EP1073671A4 - Administration de genes cibles dans des cellules au moyen de bacteriophages filamenteux - Google Patents

Administration de genes cibles dans des cellules au moyen de bacteriophages filamenteux

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Publication number
EP1073671A4
EP1073671A4 EP99919789A EP99919789A EP1073671A4 EP 1073671 A4 EP1073671 A4 EP 1073671A4 EP 99919789 A EP99919789 A EP 99919789A EP 99919789 A EP99919789 A EP 99919789A EP 1073671 A4 EP1073671 A4 EP 1073671A4
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EP
European Patent Office
Prior art keywords
phage
cells
cell
vector
antibody
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Withdrawn
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EP99919789A
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German (de)
English (en)
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EP1073671A1 (fr
Inventor
James D Marks
Marie Alix Poul
Baltazar Becerril
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University of California
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University of California
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Publication of EP1073671A1 publication Critical patent/EP1073671A1/fr
Publication of EP1073671A4 publication Critical patent/EP1073671A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2795/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2795/14045—Special targeting system for viral vectors
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

  • This invention relates to the field of cell transduction and gene delivery.
  • this invention relates to the use of filamentous phage to deliver heterologous nucleic acids into a cell.
  • BACKGROUND OF THE INVENTION Frequently gene transfection techniques require the ability to target a therapeutic gene to an appropriate "target" cell or tissue type with high efficiency (Michael and Curiel (1994) Gene Ther. 1: 223-232).
  • Targeting of retroviral vectors has been reported by inserting receptor ligands or single chain Fv (scFv) antibody fragments into the viral envelope protein (Kasahara et al. (1994) Science 266: 1373-1376).
  • Targeting of adenoviral vectors has been achieved by use of 'adapter' fusion molecules consisting of an antibody fragment which binds the adenoviral knob and a cell targeting molecule such as a receptor ligand or antibody (Douglas et al. (1996) Nat.
  • scFv single chain antibody fragments this typically requires construction of the scFv from the V-genes of a hybridoma, construction of the targeted gene delivery vehicle, and in vitro evaluation of targeting ability.
  • This invention is based, in part, on the discovery that filamentous phage displaying the an antibody that binds to an internalizing receptor (e.g. anti-ErbB2 scFv F5) as a genetic fusion with the phage minor coat protein pin can directly infect mammalian cells expressing the target receptor epitope (e.g., ErbB2) leading to expression of a heterologous gene (e.g. cDNA) contained in the phage genome.
  • this invention provides methods of transfecting (transducing) a target cell (e.g., vertebrate, invertebrate, bacteria, fungus, yeast, algal cell) with a (e.g. heterologous) nucleic acid.
  • the methods preferably involve i) providing a phage externally displaying a heterologous targeting protein (heterologous to the phage) and containing a heterologous nucleic acid (heterologous to the phage and/or to the target cell); and ii) contacting the target cell with the phage whereby said phage is internalized into said cell and wherein the heterologous nucleic acid is transcribed within the cell.
  • the heterologous nucleic acid comprises a reporter gene (or cDNA) or a selectable marker (e.g.
  • the heterologous nucleic acid transcribes a gene product (e.g., antisense molecule, ribozyme, polypeptide) other than, or in addition to, the reporter gene or selectable marker.
  • a gene product e.g., antisense molecule, ribozyme, polypeptide
  • a DNA brought into the cell by the methods of this invention is single stranded and, without being bound to a particular theory, it is believed the DNA is replicated to double stranded prior to transcription.
  • the phage used in the methods of this invention are monovalent, displaying, on average, one pill fusion protein per viral particle, while in other preferred embodiments, the phage used in the methods of this invention are multi- valent, displaying on average, at least two, more preferably at least 3, and most preferably at least 5, pill fusions per viral particle.
  • the phage used to deliver the heterologous nucleic acid into a target cell can be a member of a library of phage wherein said library comprises a number of different heterologous targeting proteins (e.g. containing, on average, at least 10 5 , preferably at least 10 6 , more preferably at least 10 7 , and most preferably at least 10 8 different members).
  • the methods can further involve selecting phage (e.g., from a library) that are internalized by the target cell.
  • the selection can be by a variety of means including, but not limited to detection of a reporter gene (e.g. GFP, Ffiux, luciferase, ⁇ -gal, etc.) or by selection via a selectable marker (e.g. an antibiotic resistance gene).
  • the method can further involve amplifying phage internalized by said cell.
  • the providing step involves i) providing an assembly cell containing the heterologous nucleic acid and a packaging signal; and ii) infecting the assembly cell with a phage expressing on its surface said heterologous targeting protein and containing the gene for the targeting protein whereby the phage acts as a helper phage and packages the heterologous nucleic acid.
  • Preferred assembly cells are prokaryotic cells (e.g. bacterial cells).
  • the heterologous targeting protein, and/or a DNA encoding the heterologous targeting protein, and/or the heterologous nucleic acid are encoded by a DNA that is a phagemid.
  • Preferred phage for use in the methods of this invention are filamentous phage.
  • Preferred heterologous targeting proteins are antibodies, more preferably single-chain Fv, or Fabs.
  • the phage can be preselected for binding to a particular internalizing cell surface receptor (e.g., erbB2).
  • Other preferred receptors include, but are not limited to receptors for platelet-derived growth factor (PDGF), epidermal growth factor- (EGF), insulin-like growth factor (IGF), transforming growth factor ⁇ (TGF- ⁇ ), fibroblast growth factors (FGF), interleukin 2 (TL2), nerve growth factor (NGF), interleukin 3 (IL3), interleukin 4 (IL4), interleukin 1 (LL1), interleukin 6 (IL6), interleukin 7 (IL7), interleukin 13, granulocyte/macrophage colony-stimulating factor (GM-CSF), granulocyte colony- stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin TGF
  • the phage can further express an endosomal escape polypeptide and/or a nuclear localization signal.
  • this invention provides a vector for (e.g., specific) transfection of a target cell.
  • Preferred vectors comprise a phage displaying a heterologous targeting protein (e.g. a single chain antibody) that specifically binds to an internalizing receptor whereby the phage binds to and is internalized into the target cell, and wherein the phage contains a heterologous nucleic acid that is transcribed inside the target cell.
  • a heterologous targeting protein e.g. a single chain antibody
  • the heterologous nucleic acid transcribes a gene product (e.g., antisense molecule, ribozyme, polypeptide) other than, or in addition to, a reporter gene or selectable marker.
  • the vector can include any of the viral particles or nucleic acids encoding the viral particles, or cells containing the nucleic acid or viral particles described herein.
  • this invention comprises a phage vector or phagemid vector encoding: a phage coat protein in fusion with a heterologous targeting protein that specifically binds to an internalizing cell surface receptor and is internalized into a cell bearing said receptor; and a heterologous nucleic acid in an expression cassette allowing transcription of the heterologous nucleic acid inside said cell as described herein.
  • the heterologous nucleic acid transcribes a gene product (e.g., antisense molecule, ribozyme, polypeptide) other than, or in addition to, a reporter gene or selectable marker
  • kits for transducing a target cell preferably a phage, and/or phage DNA, and/or phagemid DNA, and/or cell(s) containing phage and/or phagemid DNA, and/or cells containing phage particles as described herein.
  • the kits include a phage or phagemid vector encoding: a phage coat protein in fusion with a heterologous targeting protein that specifically binds to an internalizing cell surface receptor and is internalized into a cell bearing said receptor; and a pair of restriction sites that allow insertion of a heterologous nucleic acid into the phage or phagemid vector.
  • the restriction sites are preferably situated in an expression cassette such that a gene or cDNA inserted between said restriction sites is operably linked to a promoter and is transcribed, and optionally translated, when said expression cassette is transduced into a target cell.
  • an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and u constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V_) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 ⁇ a dimer of Fab which itself is a light chain joined to V H -CH1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab') 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments).
  • antibody as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
  • Preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (scFv or scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • the single chain Fv antibody is a covalently linked V H -V heterodimer which may be expressed from a nucleic acid including V H - and V L - encoding sequences either joined directly or joined by a peptide-encoding linker.
  • the first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv's (scFv), however, alternative expression strategies have also been successful.
  • Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule.
  • the two chains can be encoded on the same or on different replicons; the important point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to g3p (see, e.g., U.S. Patent No: 5733743).
  • scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and
  • Antigen-binding site refers to the part of an immunoglobulin molecule that participates in antigen binding.
  • the antigen binding site is formed by amino acid residues of the N-te ⁇ riinal variable ("V") regions of the heavy (“H”) and light (“L”) chains.
  • FR refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins.
  • the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen binding "surface". This surface mediates recognition and binding of the target antigen.
  • CDRs complementarity determining regions
  • immunological binding and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K ⁇ j) of the interaction, wherein a smaller Kd represents a greater affinity.
  • Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen- binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions.
  • both the "on rate constant” (k on ) and the “off rate constant” (k off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
  • the ratio of ko ff /k on enables cancellation of all parameters not related to affinity and is thus equal to the dissociation constant KLa (see, generally, Davies et al. (1990) Ann. Rev. Biochem., 59: 439- 473.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • F5 or Cl antibodies can be raised to the c-erbB-2 protein that bind c-erbB-2 and not to other proteins present in a tissue sample.
  • immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory - Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific irnmunoreactivity.
  • polypeptide polypeptide
  • peptide or “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • the amino acid residues are preferably in the natural "L” isomeric form. However, residues in the "D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • amino acids in addition to the 20 "standard” amino acids, include modified and unusual amino acids, which include, but are not limited to those listed in 37 CFR 31.822(b)(4).
  • binding polypeptide refers to a polypeptide that specifically binds to a target molecule (e.g. a cell receptor) in a manner analogous to the binding of an antibody to an antigen. Binding polypeptides are distinguished from antibodies in that binding polypeptides are not ultimately derived from immunoglobulin genes or fragments of immunoglobulin genes.
  • conservative amino acid substitution is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) of the molecule.
  • conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or hydrophobicity).
  • the following six groups each contain amino acids that are typical conservative substitutions for one another:
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. The term also includes peptide nucleic acids. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka et al. (1985) J. Biol Chem. 260: 2605-2608; and Cassol et al. (1992); Rossolini et al, (1994) Mol. Cell. Probes 8: 91-98).
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • isolated or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. However, the term “isolated” is not intended refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu.
  • expression cassette refers to nucleotide sequences which are capable of affecting expression of a structural gene in hosts compatible with such sequences.
  • Such cassettes include at least promoters and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein.
  • operably linked refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.
  • a fusion protein is a chimeric molecule in which the constituent molecules are all polypeptides and are attached (fused) to each other through terminal peptide bonds so that the chimeric molecule is a continuous single-chain polypeptide.
  • the various constituents can be directly attached to each other or can be coupled through one or more " peptide linkers.
  • a “target” cell refers to a cell or cell-type that is to be specifically bound by a member of a phage display library or a chimeric molecule of this invention.
  • Preferred target cells are cells for which an internalizing antibody or binding polypeptide is sought.
  • the target cell is typically characterized by the expression or overexpression of a target molecule that is characteristic of the cell type.
  • a target cell can be a cell, such as a tumor cell, that overexpresses a marker such as c-erbB-2.
  • a “targeting moiety” refers to a moiety (e.g. a molecule) that specifically binds to the target molecule.
  • the targeting moiety specifically binds the chimeric molecule to the cell bearing the target.
  • the targeting moiety is a polypeptide it can be referred to as a "targeting polypeptide”.
  • internalizing when used in reference to a cell refer to the transport of a moiety (e.g. phage) from outside to inside a cell.
  • the internalized moiety can be located in an intracellular compartment, e.g. a vacuole, a lysosome, the endoplasmic reticulum, the golgi apparatus, or in the cytosol of the cell itself.
  • An internalizing receptor or marker is a molecule present on the external cell surface that when specifically bound by an antibody or binding protein results in the intemalization of that antibody or binding protein into the cell.
  • Internalizing receptors or markers include receptors (e.g., hormone, cytokine or growth factor receptors) ligands and other cell surface markers binding to which results in intemalization. ]
  • heterologous nucleic acid refers to a nucleic acid that is not native to the cell in which it is found or whose ultimate origin is not the cell or cell line in which the "heterologous nucleic acid" is currently found.
  • the idiotype represents the highly variable antigen-binding site of an antibody and is itself immunogenic. During the generation of an antibody-mediated immune response, an individual will develop antibodies to the antigen as well as anti-idiotype antibodies, whose immunogenic binding site (idiotype) mimics the antigen. Anti-idiotypic antibodies can also be generated by immunization with an antibody, or fragment thereof,
  • a "phage display library” refers to a collection of phage (e.g., filamentous phage) wherein the phage express an external (typically heterologous) protein. The external protein is free to interact with (bind to) other moieties with which the phage are contacted. ⁇ Each phage displaying an external protein is a "member" of the phage display library.
  • an “antibody library” refers to phage display library that displays antibodies (binding proteins encoded by one or more antibody genes or cDNAs).
  • the antibody library includes the population of phage or a collection of vectors encoding such a population of phage, or cell(s) harboring such a collection of phage or vectors.
  • the library can be monovalent, displaying on average one single-chain antibody per phage particle or multi- valent displaying, on average, two or more single chain antibodies per viral particle.
  • Preferred antibody libraries comprise on average more than 10 6 , preferably more than 10 7 , more preferably more than 10 , and most preferably more than 10 different members (i.e. encoding that many different antibodies).
  • filamentous phage refers to a viral particle capable of displaying a heterogenous polypeptide on its surface.
  • the vector is, or is derived from, a filamentous bacteriophage, such as, for example, fl, fd, Pfl, Ml 3, etc.
  • the filamentous phage may contain a selectable marker such as tetracycline (e.g., "fd-tet").
  • a "viral packaging signal” is a nucleic acid sequence necessary and sufficient to direct incorporation of a nucleic acid into a viral capsid.
  • An assembly cell is a cell in which a nucleic acid can be packaged into a viral coat protein (capsid). Assembly cells may be infected with one or more different virus particles (e.g. a normal or debilitated phage and a helper phage) that individually or in combination direct packaging of a nucleic acid into a viral capsid.
  • virus particles e.g. a normal or debilitated phage and a helper phage
  • detectable label refers to any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g.
  • DynabeadsTM DynabeadsTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., H, I, 35 S, 14 C, or 32 P
  • enzymes e.g., LacZ, CAT, horse radish peroxidase, alkaline phosphatase ⁇ - and others, commonly used as detectable enzymes, either as marker gene products or in an ELISA
  • colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • Those detectable labels that can be expressed by nucleic acids are referred to as "reporter genes" or "reporter gene products”.
  • fluorescent labels are not to be limited to single species organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like.
  • CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be easily derivatized for coupling to a biological molecule (Bruchez et al. (1998) Science, 281 : 2013- 2016).
  • highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281: 2016-2018).
  • a nuclear localization signal is a nucleic acid sequence that directly or indirectly results in localization of the nucleic acid to the cell nucleus.
  • Nuclear localization sequences are well known to those of skill in the art. In most cases the NLS consists either of a short division of basic amino acids, for example as shown for the NLS of SV40 T antigen (P KKRKV). Alternatively, the NLS may have a bipartite structure comprised of two stretches of basic residues separated by a spacer of about 10 amino acids. (Dingwell et al. (1991) Trends Biochem. Sci. 16: 478). In the practice of the invention, any NLS sequences that functions to direct the localization of PUR to the nucleus may be incorporated into the phage or phagemid vectors.
  • An endosomal escape sequence is a nucleic acid sequence that directly or indirectly results in the transport of a molecule from the endosome into the cytoplasm of a cell.
  • Endosomal escape sequences e.g. viral escape mechanisms
  • examples include, but are not limited to the co-intemalization system of adenovirus (Curiel et al. (1991) Proc. Natl. Acad. Sci. USA, 8: 8850-8854), and the influenza viral peptides known to participate in endosomal escape mechanisms (Wiley and Skehel (1987) Ann. Rev. Biochem. 56: 365-394; Wagner et al. (1991) Proc. Natl. Acad. Sci. USA, 89: 7934-7938).
  • AMP ampicillin
  • c-erbB-2 ECD extracellular domain of c-erbB-2
  • CDR complementarity determining region
  • ELISA enzyme linked immunosorbent assay
  • FACS fluorescence activated cell sorter
  • FR framework region
  • Glu glucose
  • HBS hepes buffered saline, 10 mM hepes, 150 mM NaCl, pH 7.4
  • IMAC immobilized metal affinity chromatography
  • k on association rate constant
  • k off dissociation rate constant
  • MPBS skimmed milk powder in PBS
  • MTPBS skimmed milk powder in TPBS
  • PBS phosphate buffered saline, 25 mM NaH 2 P0 4 , 125 mM NaCl, pH 7.0
  • PCR polymerase chain reaction
  • RU resonance units
  • scFv or scFv single-chain Fv fragment
  • Figure 1 illustrates the method for construction of a large human scFv phage antibody library.
  • the strategy for library construction involved optimizing the individual steps of library construction to increase both the efficiency of scFv gene assembly and to increase the efficiency of cloning assembled scFv genes.
  • A First, mRNA from lymphocytes was used to generate V H and V L gene repertoires by RTPCR which were cloned into different vectors to create VH and VL gene libraries of 8.0 x 10 8 and 7.2 x 10 ⁇ members respectively. The cloned V-gene libraries provided a stable and limitless source of V H and V L genes for scFv assembly.
