • subject refers to animals, typically mammalian animals, such as a non-human primate (apes, gibbons, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.
  • Human subjects include adults, and children, for example, newborns and older children, for example, between the ages of 1 and 5, 5 and 10 and 10 and 18.
  • Human subjects may include those having or at risk of having a viral infection, such as HRV or RSV, and which develops one or more symptoms of the infection.
  • Human subjects include those having or at risk of having asthma, including asthmatics suffering from chronic asthma prior to or following suffering an acute asthma attack.
  • Subjects include disease model animals (e.g., such as mice and non-human primates) for testing in vivo efficacy of humanized antibodies of the invention (e.g., an HRV or RSV animal model, an asthma animal model, an organ transplant model, an autoimmune disorder model, cancer model, etc.).
  • a polypeptide includes a plurality of polypeptides (e.g., multimerization, chimeric, heterologous polypeptides) and reference to “a cell” can include reference to one or more such cells, and so forth.
  • This example describes the design of multimerization domains, linker sequences and chimeric polypeptides.
  • ATF ⁇ amino acids 181-211 constitute four and one-half heptad-repeats (31 amino acids). Since all the residues at the d position (position four) of each repeat are leucines, this heptad-repeat sequence is also referred to as a leucine zipper domain. ATF ⁇ amino acids 181-211 has 45% identity to the GCN4 leucine zipper domain.
  • Variants of ATF ⁇ amino acids 181-211 were produced by replacing position a and d (positions one and four in each of the heptad repeats) with each of the three hydrophobic, apolar residues: leucine, isoleucine and valine. (Table 3A).
  • ATF ⁇ variants that have leucine and isoleucine at either a or d position form trimers or tetramers; tetramer forming sequences include ATF ⁇ -LI and ATF ⁇ -IL, trimer forming sequences include ATF ⁇ -LL and ATF ⁇ -11.
  • ATF ⁇ -VL forms a mixture of dimer and tetramer; all variants with valine at a or d gave a high proportion of dimer.
  • the ATF ⁇ -LI domain forms tetramer when fused with either an antibody Fab fragment or with a reporter protein, thioredoxin.
  • Exemplary linker sequences were based on the human immunoglobulin D hinge sequence, which is the longest known flexible hinge among human immunoglobulins.
  • the human IgD hinge has a total of 63 amino acid residues, and the 57 th residue is a cysteine (Padlan, Mol. Immunol. 31:169 (1994)).
  • D30 contains the first 30 residues of the IgD hinge
  • D35 contains the first 35 residues
  • ED contains all of the 56 amino acid residues before the cysteine
  • EDC contains all of the 56 amino acid residues in ED plus the cysteine
  • D63 contains the complete hinge of 63 amino acids.
  • This example describes the cloning of an anti-ICAM humanized Fab fragment with antigen binding sites derived from Mab 1A6.
  • V H and V L domains are precisely fused on their 5′ ends to a gene segment encoding the enterotoxin III (stII) signal sequence.
  • the intervening sequence (IVS) in the dicistronic gene contains a ribosome entry site, while the 3′ end of the gene contains the bacteriophage ⁇ to transcriptional terminator (TER).
  • TER bacteriophage ⁇ to transcriptional terminator
  • a unique SacI site was inserted just before the stop codon in the C H 1 domain and a unique EcoRI site just after the stop codon to facilitate the addition of sequences encoding hinge and polymerization domains.
  • IPTG isopropyl-1-thio ⁇ -D-galactopyranoside
  • the gene segment for the light chain fragment (from SpeI to XbaI) was synthesized using PCR.
  • the PCR product was a fusion of two templates, the V L fragment encoding the variable domain of an humanized anti-ICAM antibody, and the C L template, whose sequence was derived from human ⁇ 1 light chain constant region (Palm and Hilschmann, Z. Physiol. Chem. 356:167 (1975)).
  • the V L template was synthesized using a series of overlapping oligonucleotides (Table 1). Oligonucleotides were first annealed in two groups consisting of: oligonucleotides 1, 2 and 3; and oligonucleotides 4, 5 and 6. Each annealed group was extended with Klenow fragment of DNA polymerase. Annealed and extended products were pooled as overlapping templates that were fused via PCR with P1 and P2 oligonucleotides (Table 2). The PCR product was directly cloned into the pCR2.1 vector (Invitrogen) and sequenced.