  • V H and V gene repertoires were amplified from the separate libraries and assembled into an scFv gene repertoire using overlap extension PCR. The primers used to reamplify the V H and VL gene repertoires annealed 200 bp upstream of the 5' end of the V H genes and 200 bp down stream of the V genes. These long overhangs ensured efficient restriction enzyme digestion.
  • Figures 2A 2B, and 2C show schematics illustrating antibody phage display: Cartoon of phage displaying (2 A) a single scFv (2B) a single diabody or (2C) multiple scFv.
  • scFv single chain Fv antibody fragment
  • VH Ig heavy chain variable domain
  • VL Ig light chain variable domain
  • pill phage minor coat protein pLII
  • Ag antigen bound by - scFv.
  • Figure 3 shows the effect of trypsinization on the enrichment of antigen specific phage.
  • a mixture of fd phage (5.0 x 10 11 cfu) and C6.5 scFv phagemid (5.0 x 10 8 fu) was incubated with SKBR3 cells for 2 hours at 37°C. Washes were performed either as described in Table 7 (-) or cells were trypsinized prior to cell lysis (+). Phage present in the first stripping buffer wash (cell surface phage) and the cell lysate (intracellular phage) were titered in the presence of ampicillin (C6.5 phagemid) or tetracycline (fd phage).
  • Figure 4 shows the effect of incubation time and chloroquine on the recovery of antigen specific phage.
  • SKBR3 cells were incubated in the presence ( ⁇ , •) or absence (D, O) of chloroquine (50 ⁇ M) for 2 hours prior to the addition of anti-botulinum phagemid (D, ⁇ ) or C6.5 scFv phagemid (O, •) (1.5 x 10 9 cfu/ml).
  • Cell samples were taken at 0 minutes, 20 minutes, 1 hour or 3 hours after phage addition, washed as described in Figure 4 including the trypsinization step and intracellular phages titered.
  • Figure 5 shows the effect of phage concentration on the recovery of intracellular phage.
  • Figure 6 illustrates strategies for producing anti-ErbB2 phagemids and phages packaging a eukaryotic reporter gene.
  • Helper phage are used to infect TGI containing pHEN-F5-GFP, a phagemid composed of an fl origin of replication (fl ori), the anti-ErbB2 F5 scFv gene fused to gene III and an eukaryotic GFP reporter gene driven by the CMV promoter.
  • Phage recovered from the culture supernatant display an average of 1 scFv-p ⁇ i fusion protein and 99% of them package the GFP reporter gene.
  • the anti-ErbB2 F5 scFv gene is cloned into the fd phage genome for expression as a scFv-pffl fusion.
  • fd-F5 phages are used to infect TGI containing a GFP reporter phagemid vector (pcDNA3-GFP). Phages purified from the culture supernatant display multiple scFv-pLTI fusion protein and approximately 50% package the GFP reporter gene.
  • Figure 7 shows a comparison of anti-ErbB2 phagemid and phage binding on cells.
  • 10 ⁇ ErbB2 expressing SKBR3 cells were incubated with increasing concentrations of F5-phagemids (circles) or fd-F5-phages (squares) at 4°C for 1 hour.
  • Cell surface bound " " phages were detected with biotinylated anti-M13 and strep tavidin-PE. Binding was detected by FACS and the results expressed as mean fluorescent intensity (MFI).
  • FIGS. 8A and 8B illustrate phagemid-mediated gene transfer in breast cancer cell lines.
  • Fig. 8A (1, 2, 3) 2.0 x 10 5 MCF7 (low ErbB2 expression) or (4, 5, 6) 2.0 x 10 5 SKBR3 (high ErbB2 expression) cells grown in 6-well plates were incubated with either no (1,4) no phage, (2, 5) 5,0 x 10 12 cfu/ml of helper phage packaging GFP or (3, 6) 5.0 x 10 11 cfu/ml of F5-GFP-phagemids for 48 hrs. Cells were trypsinized and GFP detected by FACS.
  • Fig. 8A (1, 2, 3) 2.0 x 10 5 MCF7 (low ErbB2 expression) or (4, 5, 6) 2.0 x 10 5 SKBR3 (high ErbB2 expression) cells grown in 6-well plates were incubated with either no (1,4) no phage, (2, 5) 5,0 x 10 12
  • Figures 9A, 9B, 9C, and 9D show concentration dependence and time course of phagemid mediated GFP expression in SKBR3 cells.
  • Figures 9A and 9B show concentration dependence of phagemid and phage mediated GFP expression in SKBR3 cells. 5.0 x 10 4 cells were grown in 24-well plates and incubated with increasing concentrations of F5-GFP-phagemid (squares), fd-F5-GFP-phage (diamonds) or GFP-helper phage (circles). After 60 hrs, the cells were trypsinized and analyzed by FACS for GFP expression.
  • Figures 9C and 9D show the time dependence of phagemid mediated GFP expression in SKBR3 cells.
  • This invention provides methods and materials for transfecting cells using targeted phage.
  • the methods involve providing a phage displaying an external binding protein or antibody and containing a heterologous nucleic acid (heterologous to the phage and/or to the cell).
  • the external binding protein preferably specifically binds to an internalizing marker (receptor/receptor epitope) which results in intemalization of the phage.
  • an internalizing marker receptor/receptor epitope
  • the antibody phage containing the heterologous nucleic acid can be prepared by a number of methods well known to those of skill in the art. In general these methods involve providing cells containing phagemid vector encoding the heterologous targeting protein or nucleic acid that is to be transfected into the cell and a corresponding page (e.g. helper phage) containing nucleic acid encoding a targeting polypeptide or the heterologous nucleic acid that is to be transfected into the target cell.
  • a bacterial e.g. E.
  • Phagemid encoding Infect with helper Phage containing both heterologous Heterologous DNA; and phage. nucleic acid sequences (targeting DNA encoding scFv protein and heterologous DNA) and expressing targeting polypeptide (e.g. scFv) on surface.
  • Phagemid encoding Infect with helper 1) Phage containing targeting protein heterologous DNA phage containing on surface and heterologous DNA nucleic acid encoding (from phagemid clone); and targeting polypeptide (scFv) on its surface 2) Phage containing targeting protein on surface and nucleic acid encoding targeting protein (from phage clone)
  • phage expressing the targeting molecule on their surface are used as helper phage (see Maniatis) to package the genome of a phagemid containing the heterologous DNA (e.g. a mammalian expression cassette).
  • this involves subcloning the targeting protein gene from the pHENl vector into a phage vector (such as FdDOGl (Clackson et al, Nature (1991) 352: 624-628) where it is located inframe with the phage gene in.
  • a phage vector such as FdDOGl (Clackson et al, Nature (1991) 352: 624-628) where it is located inframe with the phage gene in.
  • targeting scFv can be cloned as ApaLl-Notl fragments into the ApaLl-Notl sites of FdDOGl .
  • the phage genome leads to production of phage (from bacteria) which has the targeting protein on its surface and the phage genome inside. These phage are then used for superinfection of the phagemid containing bacterial cells.
  • the phagemid vector DNA by definition contains a phage origin of replication and packaging signal.
  • the phage genome products direct single stranded DNA synthesis of the phagemid DNA.
  • the phage acts as a helper phage leading to the production of two types of phage particles, those that contain the phage genome and those that contain the phagemid genome.
  • Using standard phage (such as Fd) as helper phage results in approximately an equal probability of the phage packaging either genome.
  • All of the phage will also have the targeting protein on their surface as pffl fusions. This is a simple way to generate phage that have the targeting molecule on their surface and the heterologous expression DNA inside the phage. While only a fraction (50%) of phage harbor the heterologous expression DNA, this is a large enough fraction given the high titer with which phage can be produced, to generate targeted phage.
  • Packaging is rapid, simple and most importantly can avoid tedious and time consuming subcloning steps required to insert the DNA sequence that is to be delivered to the eukaryotic cells into the phagemid or phage vector harboring the DNA sequence of the targeting gene.
  • this approach provides a generic method for packaging any DNA into the targeting phage for delivery and expression in eukaryotic cells. This makes it simple to deliver and study the effects of a large number of different genes in eukaryotic cells. It is noted that use of phage genome as a helper phage can lead to the problem of "interference" where the titer of phage generated is lower than expected (see Maniatis).
  • helper phage K07 uses a phage with a plasmid origin of replication and a partially disabled phage origin of replication (see Maniatis).
  • K07 uses a phage with a plasmid origin of replication and a partially disabled phage origin of replication (see Maniatis).
  • Use of K07 as a helper phage leads to production of higher phage titers.
  • Similar alternative phage vector backbones could be used for creation of targeted helper phage to result in higher phage titers.
  • the bacteria can be co-transformed with phage and phagemid DNA and co-selected with antibiotics.
  • the resulting cells contain both genomes and make phage containing both the heterologous targeting protein and the nucleic acid that is to be delivered into the cell.
  • the phage contains the heterologous nucleic acid that is to be delivered into the cell and the phagemid contains the nucleic acid encoding the heterologous targeting protein.
  • the phagemid genome containing the targeting molecule-p ⁇ i gene fusion is modified to contain the gene sequence that is to be delivered to the target eukaryotic cell (for example a mammalian expression cassette containing a reporter gene (or cDNA) and or another gene or (cDNA)).
  • Targeting phage are produced in the standard manner by the addition of helper phage.
  • both the heterologous nucleic acid that is to be delivered into the cell and the heterologous nucleic acid encoding the targeting protein are inserted into the phage genome.
  • the bacteria then need only the page genome inside and will make phage with targeting protein on the outside and both genomes inside.
  • phage can be delivered via an internalizing receptor into the endosome
  • the phage preferably from the endosome and uncoating facilitates exposure of the single stranded genome which then finds its way to the nucleus. There the single stranded DNA is replicated to double stranded DNA which is then transcribed and translated. It is also recognized by those skilled in the art, that methods exist to improve the efficiency of each of these steps. For example, endosomal escape sequences are known which can be incorporated into the phage coat proteins. Co incubation with . defective adenovirus would also provide endosome escape signals. Nuclear localization sequences are also known which could increase delivery to the nucleus. Inclusion of episomal replication sequences lead to amplification of the delivered DNA with an increase in the efficiency of expression.
  • Virtually any cell bearing an internalizing marker/receptor can be transfected using the methods of this invention.
  • internalizing phage display library members can be optimized for intemalization by a particular marker.
  • new, previously unknown receptors or epitopes can be identified and targeted.
  • Targets can be selected that whose distribution is restricted to particular cell types, target tissues, organs, or cells and/or tissues and/or organs displaying a particular physiological state or pathological condition.
  • targets can be selected that are characteristic of particular tumor types.
  • Tumor specific targets are well known to those of skill in the art and include, but are not limited to c-erbB-2, the IL-13 receptor, other growth factor receptors, and so forth.
  • internalizing targets can be selected that are present on most or all cell types (e.g., transferrin receptor).
  • a phage library can be selected without a subtracting cell line, or sequentially on unrelated cell lines.
  • the transfection methods allow generalized transfection of essentially any and/or all cells or an organ, tissue, or organism. Tissue specific targets can also be identified.
  • Ruoslahti et al. U.S. Patent No: 5,622,699 have identified polypeptides that specifically target particular tissues (e.g. brain, kidney, etc.).
  • ITI Transfected nucleic acids.
  • heterologous nucleic acid can be transfected into a cell. Once in the cell, the nucleic acid will optionally be transcribed, and optionally translated, depending on the nature of the particular nucleic acids.
  • the heterologous nucleic acid can encode a polypeptide gene product it - is desired to introduce into the cell.
  • a polypeptide gene product may include a reporter gene (e.g., green fluorescent protein or 5 ⁇ -galactosidase).
  • a cell such as a tumor cell
  • a toxin such as Pseudomonas exotoxin or subunits thereof, diphtheria toxin or subunits thereof, ricin, abrin, etc.
  • the nucleic acid transcript can be active in its own right (e.g. a ribozyme, an antisense molecule, etc.).
  • the heterologous nucleic acid encodes a protein product that is to be expressed in the target cell
  • the heterologous nucleic acid preferably encodes an expression cassette compatible with the target cell.
  • the expression cassette preferably includes a promoter that is inducible or constitutive in a mammalian cell, an initiation site, and a te ⁇ nination site.
  • the cassette can optionally include a selectable marker.
  • the transduction methods of this invention rely on the use of "internalizing antibodies", or “internalizing polypeptides”. Such “internalizing” molecules are internalized when they bind a target cell. Methods of identifying internalizing antibodies/target epitopes are provided herein and illustrated in the Examples. The methods generally involve contacting a "target” cell with one or more members of a phage display library displaying an antibody or a binding polypeptide.
  • the phage display library is preferably a polyvalent phage display library and it is believed that this invention provides the first description of a polyvalent antibody phage display library.
  • the cells are washed to remove externally bound phage (library members) and then internalized phage are released from the cells, e.g., by cell lysis.
  • the internalized phage are still viable (infectious).
  • the internalized phage in the cell lysate can be recovered and expanded by using the lysate containing internalized phage to infect a bacterial host. Growth of infected bacteria leads to expansion of the phage which can be used for a subsequent round of selection. Each round of selection enriches for phage which are more efficiently internalized, more specific for the target cell or have improved binding characteristics.
  • the phage display library is preferably contacted with a subtractive cell line - (i.e. a subtractive cell line is added to the target cells and culture media) to remove members of the phage display library that are not specific to the "target" cell(s).
  • the subtractive cell line is preferably added under conditions in which members of the phage display library are not internalized (e.g., at a temperature of about 4°C to about 20°C, more preferably at a temperature of about 4°C) so that non-specific binding members of the library are not internalized (sequestered) before they can be subtracted out by the subtractive cell line.
  • the "target" cells are washed to remove the subtractive cell line and to remove non-specifically or weakly-bound phage.”
  • the target cells are then cultured under conditions where it is possible for intemalization to occur (e.g. at a temperature of about 35°C to about 39°C, more preferably at a temperature of about 37°C).
  • the duration of the intemalization culture period will determine the intemalization speed of the antibodies (phage display members) for which selection takes place. With shorter intemalization periods more rapid internalizing antibodies are selected while with longer intemalization periods slower internalizing antibodies are selected.
  • the intemalization period is preferably less than about 120 minutes, more preferably less than about 60 minutes, and most preferably less than about 30 minutes or even less than about 20 minutes. It is noted that during the intemalization period the target cells are grown under conditions in which intemalization can occur. For a number of cell lines, this involves culturing the cells adherently on culture plates.
  • the target cells are washed to remove non-internalized (e.g. surface-bound phage).
  • the cells can then be moved to clean media.
  • the cells are adherent, they cells are trypsinized to free the cells from the extracellular matrix which may contain phage antibodies that bind the extracellular matrix. Freeing the cells into solution permits more through washing and moving of the cells to a new culture flask will leave behind any phage that may have stuck to the tissue culture dish.
  • the cells can then be washed with a large volume of PBS and lysed to release the internalized phage which can then be expanded e.g. used to infect E. coli to produce phage for the next round of selection. It is noted that there is no need to actually visualize the internalized phage. Simple cell lysis and expansion of the formerly internalized phage " is sufficient for recovering internalizing phage display members.
  • an antibody or polypeptide that is internalized into a cell has been identified, it is possible to probe one or more cell types with the identified antibody or polypeptide to identify the target recognized and bound by the antibody. Since the antibody is an internalizing antibody it is likely that such targets are themselves internalizing targets (e.g. members or portions of internalizing receptors).
  • the antibody can be labeled as described below.
  • the cells can then be contacted with the antibody (i.e. in vivo or in vitro) and the cells or cellular regions to which the antibody binds can then be isolated.
  • affinity chromatography involves immobilizing (e.g. on a solid support) one or more species of the internalizing antibodies identified according to the methods of this invention. Cells, cellular lysate, or cellular homogenate are then contacted with the immobilized antibody which then binds to its cognate ligand. The remaining material is then washed away and the bound/isolated cognate ligand can then be released from the antibody for further use.
  • the antibodies are used to immunoprecipitate the target from cell lysate.
  • the precipitate is then run on an SDS-PAGE gel which is Western blotted onto nitrocellulose.
  • the blot is probed with the precipitating antibody to identify the location of the target.
  • the portion of the blot containing the target can then be sent for N- terminal protein sequencing.
  • the N-terminal sequence can then be used to identify the target from standard databases, or DNA probes can be synthesized to probe genomic or cDNA libraries.
  • This approach has been used to identify the antigen bound by a phage antibody. Selections of a phage antibody library were done on intact Chlamydia trachomatis (a bacterial like organism that causes Chlamydial diseases). Selected antibodies were then used as described above to identify the antigen bound.
  • polypeptide and antibody fragments on the surface of viruses which infect bacteria makes it possible to isolate a single binding polypeptide or antibody fragment from a library of greater than 10 10 nonbinding clones.
  • phage display a polypeptide or an antibody fragment gene is inserted into the gene encoding a phage surface protein (pUi) and the antibody fragment-pin fusion protein is displayed on the phage surface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137).
  • phage bearing antigen binding polypeptides or antibody fragments can be separated from non-binding phage by antigen affinity chromatography (McCafferty et al. (1990) Nature, 348: 552-554).
  • affinity chromatography McCafferty et al. (1990) Nature, 348: 552-554
  • enrichment factors of 20 fold - 1,000,000 fold are obtained for a single round of affinity selection.
  • more phage can be grown and subjected to another round of selection. In this way, an enrichment of 1000 fold in one round can become 1,000,000 fold in two rounds of selection (McCafferty et al. (1990) Nature, 348: 552-554).
  • analysis for binding is simplified by including an amber codon between the antibody fragment gene and gene m.
  • the amber codon makes it possible to easily switch between displayed and soluble (native) antibody fragment simply by changing the host bacterial strain (Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137).
  • Human antibodies can be produced without prior immunization by displaying very large and diverse V-gene repertoires on phage (Marks et al. (1991) J. Mol. Biol. 222: 581-597).
  • VH and V L repertoires present in human peripheral blood lymphocytes were isolated from unimmunized donors by PCR.
  • the V-gene repertoires were spliced together at random using PCR to create a scFv gene repertoire which was cloned into a phage vector to create a library of 30 million phage antibodies (Id.).
  • binding antibody fragments have been isolated against more than 17 different antigens, including haptens, polysaccharides and proteins (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al (1993). Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12: 725-734; Clackson et al (1991) Nature. 352: 624-628). Antibodies have been produced against self proteins, including human thyroglobulin, immunoglobulin, tumor necrosis factor and CEA (Griffiths et al. (1993) EMBO J. 12: 725-734).
  • antibody fragments against four different erythrocyte cell surface antigens were produced by selecting directly on erythrocytes (Marks et al. (1993). Bio/Technology. 10: 779-783).
  • Antibodies were produced against blood group antigens with surface densities as low as 5,000 sites/cell. The antibody fragments were highly specific to the antigen used for selection, and were functional in agglutination and immunofluorescence assays.
  • Antibodies against the lower density antigens were produced by first selecting the phage antibody library on a highly related cell type which lacked the antigen of interest.
  • This negative selection removed binders against the higher density antigens and subsequent selection of the depleted phage antibody library on cells expressing the antigen of interest resulted in isolation of antibodies against that antigen.
  • binders against the higher density antigens With a library of this size and diversity, at least one to several binders can be isolated against a protein antigen 70% of the time.
  • the antibody fragments are highly specific for the antigen used for selection and have affinities in the 1 :M to 100 nM range (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Griffiths et al. (1993) EMBO J. 12: 725- 734). Larger phage antibody libraries result in the isolation of more antibodies of higher binding affinity to a greater proportion of antigens.
  • a phage display library can mimic this process, stimulate endocytosis, become internalized and deliver their payload into the cytosol.
  • preferred embodiments of this invention utilize a polyvalent phage display antibody library. It is believed that no multivalent phage-display antibody libraries have been created prior to this invention. Unlike the multivalently displayed peptide phage libraries, phage antibody libraries typically display monomeric single chain Fv (scFv) or Fab antibody fragments fused to pffi as single copies on the phage surface using a phagemid system (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Sheets et al. (1998) Proc. Natl. Acad. Sci. USA 95: 6157- 6162.).
  • scFv monomeric single chain Fv
  • Fab antibody fragments fused to pffi as single copies on the phage surface using a phagemid system
  • a polyvalent phage display antibody library refers to a library in which each member (e.g. phage particle) displays, on average) two or more binding domains, wherein each binding domain includes a variable heavy and a variable light region. More generally, a multivalent phage display library displays, on average, two or more pUI fusions per page particle. Polyvalent phage display can be achieved by expressing diabodies (i.e., a protein formed by fusion or conjugation of two single chain antibodies (e.g. scFv)) or by display of, on average, two or more antibodies on each phage particle. In contrast, a mono-valent library displays, on average, one single-chain antibody per viral particle.
  • diabodies i.e., a protein formed by fusion or conjugation of two single chain antibodies (e.g. scFv)
  • a mono-valent library displays, on average, one single-chain antibody per viral particle.
  • Diabodies are scFv dimers where each chain consists of heavy (VH) and light
  • VL chain variable domains connected using a linker (e.g. a peptide linker) that is too short to permit pairing between domains on the same chain. Consequently, pairing occurs between complementary domains of two different chains, creating a stable noncovalent dimer with two binding sites (Holliger et al. (1993) Proc. Natl. Acad. Sci. 90: 6444-6448).
  • the C6.5 diabody was constmcted by shortening the peptide linker between the Ig VH and V domains from 15 to 5 amino acids and binds ErbB2 on SKBR3 cells bivalently with a I j approximately 40 fold lower than C6.5 (4.0 x 10" 10 M) (Adams et al. (1998) Brit. J. Cancer. 77: 1405-1412, 1998).
  • Example 5 C6.5 diabody genes were subcloned for expression as pin fusions in the phagemid pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137). This yielded phagemid predominantly expressing a single scFv or diabody-pffl fusion after rescue with helper phage (Marks et al. (1992) J. Biol. Chem. 267: 16007-16010). Diabody phagemid display a bivalent antibody fragment resulting from intermolecular pairing of one scFv-pffl fusion molecule and one native scFv molecule. Using the teachings provided herein one of skill in the art can routinely produce other diabodies.
  • Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine.
  • antibody phage display libraries are created in which each viral particle, on average, expresses at least 2, preferably at least 3, more preferably at least 4, and most preferably at least 5 copies of a single chain antibody.
  • each copy of p ⁇ i on the page (and there is controversy as to whether there are 3 or 5 copies of pin per phage) should express an antibody.
  • proteolysis occurs and the number actually displayed is typically less.
  • preferred multivalent antibody libraries are constructed in a phage vector and not a phage mid vector. This means that helper phage need not be added to make phage. Helper phage bring into the E. coli wild-type pIH that competes with the scFv-pffl fusion.
  • this competition leas, on average, to only 1 (ore less) antibody per phage.
  • the single chain antibodies are subcloned from the phagemid vector into a phage vector.
  • No helper phage is required and there is no competition between the wild-type pffl and the fusion scFv pffl fusion, thus, on average, the phage display two or more pffl fusions.
  • Example 5 describes the subcloning of the C6.5 scFv gene into the phage vector fd-Sfi/Not. This results in phage with 3 to 5 copies each of scFv- pffl fusion protein.
  • Other phage vectors suitable for such use are well known to those of skill in the art.
  • Target cells include any cell for which it is desired to identify an internalizing polypeptide or antibody or for which it is desired to identify an internalizing marker (e.g. receptor).
  • the cells can include cells of multicellular eukaryotes, uni-cellular eukaryotes, including plants and fungi, and even prokaryotic cells.
  • Preferred target cells are eukaryotic, more preferably vertebrate cells, and most preferably mammalian cells (e.g. cells from murines, bovines, primates including humans, largomorphs, canines, felines, and so forth).
  • the cells can be normal healthy cells or cells characterized by a particular pathology (e.g. tumor cells).
  • Target cells can include any cell type where it would be useful to: 1) have an antibody specifically recognize the cell type or related cell types (for example for cell sorting, cell staining or other diagnostic procedures); 2) have a ligand which is specifically internalized into the cell type or related cell types (for example to deliver a toxic or therapeutic gene or protein).
  • Additional target cells include, but are not limited to differentiated cells (i.e. differentiated to become a tissue, e.g. prostate, breast). Thus an antibody that recognized and killed prostate cells would be good for prostate cancer even if it killed normal prostate cells (the prostate is not an essential organ).
  • Target cells may include tissue specific cells, and cells at a given developmental stage.
  • Target cells may also include precursor cells, e.g. bone marrow stem cells, would be useful for isolating, perhaps stimulating for differentiation.
  • Target cells can also include cell lines transfected with a gene for a known receptor (for example ErbB2) to which it would be useful to have internalizing antibodies. Many ErbB2 antibodies are not internalizing. Rather than immunizing with recombinant protein or selecting a phage library on recombinant protein, selection on ErbB2 transfected cells for intemalization should yield precisely antibodies with the desired characteristics (intemalization). Finally, a cDNA library could be transfected into a cell line (for example COS) from a desired target cell line or tissue and phage antibodies selected for intemalization. After several rounds of selection, the phage could be used to stain and sort (for example by FACS) transfected cells. DNA can be recovered from the cells, yielding the sequences of internalizing receptors as well as phage antibodies that bind them.
  • a cell line for example COS
  • phage antibodies selected for intemalization
  • the phage display library is contacted with cells from a "subtractive" cell line.
  • This step is intended to deplete or eliminate members of the phage display library that either bind the cells non-specifically or that bind to targets other than the target against which it is desired to obtain a binding polypeptide or antibody.
  • the contacting with the cells from a "subtractive" cell line can occur before, during, or after the target cells are contacted with members of the phage display library.
  • the contacting with cells of a subtractive cell line is simultaneous with contacting of the target cells.
  • the target cell line grown adherent to a tissue culture plate
  • the subtracting cell line in suspension in a single cell culture flask.
  • subtractive cells display all the markers on the target cell except the marker (e.g. receptor) that is to act as a target for selection of the desired binding antibodies or binding polypeptides.
  • Particularly preferred cells are thus closely related to the target cell(s), in terms of having common internalizing cell surface receptors (such as transferrin); for example fibroblasts. If one was selecting on a tumor cell line (for example a breast tumor cell line), than one could negatively select on a normal breast cell line. This may, however, deplete for antibodies that bind to overexpressed antigens, so again a parallel path would be to negatively select on fibroblasts. If one was using transfected cells, than non-transfected cell could be used as the subtractive cell line. Where the tumor is epithelial in origin, the preferred subtractive cell will also be epithelial and even more preferably from the same tissue or organ.
  • Particularly preferred subtractive cells include, but are not limited to, non- differentiated cell lines, non-transfected cells, mixtures of non-differentiated and non- transfected cells.
  • preferred subtractive cell lines are preferably the non-tumor cells of the same tissue (for example, breast tumor cells versus normal breast epithelial cells).
  • the subtractive cell line will be the non-transformed cell line used for library construction (e.g. COS, CHO, etc.).
  • the subtractive cell line is preferably the non-transformed cell line.
  • the EGF-expressing cells are used as the target cell line, and the subtractive cell line is the untransformed CHO cells.
  • the subtractive cell line is the untransformed CHO cells.
  • the subtractive cells are more effective when provided in excess over the target cells.
  • the excess is preferably at least about a 2-fold to about a 1000-fold excess, more preferably about a 3-fold to about a 100- fold excess, and most preferably about a 5- fold to about a 50-fold excess. In one embodiment, a 5-fold excess is sufficient.
  • washing steps are used in the methods of this invention.
  • a "weak” washing step can be used to remove the subtractive cells and weakly or non-specifically binding members of the phage display library.
  • a second strong washing step is preferably used after intemalization of members of the phage display library. The "strong" washing step is intended to remove tightly- and weakly-bound surface phage.
  • Buffers and methods for performing weak and strong wash steps are well known to those of skill in the art.
  • weak washes can be done with standard buffers or culture media (e.g., phosphate buffered saline (buffer) DMEM (culture media), etc.).
  • buffers or culture media e.g., phosphate buffered saline (buffer) DMEM (culture media), etc.
  • the cells are preferably cultured under "internalizing” conditions.
  • Internalizing culture conditions are conditions in which the cell when bound by a member of a phage display library at an appropriate (e.g. internalizing) site or receptor, transports the bound member into the cell. This can involve transport into a vesicle, into the endoplasmic reticulum, the golgi complex, or into the cytosol of the cell itself.
  • telomeres Internalizing conditions are most easily achieved when the cells are cultured under conditions that mimic those of the cell in its native state.
  • many cells e.g. epidermal cells, preferably grow ad adherent layers attached to a basement membrane.
  • Such cells more effectively internalize binding polypeptides and antibodies when they are cultured as adherent monolayers.
  • Chloroquine and serum free medium both avoid non specific intemalization and enhance specific intemalization (ligands in the serum that induce the intemalization of receptor of interest and take with them non specific phages being in the neighborhood).
  • the cells should be cultured at a temperature and pH that permits intemalization.
  • Suitable temperature and pH range from about 35°C to about 39°C and from pH 6 to about pH 8, more preferably from about pH 6.5 to about pH 7.5, with preferred temperature and pH being about 37°C and pH 7.5 respectively.
  • the cells are preincubated in serum culture medium for about two hours before adding the phages and the competitor (subtraction) cells.
  • the internalized phage display library members can be identified directly or indirectly. Direct identification can be accomplished simply by visualizing the phage within a cell e.g. via immunofluorescent or confocal microscopy. Phage intemalization can be identified by their ability to deliver a reporter gene that is expressed within the cell.
  • the reporter gene can be one that produces a detectable signal (e.g. a fluorescent (e.g. lux, green fluorescent protein, etc.) or colorimetric signal (e.g. HRP, ⁇ -galactosidase) or can itself be a selectable marker (e.g. an antibiotic resistance gene).
  • a detectable signal e.g. a fluorescent (e.g. lux, green fluorescent protein, etc.) or colorimetric signal (e.g. HRP, ⁇ -galactosidase)
  • HRP e.g. HRP, ⁇ -galactosidase
  • the phage display member can bear a marker (e.g. a label) and cells containing the internalized phage can be detected simply by detection of the label (e.g. in a flow cytometer).
  • a marker e.g. a label
  • the direct methods preferably used for identification of the receptors or cells that are bound after selections are performed. It is noted that cell sorting approaches (FACs) will work with identification of either surface bound or internalized phage. However, an additional level of specificity can be achieved if the cells are first sorted for the presence of internalized phage prior to lysis. Direct methods are also used during the analysis phase to demonstrate that the phage selected are indeed internalized.
  • FACs cell sorting approaches
  • the internalized phage display library members can be identified indirectly.
  • indirect detection methods the phage-display library member(s) do not need to be detected while they are present within the cell. It is sufficient that they simply have been internalized.
  • Indirect identification is accomplished for example, by isolating and expanding the phage that were internalized into the cells as described below. Indirect identification is particularly well suited where the identified phage display library members are going to be used in subsequent rounds of selection or to isolate bacteria harboring monoclonal phage genomes for subsequent monoclonal phage characterization (that is for the analysis of selection results).
  • phage display library members that have been internalized into target cells (e.g. mammalian tumor cells) remain viable and can be recovered and expanded into a "selected" library suitable for subsequent rounds of selection and/or isolation and characterization of particular members.
  • target cells e.g. mammalian tumor cells
  • the term "recovery” is intended to include recovery of the infectious phage and/or recovery of the phage antibody gene and/or recovery of a heterologous nucleic acid accompanying the antibody gene.
  • the internalized phage can be isolated and expanded using standard methods.
  • these include lysing the cells (e.g., with 100 mM triethylamine (high pH)), and using the lysate to infect a suitable bacterial host, e.g., E. coli TGI.
  • a suitable bacterial host e.g., E. coli TGI.
  • the phage-containing bacteria are then cultured according to standard methods (see, e.g., Sambrook supra., Marks et al. (1991) J. Mol. Biol. 222: 581-597).
  • this invention provides libraries and vectors for practice of the methods described herein.
  • the libraries are preferably polyvalent libraries, including diabody libraries and more preferably including multi-valent single chain antibody libraries (e.g. scFv), (e.g., expressed by phage).
  • the libraries can take a number of forms.
  • the library is a collection of cells containing members of the phage display library, while in another embodiment, the library consists of a collection of isolated phage, and in still library consists of a library of nucleic acids encoding a polyvalent phage display library.
  • the nucleic acids can be phagemid vectors encoding the antibodies and ready for subcloning into a phage vector or the nucleic acids can be a collection of phagemid already carrying the subcloned antibody-encoding nucleic acids.
  • Other preferred vectors include the phage itself carrying expressing a - heterologous binding domain (e.g. an antibody) and containing a heterologous nucleic acid that is to be delivered into the target cell(s). While in some embodiments, the heterologous nucleic acid expresses a detectable label or is itself a label (e.g. a unique sequence detectable by hybridization or amplification (e.g.
  • the heterologous nucleic acid includes a nucleic acid that encodes a molecule other than a detectable label (e.g. a polypeptide, an antisense molecule, a ribozyme, etc.).
  • a detectable label e.g. a polypeptide, an antisense molecule, a ribozyme, etc.
  • This invention provides new methods for effective transfection of cells both in vivo and ex vivo (in vitro).
  • Virtually any cell, eukaryotic or prokaryotic can be transfected according to the methods of this invention.
  • Particularly preferred cells are eukaryotic cells, more preferably vertebrate (e.g., mammalian) cells.
  • Other cells can also be transfected.
  • Such cells include, but are not limited to bacterial cells (e.g. bacteria not typically infected by phage), fungal or yeast cells (e.g. to deliver a cytotoxin in the treatment of fungal or yeast infections), algal cells, insect cells, and the like.
  • nucleic acids can be transfected into "target cells".
  • the nucleic acids can be selected to express particular polypeptide(s), or the nucleic acids can have an activity themselves (e.g. antisense molecules, ribozymes).
  • Such expressed heterologous genes (or cDNAs), antisense molecules, or ribozymes are useful in a wide variety of applications and, for example, have been used to correct acquired and inherited genetic defects, cancer, and viral infection in a number of contexts.
  • ex vivo cell transformation for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transformed cells into the host organism) is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected a heterologous gene according to the methods of this invention, and re- infused back into the subject organism (e.g., patient).
  • Various cell types suitable for ex vivo transformation are well known to those of skill in the art.
  • Particular preferred cells are progenitor or stem cells (see, e.g., Freshney et al. (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition Wiley-Liss, New York) and the references cited therein for a discussion of how to isolate and culture cells from patients).
  • stem cells are used in ex-vivo procedures for cell transformation and gene therapy.
  • the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-K and TNF-I are known (see, Inaba et al. (1992) J. Exp. Med. 176: 1693-1702, and Szabolcs et al. (1995) 154: 5851-5861).
  • Stem cells are isolated for transduction and differentiation using known methods.
  • bone marrow cells are isolated by sacrificing the mouse and cutting the leg bones with a pair of scissors.
  • Stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells).
  • T cells CD4+ and CD8+
  • CD45+ panB cells
  • GR-1 granulocytes
  • lad differentiated antigen presenting cells
  • the bone marrow aspirations is approximately 1,000 ml in quantity and is collected from the posterior iliac bones and crests. If the total number of cells collected is less than about 2 x 108/kg, a second aspiration using the sternum and anterior iliac crests in addition to posterior crests is performed. During the operation, two units of irradiated packed red cells are administered to replace the volume of marrow taken by the aspiration.
  • Human hematopoietic progenitor and stem cells are characterized by the presence of a CD34 surface membrane antigen. This antigen is used for purification, e.g., on affinity columns which bind CD34.
  • the mononuclear cells are separated from the other components by means of ficoll gradient centrifugation. This is performed by a semi-automated method using a cell separator (e.g., a Baxter Fenwal CS3000+ or Terumo machine).
  • a cell separator e.g., a Baxter Fenwal CS3000+ or Terumo machine.
  • the light density cells composed mostly of mononuclear cells are collected and the cells are incubated in plastic flasks at 370C for 1.5 hours.
  • the adherent cells (monocytes, macrophages and B-Cells) are discarded.
  • the non-adherent cells are then collected and incubated with a monoclonal anti- CD34 antibody (e.g., the murine antibody 9C5) at 40°C for 30 minutes with gentle rotation.
  • a monoclonal anti- CD34 antibody e.g., the murine antibody 9C5
  • the final concentration for the anti-CD34 antibody is 10 lg/ml.