  • the CL template was derived from oligonucleotides that had been annealed in four groups (Table 1: Fab 4,5 and 6; Fab 7 and 8; Fab 9 and 10; Fab 11 and 28) and extended with the Klenow fragment of DNA polymerase (Stratagene, Lot#0800176). The pool of DNA was then amplified using a high-fidelity polymerase mixture (Roche Expand High Fidelity, Lot #85610228), and the flanking DNA primers, Fab 29 and Fab 3S. The PCR product was cloned into the PCR 2.1 TOPO cloning vector.
  • V L and C L domains were fused using oligonucleotides Fab I and Fab 11 and a 5′ SpeI site was added with oligonucleotides Fab26 and P3.
  • the final light chain clones were sequenced in their entirety.
  • a similar approach was used to clone the gene segment containing the heavy chain and the terminator as an Xba I/Hind III fragment.
  • a C H template based on the sequence of the C H 1 domain of human IgG1 (Ellison, et al. Nucl. Acids Res. 10:4071 (1982)) was made by annealing and extending four groups of oligonucleotides (Table 1, Fab 16 and 17, Fab 18 and 19, Fab 25 and 21 and Fab 20, 22 and 23) with the C L .
  • the V H domain encoding the heavy chain variable region of a humanized anti ICAM antibody was made by first annealing oligonucleotide 7, 8 and 9; oligonucleotide 10, 11 and 12; and oligonucleotide 13 and p4. Each annealed group was extended with the Klenow fragment of DNA polymerase. The annealed and extended products were pooled as overlapping templates that were fused N-terminally to the Fab 1 oligonucleotide with oligonucleotides Fab13, 12A and 12S, and C terminally to the C H1 domain using PCR with oligonucleotides Fab 12s and Fab 24 as primers. This fragment was also completely sequenced.
  • Fab 19 The expression plasmid for the humanized, anti ICAM Fab protein, termed Fab 19, was made by ligating the SpeI/Xba light chain fragment and the XbaI/Hind III heavy chain fragment into SpeI/HindIII-digested ptac/Tet to generate pFab 19/Tet (FIG. 1).
  • Oligo Sequence Oligo 1 ACAAACGCGTACGCTGATATCCAGATGACCCAATCTCCGTC (SEQ ID NO:83) AGCCTGAGCGCCAGTGTTGGTGATCGAGTTACCATTACT Oligo 2 GGTTTTTGTTGATACCAGTGAAGATTATTACTGATAGATTGGCTGGCG (SEQ ID NO:84) CGGCAAGTAATGGTAACTCGATCACCAACACTGGCGC Oligo 3 CTTCACTGGTATCAACAAAAACCGGGTAAAGCTCCGAAACTTCTTAT (SEQ ID NO:85) CTATCACGCCTCTCAGAGCATTAGCGGCGTTCCG Oligo 4 GAGAGCTGATGGTAAGGGTAAAGTCCGTGCCCGAGCCAGA (SEQ ID NO:86) GAAGCGGCTCGGAACGCCGCTAATGCTCTGAGAG Oligo 5 CTTTACCCTTACCATCAGCTCTCTTCAGCCGGAAGACTTTGCCACCTATT (SEQ ID NO:86) GAAGCGGCTCGGAACGCCGCTAATGC
  • This example describes cloning of human ATF ⁇ leucine zipper domain variants. This example also describes cloning of the human ATF ⁇ leucine zipper domain variants with attached linker (hinge) sequences.
  • the ATF ⁇ leucine zipper domain variants (Table 3A) were made by PCR amplification using two DNA oligonucleotides “A” and “B” that are complementary at the 3′ ends as the template (Table 2).
  • the oligonucleotide pairs were annealed, extended with the Klenow fragment of DNA polymerase, and amplified by PCR using oligonucleotides P1 and P2 as primers.
  • the resulting PCR products were purified and cloned into the PCR cloning vector, TOPO pCR2.1 (Invitrogen). Plasmid DNA with the cloned insert was isolated and sequenced.
  • a junctional oligonucleotide whose 5′ end is complementary to the 3′ end of the appropriate hinge, was used in a PCR reaction against the ATF ⁇ domain variant template. This PCR product was then used in combination with the hinge template in another PCR reaction to generate the hinge-ATF ⁇ domain fragment.
  • the hinge-ATF ⁇ domain fragment has a SacI site on the N terminus and a EcoRI site on the C terminus.
  • the hinge-ATF ⁇ domain fragment was cloned into the expression vector that carries the Fab fragment and was pre-digested with SacI and EcoRI, to be fused in-frame with the C terminus of the C H 1 domain of Fab (FIG. 1).
  • ATF domain oligonucleotides for ATF Leucine Zipper Variants
  • ATF domain oligonucleotides ATF ⁇ (1)-LI: (SEQ ID NOs:126-129)
  • ATF1-LI LKSIENRLAVIENQLKTIIEELKTIKDLLSN (SEQ ID NO:9)
  • ATF1-LL LKSLENRLAVLENQLKTLIEELKTLKDLLSN (SEQ ID NO:10)
  • ATF1-IL IKSLENRIAVLENQIKTLIEEIKTLKDLISN (SEQ ID NO:11)
  • ATF1-II IKSIENRIAVIENQIKTIIEEIKTIKDLISN
  • ATF1-VL VKSLENRVAVLENQVKTLIEEVKTLKDLVSN (SEQ ID NO:13)
  • ATF1-LV LKSVENRLAVVENQLKTVIEELKTVKDLLSN (SEQ ID NO:14)
  • CJUN-LI LARIEEKLKTIKAQLSEIASTLNMIREQLAQ (SEQ ID NO:
  • the residues at positions b, c, e, f, and g form an interlocked network of inter- and intra-helical interactions that influence the geometry of the helical bundle.
  • This network of intra- and inter-helical interactions can be analyzed from three-dimensional models. To do so, a model of the multimerization domain is constructed by homology using a coiled coil of the desired multimerization state as a template. After substituting the sequence of the domain to be analyzed in the proper register relative to the heptad repeat, the structure is energy minimized. Keeping in mind the possibility of alternative side chain rotamers, the surface residues are then analyzed by inspection. If a substitution is contemplated, the propagated effect on the entire network is considered. For example, changing an e position to a residue that can make a salt bridge to a g position in a neighboring helix may cause a third residue previously interacting with the g position to also change its interacting partner(s).
  • the effect of a substitution can be predicted with frequent success by this method.
  • the substituted multimer domain can be screened for its multimerization or other properties (e.g., stability or tightness).
  • the multimeric form produced can be determined by HPLC and/or ultracentrifugation assay methods as described herein (Example 6); the effect on tightness of multimerization can be assayed by determining melting profiles of a polypeptide having the multimerization domain.
  • This example describes expression and purification of multimeric Fab protein.
  • the cell pellet was suspended in 50 ml wash buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 50 ⁇ M PMSF and 5 mM EDTA) per liter culture and repelleted by centrifugation at 4,000 g for 15 minutes at 4° C. and frozen.
  • 50 ml wash buffer 50 mM Tris, pH 8.0, 150 mM NaCl, 50 ⁇ M PMSF and 5 mM EDTA
  • Frozen cell pellets were resuspended in 20 ml/gram wet weight pellet of a lysis buffer (50 mM Tris, pH 8.0, 200 mM NaCl, 5 mM EDTA, 5 mM EGTA, 50 ⁇ M PMSF and 0.1 mg/ml lysozyme), and after incubation on ice for 30 minutes the pellets were sonicated, and the lysates clarified by centrifugation at 23,000 g for 30 minutes. The supernatant, containing soluble protein, was adjusted to 1M NaCl and loaded on a Protein A column (Amersham/Pharmacia).
  • a lysis buffer 50 mM Tris, pH 8.0, 200 mM NaCl, 5 mM EDTA, 5 mM EGTA, 50 ⁇ M PMSF and 0.1 mg/ml lysozyme
  • the yield was 1-2 mg/liter of culture in shake flask.
  • the purity of protein preparations were determined by size exclusion chromatography and SDS-PAGE gel (FIG. 2).
  • Sedimentation velocity measures the movement of a solute boundary as the solute moves through solvent under the influence of a centrifugal force.
  • the data are a series of boundary positions recorded over time.
  • the raw data is then processed to give a distribution of sedimentation coefficients (FIG. 3).
  • the large peak shows the sedimentation coefficient for the major species in the sample.