  • paramagnetic microspheres (DynaBeads, supplied by Baxter Immunotherapy Group, Santa Ana, California) coated with sheep antimouse IgG (Fc) antibody are added to the cell suspension at a ratio of 2 cells/bead.
  • Fc sheep antimouse IgG
  • the rosetted cells with magnetic beads are collected with a magnet.
  • Chymopapain supplied by Baxter Immunotherapy Group, Santa Ana, California at a final concentration of 200 U/ml is added to release the beads from the CD34+ cells.
  • an affinity column isolation procedure can be used which binds to CD34, or to antibodies bound to CD34 (see, the examples below). See, Ho et al. (1995) Stem Cells 13 (suppl. 3): 100-105. See also, Brenner (1993) Journal of Hematotherapy 2: 7-17.
  • hematopoietic stem cells are isolated from fetal cord blood. Yu et al. (1995) Proc. Natl. Acad. Sci. USA 92: 699-703 describe a preferred method of transducing CD34+ cells from human fetal cord blood using retroviral vectors.
  • the vectors of this invention can be administered directly to the organism for transduction of cells in vivo. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells.
  • the phage packaged nucleic acids are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such packaged nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • compositions of the present invention are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
  • the vectors of this invention are compounded in a formulation in combination with a pharmaceutically acceptable excipient (i. e., a pharmaceutical formulation).
  • a pharmaceutically acceptable excipient i. e., a pharmaceutical formulation.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the vector(s) of this invention suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, com starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • the vectors of this invention alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptabl propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged nucleic acid with a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the packaged nucleic acid with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • Parenteral administration and intravenous aclministration are the preferred methods of administration.
  • the formulations of packaged nucleic acid can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Cells transduced by vectors of this invention as described above in the context of ex vivo therapy can also be administered intravenously or parenterally as described above.
  • the dose administered to a patient should be sufficient to effect detectable transformation, more preferably sufficient to effect a beneficial therapeutic response in the patient over time.
  • the dose will be determined by the efficacy of the particular vector employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient.
  • the physician evaluates circulating plasma levels of the vector, vector toxicities, progression of the disease, and the production of anti- vector antibodies.
  • the dose equivalent - of a naked nucleic acid from a vector is from about 1 ⁇ g to 1 g for a typical 70 kilogram patient, and doses of vectors which include a phage particle are calculated to yield an equivalent amount of therapeutic nucleic acid.
  • inhibitors and transduced cells of the present invention can be administered at a rate determined by the LD-50 of the inhibitor, vector, or transduced cell type, and the side-effects of the inhibitor, vector or cell type at various concentrations, as applied to the mass and overall health of the patient.
  • Administration can be accomplished via single or divided doses.
  • blood samples are obtained and saved for analysis. Between 1 x 10 and 1 x 10 transduced cells are infused intravenously over 60-200 minutes. Vital signs and oxygen saturation by pulse oximetry are closely monitored. Blood samples are obtained 5 minutes and 1 hour following infusion and saved for subsequent analysis. Leukopheresis, transduction and reinfusion can be repeated are repeated every 2 to 3 months.
  • infusions can be performed on a outpatient basis at the discretion of the clinician. If the reinfusion is given as an outpatient, the participant is monitored for at least 4, and preferably 8 hours following the therapy.
  • Transduced cells are prepared for reinfusion according to established methods. See, Abrahamsen et al. (1991) J. Clin. Apheresis, 6: 48-53; Carter et al. (1988) J. Clin. Apheresis, 4:113-117; Aebersold et al. (1988) J. Immunol Meth., 112: 1-7; Muul et al. (1987) J. Immunol. Methods 101:171-181 and Carter et al. (1987) Transfusion 27: 362-365. After a period of about 2-4 weeks in culture, the cells should number between 1 x 108 and 1 x 1012. In this regard, the growth characteristics of cells vary from patient to patient and from cell type to cell type. About 72 hours prior to reinfusion of the transduced cells, an aliquot is taken for analysis of phenotype, and percentage of cells expressing the therapeutic agent.
  • kits for practice of the methods described herein preferably include phage expressing a heterologous binding domain and containing a heterologous nucleic acid (e.g., an expression cassette) that is to be delivered inside a target cell or a nucleic acid encoding such a phage.
  • the nucleic acid can include restriction sites to facilitate insertion of a heterologous nucleic acid into an expression cassette.
  • the assay kits can additionally include any of the other components - described herein for the practice of the assays of this invention.
  • Such materials preferably include, but are not limited to, helper phage, one or more bacterial or mammalian cell lines, buffers, antibiotics, labels, and the like.
  • kits may optionally include instructional materials containing directions (i.e., protocols) disclosing the transformation methods described herein.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • Example 1 Creation of a non-immune human Fab phage antibody library containing 10 9 -10 ⁇ members
  • V H and V L library on separate replicons, combine them into an scFv gene repertoire by splicing by overlap extension, and clone the scFv gene repertoire into the phage display vector pHENl .
  • Human peripheral blood lymphocyte and spleen RNA was primed with IgM heavy chain constant region and, kappa and lambda light chain constant region primers and first strand cDNA synthesized. 1st strand cDNA was used as a template for PCR amplification of VH VKk and V ⁇ gene repertoires.
  • VH gene repertoires were cloned into the vector pUCl 19Sfi-Not as -Ncol- Notl fragments, to create a library of 8.0 x 108 members.
  • the library was diverse by PCR fingerprinting. Single chain linker DNA was spliced onto the V L gene repertoires using PCR and the repertoire cloned as an Xhol-Notl fragment into the vector pHENTXscFv to create a library of 7.2 x 10 6 members.
  • the V H and V L gene repertoires were amplified from their respective vectors and spliced together using PCR to create an scFv gene repertoire.
  • the scFv gene repertoire was cloned as an Ncol-Notl fragment into the vector to create an scFv phage antibody library of 7.0 x 10 9 members.
  • the library was diverse as determined by BstNl fingerprinting. To verify the quality of the library, phage were prepared and selected on 14 different protein antigens. The results are shown in Table 2.
  • scFv antibodies were obtained against all antigens used for selection, with between 3 and 15 unique scFv isolated per
  • Protein antigen used for selection Percentage (number) of Number of different ELISA positive clones antibodies isolated
  • Chlamydia trachomatis elementary bodies yielded seven that specifically recognized chlamydia (Table 2).
  • the scFv could be successfully used in a number of immuno logic assays including ELISA, immunofiuorescence, Western blotting, epitope mapping and irnmunoprecipitation.
  • the number of binding antibodies for each antigen, and the affinities of the binding scFv are comparable to results obtained from the best phage antibody libraries (Table 4).
  • the library was established as a source of panels of human antibodies against any antigen with affinities at least equivalent to the secondary murine response.
  • N V-gene repertoires obtained from V-genes rearranged in vivo;
  • SS semi-synthetic V-genes constructed from cloned V-gene segments and synthetic oligonucleotides encoding VJJ CDR3.
  • ND not determined.
  • Example 2 Uptake of scFV into cells bv receptor mediated endocvtosis and subsequent recovery.
  • the 7.0 x 10 9 member scFv phage antibody library described above was selected on the malignant breast tumor cell lines MB231 and ZR-75-1, both with and without negative selections on the normal breast cell line HBLIOO. Similar results were obtained as described in section above. scFv were isolated that could not distinguish malignant from non-malignant cell lines.
  • phage binding cell surface receptors could be taken up into cells by receptor mediated endocytosis and could then be recovered from cells by lysing the cells. This assumed: 1) that phage could be internalized by receptor mediated endocytosis and 2) that phage could be recovered in the infectious state from within cells prior to lysosomal degradation.
  • the ability to select for intemaiized phage antibodies would have two major benefits: 1) the identification of antibodies that bind to receptors capable of intemalization and 2) an added level of specificity in the selection process. Identification of antibodies which are internalized would be highly useful for many targeted therapeutic approaches where intemalization is essential (e.g. irnmunotoxins, targeted liposomes, targeted gene therapy vectors and others).
  • C6.5 phage and C6.5 diabody phage were incubated with SKBR3 cells (ErbB2 expressing breast tumor cell line) at either 37° C or 4° C and non-internalized phage removed by sequential washing with PBS and low pH glycine buffer.
  • the cells were then permeabilized and biotinylated anti-M13-antibody added followed by streptavidin Texas Red. Cells were then examined by using a confocal microscope. Both C6.5 phage and C6.5 diabody phage were observed within the cytoplasm). Approximately 1% of cells had internalized C6.5 phage and 20% of the cells had intemaiized C6.5 diabody phage. There was no intemalization of the anti-Botulinum phage.
  • C6.5 phage or C6.5 diabody phage were incubated with SKBR3 cells at 37° C. Non bound phage were removed by washing with PBS and phage bound to the cell surface were eluted by washing twice with low pH glycine. The cells were then lysed and each fraction (the first and second glycine washes and the cytoplasmic fraction) used to infect E. coli TGI. Twenty times (C6.5) or 30 times (C6.5 diabody) more phage were bound to the cell surface than the anti-Botulinum phage (glycine 1 wash) (Table 5).
  • Anti-ErbB2 (C6.5 scFv) 1.2 xlO 7 5.2 x 10 6 6.8 x 10 7
  • Anti-ErbB2 (C6.5 diabody) 1.8 xlO 7 2.8 x 10 6 1.7 xlO 7
  • the results indicate that: 1) phage binding cell surface receptors can be taken up by cells and the infectious phage recovered from the cytoplasm. The amount of uptake is significantly greater than uptake of non-binding phage, and the 100 to 200 fold difference is well within the range that would allow enrichment from a library. What is unknown from the results is whether the phage antibodies are mediating receptor mediated intemalization or whether they are merely taken up after binding by membrane turnover.
  • Phage antibodies were rescued from the library and selected on SKBR3 cells. For selection, phage were incubated with cells at 37°C, non-binding phage removed by washing cells with PBS and phage bound to cell surface antigens removed by sequential washes with low pH glycine. Cells were then lysed to release internalized phage and the lysate used to infect E. coli TGI to prepare phage for the next round of selection. Three rounds of selection were performed. One hundred clones from each round of selection were analyzed for binding to SKBR3 cells and to ErbB2 extracellular domain by ELISA. We hypothesized that we were likely to obtain binders to ErbB2 since SKBR3 cells are known to express high levels and
  • ErbB2 is a receptor which is known to be internalized. After each round of selection, the titer of phage recovered from the cytoplasm increased (Table 6). After the third round, 45% of the clones were positive SKBR3 cell binding and 17% bound ErbB2 (Table 6).
  • the scFv gene from ELISA positive clones was PCR amplified and fingerprinted by digestion with BstNl. Two unique restriction patterns were identified. The scFv genes were sequenced and 2 unique ErbB2 binding scFv identified. Similar analysis of SKBR3 ELISA positive clones that did not bind ErbB2 identified an additional 11 unique scFv.
  • phage were prepared from each unique clone and analyzed for binding to SKBR3 cells (high ErbB2 expression) as well as 2 other epithelial tumor cell lines (SK-OV-3, moderate ErbB2 expression and MCF7, low ErbB2 expression) and a normal breast cell line (HS578B). Each unique clone specifically stained tumor cell lines but not the normal breast cell line.
  • SKBR3 and MCF7 cells were incubated with phage antibodies C6.5 (positive control), 3TF5 and 3GH7.
  • C6.5 positive control
  • 3TF5 3TF5 binding ErbB2
  • 3GH7 3GH7 unknown.
  • All 3 phage antibodies intensely stain SKBR3 cells (the selecting cell line and high ErbB2 expresser.
  • C6.5 phage weakly stain MCF7 cells (low ErbB2 expressor).
  • the anti-ErbB2 clone 3TF5 from the library stains MCF7 cells much more intensely then C6.5, as does 3GH7.
  • SKBR3, SK-OV-3, MCF7 and HST578 cells were studied using native purified scFv 3TF5 and 3GH7.
  • the scFv genes were subcloned into a vector which fuses a hexahistidine tag to the scFv C-terminus.
  • scFv was then expressed, harvested from the bacterial periplasm and purified by immobilized metal affinity chromatography.
  • the two scFv intensely stain SKBR3 cells, and do not stain the normal breast cell line HST578.
  • the intensity of staining is less than seen with phage. This is to be expected since the secondary antibody for phage staining recognizes the major coat protein (2500 copies/phage) resulting in tremendous signal amplification.
  • the anti-ErbB2 phage antibody 3TF5 was studied further to determine if it was indeed internalized. This antibody was selected for initial study since its intemalization could be compared to ErbB2 binding C6.5.
  • 5.0 xlO 11 3TF5 or C6.5 phage were incubated with SKBR3 cells at 37°C or at 4°C. After washing with PBS, 3TF5 phage stained cells more intensely than C6.5 phage. After washing with low pH glycine, confocal microscopy revealed that 3TF5 phage were internalized by greater than 95% of cells, while C6.5 was intemalized by only a few percent of cells. Incubation of either antibody at 4°C led to no - intemalization.
  • the native purified 3TF5 scFv was similarly analyzed and was also efficiently internalized by SKBR3 cells. It should be noted that the native 3TF5 scFv existed only as a monomer with no appreciable dimerization or aggregation as determined by gel filtration.
  • Phage that bind an internalizing cell surface receptor can be enriched more than 100 fold over non-binding phage. This level of enrichment is greater than that achieved by selecting on the cell surface.
  • Example 3 Increasing the affinity of antibody fragments with the desired binding characteristics by creating mutant phage antibody libraries and selecting on the appropriate breast tumor cell line.
  • Phage display has the potential to produce antibodies with affinities that cannot be produced using conventional hybridoma technology.
  • Ultra high affinity human antibody fragments could result in excellent tumor penetration, prolonged tumor retention, and rapid clearance from the circulation, leading to high specificity. We therefore undertook a series of experiments to develop methodologies to generate ultra high affinity human antibody fragments.
  • Example 4 Preclinical development of C6.5 based breast cancer therapies
  • C6.5 based molecules are being engineered for radioimmunotherapy.
  • dimeric scFv 'diabodies' were created by shortening the linker between the VH and VL domains from 15 to 5 amino acids. Consequently, pairing occurs between complementary domains of two different chains, creating a stable noncovalently bound dimer with two binding sites.
  • diabodies produced from the V-genes of C6.5 have a significantly higher apparent affinity and longer retention on the surface of SK-OV-3 cells compared to C6.5 scFv (T1/2 > 5 hr vs. 5 min) (Adams et al.
  • C6.5 based molecules are being used to target doxorubicin containing stealth liposomes to ErbB2 expressing breast cancers (Kirpotin et al. (1997) Biochemistry. 36: 66-75).
  • the C6.5 gene was subcloned into an E. coli expression vector resulting in addition of a free cysteine residue at the C-terminus of the scFv.
  • Purified C6.5cys scFv was conjugated to liposomes and in vitro uptake determined using SKBR3 cells. Total uptake was 3.4 mmol phospholipid/10 6 cells at 6 hour, with 70% of the uptake internalized.
  • a large (7.0 x 10 ⁇ member) phage antibody library has been created which can provide panels of human antibodies to purified antigens with affinities comparable to the affinities of antibodies produced by murine immunization.
  • the phage antibodies binding cell surface receptors can be can be internalized by cells and recovered in an infectious state from within the cell. Methodologies were developed which permit enrichment of internalizing phage antibodies over non-internalizing antibodies more than 100 fold. These methodologies were then applied to select new scFv antibodies that bind to internalizing receptors on SKBR-3 cells. Several of these antibodies bind to ErbB2, but are internalized more efficiently than C6.5 based scFv. Many more antibodies bind to unknown internalizing receptors.
  • Example 5 Selection of internalizing antibodies from phage libraries
  • Antibodies that bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins or DNA into the cytosol of mammalian cells for therapeutic applications.
  • internalizing antibodies have been identified by screening hybridomas.
  • C6.5 human scFv
  • ErbB2 scFv
  • affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage
  • affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage
  • Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine.
  • HER2/neu gene for example, is amplified in several types of human adenocarcinomas, especially in tumors of the breast and the ovary (Slamon et al. (1989) Science 244: 707-712) leading to the overexpression of the corresponding growth factor receptor ErbB2.
  • Targeting of ErbB2 overexpressing cells has been accomplished primarily using anti-ErbB2 antibodies in different formats, including conjugation to liposomes containing chemotherapeutics (Kirpotin et al. (1997). Biochem.
  • phage antibody libraries typically display monomeric single chain Fv (scFv) or Fab antibody fragments fused to pffl as single copies on the phage surface using a phagemid system (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Sheets et al (1998) Proc. Natl. Acad. Sci. USA 95: 6157-6162.). We hypothesized that such monovalent display was unlikely to lead to efficient receptor crosslinking and phage intemalization.
  • the SKBR3 breast tumor cell line was obtained from ATCC and grown in RPMI media supplemented with 10% FCS (Hyclone) in 5% CO 2 at 37°C.
  • the C6.5 scFv phage vector was constmcted by subcloning the C6.5 gene as a Sfi VNot I fragment from scFv C6.5 pHENl (Schier et al. (1995) Immunotechnology 1: 63- 71) into the phage vector f ⁇ /Sfi VNot I (a gift of Andrew Griffiths, MRC Cambridge, UK).
  • the C6.5 diabody phagemid vector was constmcted by subcloning the C6.5 diabody gene (Adams et al. (1998) Brit. J. Cancer.
  • Phage were prepared (Sambrook et al. (1990). Molecular cloning- a laboratory manual, New York: Cold Spring Harbor Laboratory) from the appropriate vectors and titered on E. coli TGI as previously described (Marks et al. (1991) J. Mol. Biol. 222: 581-597) using ampicillin (100 ⁇ g/ml) resistance for titration of constructs in pHENl and tetracyline (50 ⁇ g/ml) for titration of constructs in fd. Soluble C6.5 scFv, C6.5 diabody and anti-botulinum scFv were expressed from the vector pUCl 19mycHis (Schier et al. (1995) Immunotechnology 1 : 63-71) and purified by immobilized metal affinity chromatography as described elsewhere (Id.)).
  • SKBR3 cells were grown on coverslips in 6-well culture plates (Falcon) to 50% of confluency. Culture medium was renewed 2 hours prior to the addition of 5.10 11 cfu/ml of phage preparation (the phage preparation representing a maximum of 1/10 of the - culture medium volume) or 20 ⁇ g/ml of purified scFv or diabody in phosphate buffered saline, pH 7.4 (PBS).
  • PBS phosphate buffered saline
  • Phage particles were detected with biotinylated anti-M13 immuno globulins (5 Prime-3 Prime, Ine, diluted 300 times) (45 min. at RT) and Texas red-conjugated streptavidin (Amersham, diluted 300 times) (20 min. at RT). Soluble scFv and diabodies containing a C-terminal myc peptide tag were detected with the mouse mAb 9E10 (Santa Cruz Biotech, diluted 100 times) (45 min. at RT), anti-mouse biotinylated immunoglobulins (Amersham, diluted 100 times) and Texas red-conjugated streptavidin. Optical confocal sections were taken using a Bio-Rad MRC 1024 scanning laser confocal microscope. Alternatively, slides were analyzed with a Zeiss Axioskop UV fluorescent microscope.