  • the number of peaks is an indication of purity, and the broadness of the major peak indicates the homogeneity of the major species.
  • the oligomeric state of purified multimeric Fab fusion proteins was determined by sedimentation velocity using analytical ultracentrifugation (Modem Analytical Ultracentrifugation, T M Schuster and T M Laue, eds. (1994) Birkhauser, Boston) in combination with light scattering data (Wen, et al., Analytical Biochem 240:155 (1996)).
  • Trimeric Fab-ATF ⁇ domain fusion proteins sediment at about 5.5 S, and tetrameric proteins sediment at 6.5 S.
  • Table 6 The results of sedimentation studies on Fab-ATF ⁇ domain fusion proteins are summarized in Table 6.
  • the multimerization state of additional ATF ⁇ -based domains was determined by size exclusion HPLC.
  • HPLC size exclusion chromatography was performed on a column selected to resolve species of chimeric multimers ranging from dimers through pentamers. The selected column also resolved species including the Fab moiety of the chimera: Tetramers (6.5S by ultracentrifugation) consistently eluted from the column at 13.7 ⁇ 0.1 min.; trimers (5.5S) eluted at 14.0 ⁇ 0.1; and dimers (4.5S) eluted at 15.0 ⁇ 0.1 min.
  • Data on additional domains is listed in Table 7 along with those determined by sedimentation.
  • Sedimentation data also allows distinguishing between tetramers.
  • CFY 192B, 196 and 484 have the same a and d amino acids, and all form predominantly tetramers. These proteins, however, bear variations in sequence elsewhere. Examination of the c(s) vs. S plots for CFY196 and CFY192B reveals that the former is superior (FIG. 5). The distribution of sedimentation coefficient fitted for CFY196 is much narrower than that for CFY192B indicating a more homogeneous tetrameric population for CFY196.
  • This example describes biological activities of monovalent Fab19 anti human ICAM-1 protein and multivalent Fab19-EDATF ⁇ domain fusion proteins. This example also describes data indicating that trimeric and tetrameric Fab 19-EDATF ⁇ proteins provide greater protection of cells from HRV infection than monovalent Fab19 and bivalent monoclonal antibody.
  • HRV protection assay was performed to compare the abilities of the monovalent Fab19 protein and multivalent Fab19-based proteins to protect cells from infection.
  • HeLa cells were plated 1 ⁇ 10 5 per well in a 48-well tissue culture dish 24 hours before the assay. Growth medium was removed and 100 ⁇ l of multivalent or monovalent proteins diluted to the indicated concentrations added to each well. The plates were incubated for one hour in a 37° C. incubator, the solution was removed and 200 ⁇ l HRV15 (at MOI of 1) added and then incubated for one hour at 33° C. Cells were washed and 1 ml/well growth medium added. Infected cells were incubated at 33° C. for 48 hours, the medium removed and the remaining viable cells stained with crystal violet. Bound crystal violet was extracted with 3 ml methanol per well, then the amount of cell staining was quantified by measuring the A 570 .
  • % ⁇ ⁇ protection ( 100 ) ⁇ ( Absorbance ⁇ ⁇ of ⁇ ⁇ sample - Absorbance ⁇ ⁇ of ⁇ ⁇ virus ⁇ ⁇ only ) ( Absorbance ⁇ ⁇ of ⁇ ⁇ uninfected ⁇ ⁇ cells - Absorbance ⁇ ⁇ of ⁇ ⁇ virus ⁇ ⁇ only )
  • the protective efficacy is quantitated as EC 50 , the dose of antibody that gives 50% protection.
  • the EC 50 of Fab19 is 76 nM.
  • the tetrameric proteins CFY192B and CFY196 are more effective than the trimeric CFY192B and CFY195.
  • CFY196 (Fab19-EDATF ⁇ (1) ⁇ LI+S) demonstrated the highest protection (EC 50 of 0.73 nM), which is 104 times greater protection than monovalent Fab19 protein.
  • This example describes the construction and multimeric characterization of a non-Fab chimeric polypeptide of the invention, a thioredoxin-ATF ⁇ LI fusion protein.
  • the hinge-ATF ⁇ (1) ⁇ LI-+SG 3 H 6 fragment was fused to the C-terminus of thioredoxin in the expression vector pBAD-thio (Invitrogen).
  • the expression construct, pBAD-thio-hinge-ATF ⁇ (1) ⁇ LI-+SG 3 H 6 was transferred into the E. coli strain TOP-10 and grown in selective TB medium to an OD 600 of 0.8. After induction by addition of arabinose to 0.02% final concentration, and incubation overnight at room temperature, cells were harvested by centrifugation at 4,000 g for 15 minutes at 4° C.
  • the cell pellets were suspended in lysis buffer (20 mM sodium phosphate, pH 8.0, 1% Triton X-100, 500 mM NaCl, 40 mM imidazole, 1 mM ⁇ -mercaptoethanol), 0.2 mM PMSF, 0.5 mg/ml lysozyme and incubated on ice for 20 minutes.
  • the cells were sonicated, and another aliquot of PMSF was added.
  • Cell debris was pelleted by centifugation at 23,000 g and the clarified sonicate was filtered and fractionated by metal affinity chromatography.
  • the molecular weight of the thioredoxin-ATF ⁇ (1)-LI fusion protein was determined by size exclusion chromatography.
  • a Superdex 200 column (7.5 mm ⁇ 25 cm) was equilibrated in TBS buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) at a flow rate of 0.4 ml/min.
  • the column was calibrated with standard proteins (ribonuclease A, 13.7; chymotrypsinogen A, 25 kDa; ovalbumin, 43 kDa; bovine serum albumin, 66 kDa; alcohol dehydrogenase, 150 kDa; and ⁇ -amylase, 200 kDa).
  • the purified thioredoxin-ATF ⁇ LI1 fusion protein was analyzed separately at the same conditions as standard proteins.
  • the calculated molecular weight of a monomeric thioredoxin-ATF ⁇ (1)-LI-+SG 3 H 6 fusion protein is 19.6 kDa.
  • the calculated molecular mass of purified fusion protein is about 78 kDa. This result demonstrates that the thioredoxin-ATF ⁇ (1)LI-+SG 3 H 6 fusion protein exists as a tetramer.
  • This example describes inhibiting and treating respiratory syncytial virus (RSV) infection by multivalent ICAM-1 binding proteins of the invention.
  • RSV respiratory syncytial virus
  • RSV appears to bind to ICAM-1 via F protein on the surface of RSV; F protein is also the target of neutralizing antibodies against RSV.
  • RSV respiratory syncytial virus
  • very high antibody concentration 200-400 microgram/ml, >1 mM was required to achieve the apparent inhibition of RSV infection indicating that a monoclonal antibody is unlikely to be an effective therapy against RSV.
  • RSV may have relatively high binding affinity, or avidity, for ICAM-1. If so, a multimeric (dimer, trimer, tetramer, pentamer or even higher order oligomer) ICAM-1 binding protein will have better efficacy against RSV infection than a monovalent ICAM-1 antibody.
  • a full-blown RSV infection is a systemic disease, the virus first invades the body at the upper respiratory tract, especially at the nasopharynx area. Blocking RSV from the upper respiratory tract will prevent the entry of RSV, and therefore prevent RSV from invading the lower respiratory tract.
  • multivalent ICAM-1 binding protein appears to block the RSV foothold or entryway into cells of the upper respiratory tract. Consequently, free-floating RSV particles are re-routed by the normal mucocilliary clearing system into the gastrointestinal tract and become harmless.
  • the multimerization domain can be at the center of a molecule linking Fab from two different antibody molecules (see, e.g., FIG. 6A).
  • a trimerization or tetramerization domain could be used to produce hexavalent and octavalent molecules, respectively.
  • the Fab moities illustrated with different hatching could have different specificities, such as anti-CD-3 and anti-CD-19.
  • the Fab portion of each protein, consisting of the heavy and light chains, would be linked by a linker sequence to a dimerization domain, illustrated as a hexagon in FIG. 6A.
  • This polypeptide is produced from one tricistronic RNA molecule.
  • three promoters could drive the expression of three distinct polypeptides.
  • the coding sequence for the first polypeptide chain translated from this message is illustrated by the hatched box in FIG. 6B, representing the anti-CD-3 light chain (LC-1).