  • Subconfluent SKBR3 cells were grown in 6-well plates. Culture medium was renewed 2 hours prior to the experiment. Cells were incubated for varying times with different concentrations of phage preparation at 37°C. Following PBS and stripping buffer washes, performed exactly as described above for detection of intemaiized native antibody fragments and phage antibodies, the cells were washed again twice with PBS and lysed with 1 mL of 100 mM triethylamine (TEA). The stripping buffer washes and the TEA lysate were neutralized with 1/2 volume of Tris-HCl 1M, pH 7.4.
  • TEA triethylamine
  • the human anti-ErbB2 scFv C6.5 was obtained by selecting a human scFv phage antibody library on recombinant ErbB2 extracellular domain (13).
  • affinity on intemalization we studied a scFv (C6ML3-9) which differs from C6.5 by 3 amino acids (Schier et al. (1996) J. Mol. Biol. 263: 551-567).
  • C6ML3-9 scFv is also a stable monomer in solution and binds the same epitope as C6.5 scFv but with a 16 fold lower K d (1.0 x 10" 9 M) (Schier et al. (1996) J. Mol. Biol. 263: 551-567; Adams et al. (1998) Cancer Res. 58: 485-490). Since receptor homodimerization appears to typically be requisite for antibody intemalization we also studied the dimeric C6.5 diabody (Adams et al. (1998) Brit. J. Cancer. 77: 1405-1412, 1998).
  • Diabodies are scFv dimers where each chain consists of heavy (VH) and light (VL) chain variable domains connected using a peptide linker which is too short to permit pairing between domains on the same chain. Consequently, pairing occurs between complementary domains of two different chains, creating a stable noncovalent dimer with two binding sites (Holliger et al. (1993) Proc. Natl. Acad. Sci. 90: 6444-6448).
  • the C6.5 diabody was constructed by shortening the peptide linker between the Ig VH and VL domains from 15 to 5 amino acids and binds ErbB2 on SKBR3 cells bivalently with a K d approximately 40 fold lower than C6.5 (4.0 x 10" 10 M) (Adams et al. (1998) Brit. J. Cancer. 11: 1405-1412, 1998).
  • Native C6.5 scFv and C6.5 diabody was expressed and purified from E. coli and analyzed for endocytosis into ⁇ rbB2 expressing SKBR3 breast tumor cells by immunofluorescent confocal microscopy. As expected, monomeric C6.5 scFv is not significantly internalized whereas the dimeric C6.5 diabody can be detected in the cytoplasm of all cells visualized.
  • the human breast cancer cell line SKBR3 was used as a target cell line for endocytosis. Its surface ErbB2 density is approximately 1.0 x 10 6 per cell (Hynes et al. (1989) J. Cell. Biochem 39: 167-173).
  • C6.5 scFv phagemids were incubated for 2 hours with SKBR3 cells grown on coverslips at 37°C to allow active intemalization. Cells were extensively washed with PBS to remove non specific binding and washed an additional three times with high salt and low pH (stripping) buffer to remove phage specifically bound to cell surface receptors. Internalized phagemid were detected with a biotinylated Ml 3 antiserum recognizing the major coat phage protein pVffl. An anti-botulinum toxin phagemid was used as a negative control. Staining was analyzed by using immunofluorescent microscopy.
  • Table 7 Titration of membrane bound and intracellular phage. 3.0 x 10 1 * ⁇ cfu of monovalent C6.5 scFv phagemid, 16 fold higher affinity monovalent C6ML3-9 scFv phagemid, bivalent C6.5 diabody phagemid or multivalent C6.5 fd phage were incubated with sub confluent SKBR3 cells for 2 hours at 37'C. Cells were washed 6 times with PBS, 3 times with stripping buffer and then lysed to recover intracellular phage. The various fractions were neutralized and the phage titered. The total number of cfu of each fraction is reported. Non specific anti-botulinum phagemid were used to determine non specific recovery.
  • Table 9 Specific enrichment of anti-ErbB2 phage compared to anti-botulinum phage.
  • the titers of anti-ErbB2 phage are divided by the titers of the anti-botulinum phage (Table 7) to derive an enrichment ratio for specific vs nonspecific binding or intemalization. **The titer of intracellular phage is divided by the titer of cell surface bound phage (Table 7) to derive the ratio of internalized phage vs surface bound phage.
  • Some anti-botulinum phage might also be non-specifically endocytosed by cells, but this is likely to be a small amount given the immunofluorescence results.
  • the titer of phage in the TEA fraction increased with increasing affinity and avidity of C6.5, with the highest titers observed for the dimeric C6.5 diabody phagemid and the multivalent C6.5 scFv phage (Table 7).
  • the values represent a 30 fold (C6.5 diabody phagemid) and 146 fold (C6.5 scFv phage) increase in titer compared to the anti-botulinum phage (Table 7).
  • the increase in the phage titer in the cell lysate compared to the last stripping wash is due to endocytosed phage.
  • some of these phage could have come from the cell surface or intracellular matrix. While this could be true for a fraction of the phage from the cell lysate, the immunofluorescence results indicate that at least some of the phage are endocytosed.
  • One indicator of the relative fraction of endocytosed phage for the different C6.5 molecules is to compare the amount of phage remaining on the cell surface prior to cell lysis (last stripping wash) with the amount recovered after cell lysis.
  • phage antibodies can undergo receptor mediated endocytosis and remain infectious in a cell lysate.
  • Selection of internalized phages from a phage library requires the optimization of the method to increase enrichment of specifically internalized phages over non-intemalized phage.
  • Two parameters can be improved: (1) reduction of the recovery of non-specific or non-intemalized phage and (2) preservation of the infectivity of intemaiized phage.
  • wild-type C6.5 scFv phagemid We chose this molecule because it was clearly endocytosed based on confocal microscopy, yet was the molecule undergoing the least degree of specific endocytosis.
  • C6.5 scFv phagemid also represents the most commonly utilized format for display of non-immune phage antibody libraries (single copy pffl in a phagemid vector) and has an affinity (16 nM) more typical of Kd's of scFv from such libraries than the affinity matured C6ML3-9 scFv (Sheets et al. (1998) Proc. Natl. Acad. Sci. USA 95: 6157-6162; Vaughan et al (1996) Nature Biotech. 14: 309-314).
  • the ratio of fd phage/C6.5 scFv phagemid in the cell surface fractions yields a 4 to 6 fold enrichment achieved by specific cell surface binding from the initial 1000 fold ratio.
  • the ratio of fd phage /C6.5 scFv phagemid in the cell lysate increases only 6.1 fold; in contrast, the ratio increases 209 fold with trypsinization (Figure 3). This results from a 60 fold reduction in non-specific binding with only a minor reduction in the amount of specific phage recovery ( Figure 3).
  • SKBR3 cells were incubated with chloroquine and either C6.5 scFv phagemid or anti- botulinum phagemid. Cell lysates were titered at various time points to determine the number of intracellular phagemid. C6.5 scFv phagemid were present at the 20 minute time point and the amount of phagemid was comparable with or without the addition of chloroquine.
  • SKBR3 cells were incubated with C6.5 scFv, C6ML3-9 scFv or C6.5 diabody phagemids or C6.5 scFv phage for 2 hours at 37°C. Cells were washed three times with stripping buffer, trypsinized and washed twice with PBS. Cells were lysed and intracellular phage titered on E. coli TGI. Phage recovery increased with increasing phage titer for all phage studied ( Figure 5).
  • phagemid could not be recovered from within the cell at input titers less than 3.0 x 10 5 (C6.5 scFv) to 3.0 x 10 6 (C6ML3-9 scFv) This threshold decreased for bivalent and multivalent display (3.0 x 10 4 for C6.5 diabody phagemid and C6.5 scFv phage).
  • phage displaying an anti-receptor antibody can be specifically endocytosed by receptor expressing cells and can be recovered from the cytosol in infectious form.
  • the results demonstrate the feasibility of directly selecting internalizing antibodies from large non-immune phage libraries and identify the factors that will lead to successful selections.
  • Phage displaying anti- ⁇ rbB2 antibody fragments are specifically endocytosed by ErbB2 expressing SKBR3 cells, can be visualized within the cytosol and can be recovered in an infectious form from within the cell.
  • monovalent scFv antibody fragments were displayed monovalently in a phagemid system, recovery of internalized phage was only 3.5 to 18 fold above background.
  • scFv monovalent scFv
  • scFv could be especially useful for the construction of fusion molecules for the delivery of drugs, toxins or DNA into the cytoplasm. Since antibodies which mediate receptor intemalization can cause receptor down regulation and growth inhibition (Hurwitz et al. (1995) Proc. Natl. Acad. Sci. USA 92: 3353-3357; Hudziak et al. (1989) Mol. Cell. Biol. 9: 1165-1172; Stancovski et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8691-8698; Lewis et al. (1993) Cancer Immunol. Immunother. 37: 255-263), selection for endocytosable antibodies may also identify antibodies which directly inhibit or modulate cell growth.
  • the F5 scFv gene was removed from pHENl-F5 by digestion of phagemid DNA with the restriction enzymes Sfil and Notl.
  • E. coli containing the F5 recombinant phage were grown in culture to produce F5-Fd phage (see Maniatis for phage preparation). F5 phages were then used to infect E.
  • coli harboring a phagemid which contains a mammalian promoter (CMV) followed by either the gene for 3-galactosidase (pcDNA3.1/HisB/LacZ, In Vitrogen) or the gene for the enhanced green fluorescent protein (pN2EGFP, Clonetch plasmid) and a eucaryotic polyadenylation sequence.
  • CMV mammalian promoter
  • pcDNA3.1/HisB/LacZ In Vitrogen
  • pN2EGFP enhanced green fluorescent protein
  • Bacteria were grown overnight in the presence of tetracycline 15 ug/rnL and either ampicillin 100 ug/mL (pcDNA3.1/HisB/LacZ containing bacteria) or Kanamycine 30 ug/mL (pN2EGFP containing bacteria).
  • the phage prepared from the supernatant a mixture of F5-Fd coat contains either the reporter gene (about 50% of the phages) in a single strand format or the F5-Fd phage genome (about 50% of the phages).
  • Incubation of ErbB2 positive cells 5.105 SKBR3 with 10 7 pfu the phage mix (Filtered twice through a 0.45 nm filter to sterility) allowed expression of the reporter gene in 1% of the cells.
  • Example 7 Targeted gene delivery to mammalian cells by filamentous bacteriophage
  • prokaryotic viruses can be re-engineered to infect eukaryotic cells resulting in expression of a portion of the bacteriophage genome.
  • Phage capable of binding mammalian cells expressing the growth factor receptor ErbB2 and undergoing receptor mediated endocytosis were isolated by selection of a phage antibody library on breast tumor cells and recovery of infectious phage from within the cell.
  • F5 phage were efficiently endocytosed into 100% .of " - ErbB2 expressing SKBR3 cells.
  • F5 phage were engineered to package the green fluorescent protein (GFP) reporter gene driven by the CMV promoter.
  • GFP green fluorescent protein
  • This approach can also be adapted to directly select, rather than screen, phage antibodies for targeted gene expression.
  • the results demonstrate the potential of phage antibodies as an in vitro or in vivo targeted gene delivery vehicle.
  • pHEN-F5 Nucleic Acids Res. 19(15): 4133-4137
  • pHEN-F5 was obtained by selecting a non-immune phage antibody library (Sheets et al. (1998) Proc. Natl. Acad. Sci. USA 95(11): 6157-6162) on ErbB2 expressing SKBR3 cells followed by screening for binding on recombinant ErbB2 extracellular domain (ECD).
  • ECD extracellular domain
  • Phage and phagemid vectors pcDNA3-GFP (6.1 Kbp) was obtained by subcloning the Hind ffl/Not I fragment of p ⁇ 2EGFP (4.7 Kbp) (Clontech) into the pcDNA3-HisB/LacZ (Invitrogen) Hind Til/Not I backbone.
  • a fd-F5-phage vector was constmcted by subcloning the Sfi VNot I scFv-F5 insert from pHEN-1 into the Sfi VNot I sites of fd-Sfi/Not (constructed from fd-tet- " - DOG (Clackson et al.
  • the pHEN-F5-GFP phagemid vector (6.8 Kbp) was obtained by subcloning the 1.6 Kbp pN2EGFP blunted Ase VAfl II fragment into the blunted EcoR I site of pHEN-F5. The orientation of the insert was analyzed by Not I restriction digest.
  • SKBR3 and MCF7 were grown in RPMI complemented with 10% fetal bovine serum (FBS) (Hyclone). 50 % confluent SKBR3 cells grown in 6-well plates were transfected with 1 ⁇ g of D ⁇ A per well using Lipofectamine (GIBCO BRL) as recommended by the manufacturer.
  • p ⁇ 2EGFP dsDNA was prepared by alkaline lysis using the Maxiprep Qiagen Kit (Qiagen Inc.). ssDNA was extracted from 500 ⁇ l of phagemid preparation (see below) by 2 phenol extractions followed by ethanol precipitation.
  • DNA was quantified by spectophotometry with 1.0 A26O nm equal to 40 ⁇ g/ml for ssDNA or 50 ⁇ g/ml for dsDNA.
  • 1.0 A26O nm equal to 40 ⁇ g/ml for ssDNA or 50 ⁇ g/ml for dsDNA.
  • cells were detached using a trypsin-EDTA mix (GIBCO BRL) and analyzed on a FACScan (Becton Dickinson).
  • Phagemid and phage preparation pHEN-F5, pHEN-F5-GFP, pcDNA3-GFP or pN2EGFP phagemids were prepared from E. coli TGI by superinfection with VCS-M13 helper phage (Stratagene) as previously described (Marks et al. (1991) J. Mol Biol. 222(3): 581-597). Fd-F5-phage were prepared from E. coli TGI as previously described (McCafferty et al. (1990) Nature 348(6301): 552-554).
  • F5-GFP-phage and F5-LacZ-phage were prepared by superinfection of E coli TGI containing pcDNA3-GFP with fd-F5-phage.
  • Virus particles were purified from the culture supernatant by 2 polyethylene giycol precipitations (Sambrook et al. (1990). Molecular cloning- a laboratory manual, Cold Spring Harbor Laboratory, New York) resuspended in phosphate buffered saline, pH 7.4 (PBS), filtered through a 0.45 ⁇ m filter and stored at 4°C. Alternatively, the preparations were submitted to an additional CsCl ultracentrifugation step (Smith and Scott (1993) Meth. Enzymol.
  • the ratio of packaged helper phage DNA versus phagemid DNA was determined by titering (Sambrook et al, supra.) the phage for ampicillin and kanamycin resistance (for helper phage rescued pHEN-F5) or ampicillin and tetracycline resistance (for fd-F5 phage rescued - pcDNA3-GFP).
  • Cells were grown on coverslips to 50% confluency in 6 well-plates. Phage preparation (less than 10% of the culture medium) was added and the cells were incubated for 16 hours. The coverslips were washed 6 times with PBS, 3 times for 10 min with Glycine buffer (50 mM glycine, pH 2.8, NaCl 500 mM), neutralized with PBS and fixed with PBS- 4% paraformaldehyde for 5 min at room temperature. Cells were permeabilized with cold acetone for 30 sec, saturated with PBS-1% BSA and incubated with anti-M13 antibody (d: 1/300 in the saturation solution) followed by streptavidin-Texas Red (Amersham) (d: 1/300 in the saturation solution). Coverslips were analyzed with an Axioskop fluorescent microscope (Zeiss).
  • CsCl phage preparations were diluted at least 10 fold in cell culture medium, filtered through a 0.45 ⁇ m filter and added to 30% to 80% confluent cells. After incubation, the cells were trypsinized, washed with FACS buffer and analyzed for GFP expression by FACS.
  • ErbB2 expression was quantitated by FACS using the anti-ErbB2 mouse mAb 4D5 which binds ErbB2 ECD (10 ⁇ g/ml) (1 hr), biotinylated sheep anti-mouse immunoglobulins (Amersham) and sfreptavidin-Phycoerythrin.
  • the resulting vims particles display an average of 1 copy of scFv-pffl fusion protein and 3 to 4 copies of the wild type pffl minor coat protein from the helper phage (Marks et al. (1992) J. Biol. Chem. 267(23): 16007-16010). As a result, the phagemid bind monovalently.
  • multivalent phage antibodies were created by subcloning the F5 scFv DNA into the phage vector fd-Sfi/Not for fusion with the pill protein.
  • Vims particles referred to as fd-F5 phage, display 4 to 5 copies of scFv-pffl fusion protein (Id.).
  • F5 phage antibodies could be internalized by mammalian cells
  • SKBR3 cells overexpressing ErbB2 were incubated for 16 hrs with fd-F5 phage (10 9 colony forming unit/ml, cfu/ml), F5 phagemid (10 1 cfu/ml), or with phagemids displaying an irrelevant anti-botulinum scFv-pffl fusion protein (10*2 cfu/ml) (Amersdorfer et al., 1997) as a negative control.
  • the cell surface was stripped of phage antibodies using low pH glycine buffer, the cells permeabilized and fixed, and intracellular phage detected with anti-M13 antibody.
  • the ratio of packaged phagemid DNA versus helper phage DNA was determined to be 100:1.
  • fd-F5-GFP phage were generated by infecting E. coli TGI carrying the pcDNA3-GFP phagemid (ampicillin resistant) with fd-F5 phage (tetracycline resistant), thus using fd-F5 phage as a helper phage.
  • the fd-F5-GFP phage titer was approximately 5.0 x 10 8 ampicillin resistant cfu/ml of culture supernatant.
  • the amount of GFP expressed per cell also increased with phage concentration, with a small number of cells showing expression with phage titers as low as 2.0 x 10 7 cfu ml (fd-F5-GFP phage) to 1.0 x 10 10 cfu/ml (F5-GFP phagemid).
  • ssDNA single stranded
  • dsDNA double stranded
  • F5-phagemid 15 ⁇ g 3.84 Mediated pHEN-F5-GFP 3-1 ⁇ g 1.44 0.78 ⁇ g 0.64
  • filamentous phage displaying an anti-ErbB2 scFv antibody fragment as a genetic fusion with the minor coat protein pffl can be directly targeted to mammalian cells expressing the specificity of the scFv.
  • Such phage undergo receptor mediated endocytosis and enter an intracellular trafficking pathway which ultimately leads to reporter gene expression.
  • prokaryotic viruses can be re-engineered to infect eukaryotic cells resulting in expression of a portion of the bacteriophage genome.
  • Gene expression was detected with as few as 2.0 x 10 7 cfu of phage and increased with increasing phage titer up to 4% of cells.
  • Multivalent display decreased the threshold for detectable gene expression approximately 500 fold compared to monovalent display, most likely due to an increase in the functional affinity and an increased rate of receptor mediated endocytosis from receptor crosslinking.
  • the lower titer of multivalent phage is due to interference of the fl origin of replication on the reporter phagemid with the fd phage antibody origin of replication (Cleary and Ray (1980) Proc. Natl. Acad. Sci. USA 77(8): 4638-4642).
  • Targeted infection of mammalian cells using phage which bind endocytosable receptors is likely to be a general phenomenon.
  • fusing an anti-transferrin receptor scFv to gene ffl of pHEN-GFP results in GFP expression in 10% of MCF7 cells, 4% of SKBR3 cells, 1% of LNCaP cells and 1% of primary melanoma cells.
  • targeted GFP gene delivery to FGF receptor expressing cells using biotinylated phage and a streptavidin-FGF fusion molecule was recently reported (Larocca et al. (1998) Hum. Gene Ther. 9: 2393-2399).
  • Phage antibody or peptide libraries are first selected for endocytosis by mammalian cells (Barry et al. (1996) Nat. Med. 2: 299-305) or for binding to purified antigen, cells, tissues or organs. After subcloning the selected scFv genes into the pHEN-GFP vector, phage produced from individual colonies can be directly screened for gene expression. This is possible since expression can be detected with as little as 1.0 x 10 10 cfu of phagemids. This permits not only direct identification of endocytosed scFv but also the subset of receptor antibodies which undergo proper trafficking for gene expression.
  • the scFv genes can be subcloned into fd-Sfi-Not which is then used to rescue the reporter phagemid.
  • Use of scFv-fd phage also allows the targeting of a large number of different reporter genes in various expression vectors since many commercially available mammalian vectors contain fl origins of replication. As such, antibody targeted phage might prove useful transfection reagents, especially for cells difficult to transfect by standard techniques.
  • mammalian cells are incubated with a phage antibody library containing the GFP gene, and then sorted based on GFP expression using FACS.
  • Phage antibody DNA would be recovered from the mammalian cytoplasm by cell lysis and used to transfect E. coli and prepare more phage for another round of selection. If the quantities of recoverable phage DNA are inadequate, inclusion of the neomycin gene in the pHEN-GFP vector would permit selection of GFP expressing mammalian cells using G418 (Larocca et al. supra).
  • this system has promise as a targetable in vitro or in vivo gene therapy vehicle.
  • the main limitations are infection efficiency, pharmacokinetics and immunogenicity.
  • values achieved by targeted phage in this report (8.0 x 10 4 /ml of phage preparation) are not dissimilar to values reported for targeted retrovims (10 3 -10 5 /ml of vims) (Kasahara et al. (1994) Science 266: 1373-1376; Somia et al. (1995) Proc. Natl. Acad. Sci. USA 92(16): 7570-7574) but less than reported for adenovims targeting strategies (Douglas et al. (1996) Nat. Biotechnol.
  • endosomal escape could be enhanced by co-administering replication defective adenovims (Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88(19): 8850-8854) or incorporating endosomal escape peptides (Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89(17): 7934-7938) or proteins (Fominaya and Wels (1996) J. Biol. Chem. 271(18): 10560-10568) into the phage major coat protein pV I.
  • infection efficiency could be increased combinatorially by creating scFv targeted libraries of pVffl mutants and selecting for increased gene expression.
  • biodistribution of phage is limited to the intravascular space. This would not affect in vitro phage gene therapy, but might limit in vivo uses to those targeting the vasculature. This still leaves numerous applications including those where neovascularization plays a role, such as cancer.
  • immunogenicity it is likely that phage will be immunogenic, thus limiting the number of times that phage could be administered in vivo.