  • the second DNA fragment encodes the central chimeric polypeptide consisting of the anti-CD-3 heavy chain (HC-1) linked to a long hinge derived from IgD, followed by a dimerization domain, a second hinge, followed by the anti-CD 19 light chain (LC-2).
  • the third RNA illustrated by the white box in FIG. 6B, would encode the anti-CD-19 heavy chain (HC-2).
  • the first is an Nde I/Xba I fragment consisting of the light chain from the anti-CD-3 Fab and the bulk of an intervening sequence (IVS1) which includes a ribosome binding site. Downstream from this fragment the XbaI/SpeI restriction fragment consisting of six pieces fused together using PCR is ligated: the remainder of the IVS1, the heavy chain of Fab, a hinge derived from IgD, the CREB-IL dimerization domain, a second hinge derived from IgD, the light chain of anti-CD-19, and the bulk of the second intervening sequence (IVS2).
  • the third SpeI/HindIII restriction fragment would contain the remainder of IVS2, the heavy chain of anti-CD-19, and a terminator sequence.
  • Each of the IVS contain a ribosome binding site, and the 3′ end of the construction contains the bacteriophage ⁇ t 0 terminator (TER).
  • TER bacteriophage ⁇ t 0 terminator
  • the multimerization domain indicated by the filled box would be replaced by alternative multimerization sequences to form tetravalent, hexavalent, octavalent or higher order binding arms.
  • These three DNA coding sequences could be driven by multiple promoters, or be encoded on distinct vectors.
  • a similar constuct could also be used for bispecific antibodies when two Fab molecules of different specificity share a common light chain.
  • the sequences encoding HC-2 in FIG. 6B would replace those of LC-2, and the TER sequences would replace IVS2. This expression vector would then produce two polypeptide chains in a biscistronic message.
  • ATF ⁇ (1)-LI (SEQ ID NO:1) was modified by the addition of a C terminal serine (SEQ ID NO:154).
  • this multimerization domain was used in an anti-ICAM antibody chimera (CFY196)
  • the purified protein multimer (tetramer) was found to give an exceptionally narrow distribution of sedimentation coefficient (FIG. 3C, Example 6), which indicates tighter multimerization, (i.e., the K D decreases) for the subunits that comprise the multimer.
  • the multimerization domain ATF ⁇ -II (SEQ ID NO:6), which forms trimers when used in a chimeric anti-ICAM antibody, termed CFY195, was selected.
  • This sequence was chosen because the beta branched isoleucine residues can pack efficiently in the hydrophobic core of a pentamer. Residues at the interface between the hydrophobic core and the solvent exposed exterior of the domain must be changed to be more hydrophobic in order to change the trimer to a pentamer. Accordingly, four residues at e and g positions were changed to leucine.
  • both ATF ⁇ -II-g and several variants (Table 9) containing subsets of the mutations in ATF ⁇ -II-g were generated by PCR, subcloned, and sequenced in their entirety. These all have isoleucine in their a and d positions, but differ in the six amino acids that are underlined in Table 9. These domains were each fused to Fab19 and the ED hinge and assessed for their multimerization state by size-exclusion chromatography as described in Example 6.
  • Multimeric proteins composed of Fab 19, an ED hinge and the multimerization domains ATF ⁇ -II-a through ATF ⁇ -II-g (SEQ ID NO:155-159) exhibited a retention time greater than trimeric CFY195, but a, b, and c eluted from the HPLC column as broad peaks, indicating substantial heterogeneity. Only the ATF ⁇ -II-f and ATF ⁇ -II-g multimers produced the homogeneous species and eluted before tetramer, at a retention time expected for pentamer.
  • the activity of ATF ⁇ -II-g multimer was measured in a cell protection assay as described in Example 7.
  • ATF-1 a pentamer in the context of another coiled-coil domain, ATF-1.
  • Table 10 is the sequence of ATF1-II and a variant, ATF1-IIa, designed to form pentamers. The two sequences differ at the positions indicated by underlined amino acids. Comparison by HPLC of two chimeric proteins prepared from these domains indicate that the amino acid alterations changed the trimeric protein prepared from ATF1-II into a higher order multimer.
  • This example describes a competition ELISA assay for evaluation of binding affinity of monomeric and multimeric Fab antibodies.
  • a tetrameric Fab 19, termed 196TGC, conjugated with horesradish peroxidase (HRP) was developed for use as a tracer.
  • a cysteine was introduced into a tetratmeric anti-ICAM antibody by adding the sequence TGC to the C terminus of a chimeric Fab 19-ED protein containing the ATF(1) ⁇ -LI multimeriztion domain (196TGC).
  • HRP was chemically coupled to 196TGC using EZ-Link maleimide activated HRP (Pierce).
  • a 96-well EIA plate (Corning, Inc.) was coated with 100 ⁇ l/well soluble ICAM-1 (Bender MedSystems) at 1 ⁇ g/ml in 0.1 M NaHCO 3 . After washing with TBST (50 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween-20), the plate was blocked with 3% non-fat milk in TBST at room temperature for 1 hour. After washing with TBST, anti-ICAM-1 Fab samples (monomer or multimer) diluted serially in 1% non-fat milk/TBST solution were added and incubated at room temperature for 1 hour.
  • TBST 50 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween-20
  • the plate was incubated with the horseradish peroxidase-conjugated anti-ICAM-1 tetrameric antibody (196TGC-HRP) diluted 1:50,000 in 1% non-fat milk/TBST at room temperature for 2 hours.
  • the plate was washed thoroughly with TBST and 100 ⁇ l/well 3,3′,5,5′-tetramethybenzidine substrate solution (Kirkegaard and Perry Laboratories) was added. After 15 min incubation, the color development was stopped by adding 100 ml/well 0.12 N HCl and the absorbance of the wells at 450 nm was measured by a plate reader (ICN).
  • a 0 is OD 450 of the reference well without samples (196TGC-HRP only);
  • a s is the OD 450 reading from the diluted sample.
  • the relative binding affinity of the anti-ICAM-1 antibodies were represented by the protein concentration that blocks tracer antibody (TGC-HRP) at 50% (IC 50 ).
  • FIG. 8 shows that monomeric Fab (Fab19) only inhibits 25% tracer antibody (TGC-HRP) binding at the highest concentration tested. However, its trimer (Fab19-EDATF ⁇ -II) or its tetramer (Fab 19-EDATF ⁇ LI) gave a much higher percentage inhibition.
  • the IC 50 for these three molecules are as follows: Samples IC 50 ( ⁇ /ml) Fab19 below IC 50 Fab19-EDATF ⁇ -II 0.54 Fab19-EDATF(1) ⁇ -LI + S 0.069
  • Multimers may be used in any context where greater binding affinity or multivalent binding is desired.
  • the only requirement is the availability of a moiety that can be adapted for use in the construction of a chimeric protein of the invention.
  • the moiety may be a binding protein or any synthetic or natural molecule that can be coupled by a chemical bond to form the chimera. Examples of such moieties include chelators, binding peptides, binding proteins, and the like.
  • Multimeric molecules of the invention could be used for various environmental applications. For example, invention multimers that bound heavy metals or toxins could be used in bioremediation. Multimers that bound a protein on the surface of a pathogen can be used to neutralize that pathogen in the environment, for example, in a field where cattle graze. Multimers of the invention can also be constructed that act as insecticides or herbicides free of the unintended toxicity of many small molecules.
  • Multimeric molecules of the invention can also have industrial applications.
  • a multimer could be used in industrial chemistry to either remove a product of a reaction, thus speeding the reaction or driving it to completion, or to remove a reactant or catalyst, thus stopping a reaction.
  • multimeric molecules can be used to recover substances from dilute solutions.
  • bispecific molecules can be constructed from two successive enzymes in a synthetic pathway in order to increase the reaction rate for the combined steps.
  • Binding molecules such as antibodies against a target can be identified by any means known in the art.
  • CDR regions of an antibody that binds to a target of interest can be transferred to a humanized framework using methods known in the art.
  • the humanized antibody can optionally be expressed as either ScFv or Fab fragments as part of a chimeric multimeric protein.