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  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Oncology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur des procédés visant à administrer et exprimer spécifiquement des acides nucléiques hétérologues dans des cellules cibles. Ces procédés consistent généralement à produire un phage présentant extérieurement une protéine de ciblage hétérologue (telle que scFv) et contenant un acide nucléique hétérologue ; et mettre en contact la cellule cible avec le phage, ce dernier étant internalisé dans la cellule, et l'acide nucléique hétérologue est transcrit, puis éventuellement traduit, dans la cellule.
EP99919789A 1998-04-24 1999-04-23 Administration de genes cibles dans des cellules au moyen de bacteriophages filamenteux Withdrawn EP1073671A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US8295398P 1998-04-24 1998-04-24
US82953P 1998-04-24
US24940299A 1999-02-12 1999-02-12
US249402 1999-02-12
PCT/US1999/007398 WO1999055720A1 (fr) 1998-04-24 1999-04-23 Administration de genes cibles dans des cellules au moyen de bacteriophages filamenteux

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EP1073671A1 EP1073671A1 (fr) 2001-02-07
EP1073671A4 true EP1073671A4 (fr) 2004-08-18

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EP (1) EP1073671A4 (fr)
AU (1) AU746040B2 (fr)
CA (1) CA2329908A1 (fr)
WO (1) WO1999055720A1 (fr)

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Publication number Priority date Publication date Assignee Title
GB0028130D0 (en) 2000-11-17 2001-01-03 Phico Therapeutics Ltd Polypeptide and uses thereof
GB0107319D0 (en) 2001-03-23 2001-05-16 Moredun Res Inst Bacteriophage-mediated immunisation
GB0222824D0 (en) * 2002-10-02 2002-11-06 Moredun Res Inst Bacteriophage-mediated immunisation II
EP2192922A4 (fr) 2007-09-17 2010-09-29 Univ California Anticorps monoclonaux humains internalisants ciblant des cellules du cancer de la prostate<i>in situ</i>
WO2015002939A1 (fr) 2013-07-01 2015-01-08 Massachusetts Institute Of Technology Fonctionnalisation de bactéries endogènes
EP3383920B1 (fr) 2015-11-30 2024-01-10 The Regents of the University of California Administration de charge utile spécifique de tumeur et activation immunitaire au moyen d'un anticorps humain ciblant un antigène de surface de cellule tumorale très spécifique
EP3538153A4 (fr) 2016-11-11 2020-06-24 The Regents of the University of California Anticorps anti-cd46 et méthodes d'utilisation
IL300528A (en) 2020-08-07 2023-04-01 Fortis Therapeutics Inc Immune conjugates targeting cd46 and methods of using them