  • target proteins of interest along with an indication of the medical condition that can be treated by binding of a multimeric protein to the target:
  • Beta tryptase allergy, inflammation
  • CD 105 CD 105
  • VEGF macular degeneration, cancer
  • CD154 (lupus, transplant rejection)
  • Folate receptor alpha (filovirus infection, e.g., Ebola and Marburg viruses)
  • nectin-1 also known as CD111-(human herpesviruses)
  • anti-CD-20 non Hodgkin's lymphoma

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US20080299121A1 (en) * 1997-12-01 2008-12-04 Fang Fang Multivalent recombinant antibodies for treating HRV infections
WO2009111304A2 (fr) * 2008-02-29 2009-09-11 President And Fellows Of Harvard College État de fusion intermédiaire de hiv-1 gp41 ciblé par des anticorps neutralisant à grande échelle
US8394771B1 (en) 2001-07-19 2013-03-12 Perlan Therapeutics, Inc. Multimeric proteins and methods of making and using same
US20130287780A1 (en) * 2008-01-22 2013-10-31 Multimerics Aps Products and methods to prevent infections
US9796788B2 (en) 2010-02-08 2017-10-24 Regeneron Pharmaceuticals, Inc. Mice expressing a limited immunoglobulin light chain repertoire
US9969814B2 (en) 2010-02-08 2018-05-15 Regeneron Pharmaceuticals, Inc. Methods for making fully human bispecific antibodies using a common light chain
US10130081B2 (en) 2011-08-05 2018-11-20 Regeneron Pharmaceuticals, Inc. Humanized universal light chain mice
US10143186B2 (en) 2010-02-08 2018-12-04 Regeneron Pharmaceuticals, Inc. Common light chain mouse
US10329350B2 (en) 2012-12-26 2019-06-25 Industrial Technology Research Institute Method for producing a multivalent fab fragment with collagen-like peptide
WO2020181062A1 (fr) 2019-03-06 2020-09-10 Cue Biopharma, Inc. Polypeptides multimères modulateurs des lymphocytes t et leurs méthodes d'utilisation
US10881085B2 (en) 2014-03-21 2021-01-05 Regeneron Pharmaceuticals, Inc. Non-human animals that make single domain binding proteins
US11111314B2 (en) 2015-03-19 2021-09-07 Regeneron Pharmaceuticals, Inc. Non-human animals that select for light chain variable regions that bind antigen

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WO2013078425A1 (fr) * 2011-11-22 2013-05-30 University Of Maryland, Baltimore Anticorps monoclonaux de la lamproie avec une affinité et une sélectivité élevées pour des glycanes et leurs utilisations
US20150038682A1 (en) * 2013-08-02 2015-02-05 Jn Biosciences Llc Antibodies or fusion proteins multimerized via homomultimerizing peptide
WO2016055656A1 (fr) * 2014-10-10 2016-04-14 Ablynx N.V. Méthodes de traitement d'infections à rsv
KR20170123849A (ko) 2016-04-29 2017-11-09 주식회사유한양행 Ccl3 변이체를 포함하는 융합 단백질 및 이의 용도

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Cited By (24)

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US20080299121A1 (en) * 1997-12-01 2008-12-04 Fang Fang Multivalent recombinant antibodies for treating HRV infections
US20070071742A1 (en) * 1998-11-30 2007-03-29 Fang Fang Humanized antibodies
US7696324B2 (en) 1998-11-30 2010-04-13 Perlan Therapeutics, Inc. Humanized antibodies
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US8394771B1 (en) 2001-07-19 2013-03-12 Perlan Therapeutics, Inc. Multimeric proteins and methods of making and using same
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US9259440B2 (en) * 2008-01-22 2016-02-16 Multimerics Aps Methods for passive immunization
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US10167344B2 (en) 2010-02-08 2019-01-01 Regeneron Pharmaceuticals, Inc. Mice expressing a limited immunoglobulin light chain repertoire
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US8394771B1 (en) 2013-03-12
JP5264518B2 (ja) 2013-08-14
CA2454358A1 (fr) 2003-07-31
AU2002365196B2 (en) 2009-01-15
AU2002365196C1 (en) 2009-08-13
JP2009136292A (ja) 2009-06-25
US20130243768A1 (en) 2013-09-19
CA2898314A1 (fr) 2003-07-31
JP2005522192A (ja) 2005-07-28
JP2013013417A (ja) 2013-01-24
AU2009201466B2 (en) 2012-05-03
WO2003062370A3 (fr) 2005-12-15
AU2009201466A1 (en) 2009-05-07
AU2002365196B8 (en) 2009-01-29
WO2003062370A2 (fr) 2003-07-31
EP1578917A2 (fr) 2005-09-28

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