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WO1994013804A1 (fr) * 1992-12-04 1994-06-23 Medical Research Council Proteines de liaison multivalentes et multispecifiques, leur fabrication et leur utilisation
WO1996021007A2 (fr) * 1994-12-30 1996-07-11 Chiron Viagene, Inc. Systemes de transfert genique a mediation par bacteriophage capables d'effectuer une transfection des cellules eucaryotiques
WO1999010014A2 (fr) * 1997-08-29 1999-03-04 Selective Genetics, Inc. Administration de gene effectuee au moyen de recepteurs, et utilisant des vecteurs bacteriophages

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ATE408012T1 (de) * 1991-12-02 2008-09-15 Medical Res Council Herstellung von autoantikörpern auf phagenoberflächen ausgehend von antikörpersegmentbibliotheken

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WO1994013804A1 (fr) * 1992-12-04 1994-06-23 Medical Research Council Proteines de liaison multivalentes et multispecifiques, leur fabrication et leur utilisation
WO1996021007A2 (fr) * 1994-12-30 1996-07-11 Chiron Viagene, Inc. Systemes de transfert genique a mediation par bacteriophage capables d'effectuer une transfection des cellules eucaryotiques
WO1999010014A2 (fr) * 1997-08-29 1999-03-04 Selective Genetics, Inc. Administration de gene effectuee au moyen de recepteurs, et utilisant des vecteurs bacteriophages

Non-Patent Citations (3)

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Title
BARRY M A ET AL: "Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries", NATURE MEDICINE, NATURE PUBLISHING, CO, US, vol. 2, no. 3, March 1996 (1996-03-01), pages 299 - 305, XP002128875, ISSN: 1078-8956 *
SCHIER R ET AL: "ISOLATION OF HIGH-AFFINITY MONOMERIC HUMAN ANTI-C-ERBB-2 SINGLE CHAIN FV USING AFFINITY-DRIVEN SELECTION", JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 255, 1996, pages 28 - 43, XP000921474, ISSN: 0022-2836 *
See also references of WO9955720A1 *

Also Published As

Publication number Publication date
AU3743299A (en) 1999-11-16
WO1999055720A1 (fr) 1999-11-04
WO1999055720A9 (fr) 2000-04-13
EP1073671A1 (fr) 2001-02-07
CA2329908A1 (fr) 1999-11-04
AU746040B2 (en) 2002-04-11

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