MXPA00010434A - Methods of selecting internalizing antibodies - Google Patents

Abstract

This invention provides methods of selecting antibodies that are internalized into target cells. The methods generally involve contacting target cells with one or more members of an antibody phage display library, shown in the figure. The members of the phage display library are also contacted with cells of subtractive cell line. The target cells are then washed to remove the subtractive cell line cells and members of phage display library that are non-specifically bound or weakly bound to the target cells. The target cells are cultured under conditions where members of the phage display library can be internalized if bound to an internalizing marker and internalized members of the phage display library are then identified.

Description

METHODS OF SELECTION PE ANTIBODIES INCORPORAB ES RECIPROCAL REFERENCE TO RELATED REQUESTS This application claims the benefit according to 35 Code of E.U .: §119 (e) of provisional application USSN 60 / 082,953, filed on April 24, 1998, which is hereby incorporated by reference in its entirety for all purposes.

DECLARATION IN CONFORMITY WITH THE RIGHTS TO THE INVENTIONS CARRIED OUT UNDER SPONSORED RESEARCH AND DEVELOPMENT FOR THE FEDERAL GOVERNMENT This work was sponsored, in part, by the Department of Defense Rights DAMD17-96-1 -6244 j and DAMD 17-94-4433. The government of the United States of America may have some rights in this invention. FIELD OF THE INVENTION This invention belongs to the fields of immunodiagnostics and immunotherapy. The invention provides methods for identifying internalizable antibodies and internalizable receptor ligands, as well as the binding of internalizable receptors.

BACKGROUND OF THE INVENTION Growth factor receptors and other signal transduction receptors are frequently overexpressed in human carcinomas and other diseases, and have therefore been used for the development of target therapy. The HER2 / neu gene, for example, is amplified in various types of human adenocarcinomas, especially in breast and ovarian tumors (Slamon et al. (1989) Science 244: 707-712) that lead to overexpression of the ErbB2 receptor corresponding to the growth factor. The selection of cells that overexpress ErbB2 has been achieved mainly using anti-Erb2 antibodies in different formats, including conjugation to liposomes containing chemotherapeutics (Kirpotin et al. (1997). ß oc /? Em.36: 66-75) , the fusion to the proteins that transport DNA that supply a toxic gene (Forminaya and Wels (1996) J. Biol. Che. T ??: 10560-10568), and the direct fusion to a toxin (Altenschmidt et al. (1997 Int. J. Cancer 73: 117-124). For many of these targeted approaches, it is necessary to deliver the effector molecule through the cell membrane and into the cytosol. In some cases, this can be facilitated by taking advantage of receptor-mediated endocytosis (Ullrich and Schlessinger (1990) Cell 61: 203-212). The receptor-mediated endocytosis is often caused when the binding of the ligand causes receptor activation by homo- or heterodimerization, either directly to the bivalent ligand or causing a change in the configuration at the receptor for the monovalent ligand. Antibodies can mimic that procedure, stimulate endocytosis, enter and act within the cytosol. In addition, the efficiency with which antibodies mediate hospitalization differ significantly depending on the type of antibody (eg, the complete antibody, a fragment, single chain, dimeric, monomeric, etc.), and in the recognized epitope (Yarden (1990 ) Proc. Nati, Acad. Sci. USA 92: 3353-3357). Therefore, for some applications, such as the liposomal direction, only the bodies that bind to specific epitopes are rapidly interned and produce a functional steering vehicle. It has also been shown that the internable antibodies cause the inhibition of cell growth or increased cell growth, depending on the recognized epitope. Therefore, selection for hospitalization should lead to isolation of growth inhibitory or growth-stimulating antibodies (agonists). Said inhibitory antibodies can be used as treatments for cancer or for treatment of other conditions characterized by cell hyperproliferation, and for the treatment of inflammations (anti-inflammatories). Agonist antibodies could be used to stimulate the growth of relevant cells (e.g., stem cells). The selection of cells in addition to cancer cells for gene delivery will also have many applications.

Currently, antibodies that mediate hospitalization are identified by selecting hybridomas. However, the selection of antibodies produced by hybridomas is laborious, wasteful of time and costly.

BRIEF DESCRIPTION OF THE INVENTION This invention is based, in part, on the discovery that it is possible to directly select antibodies that can be internalized from broad libraries of non-immune phages, by recovering infectious phage particles from the interior of cells after receptor-mediated endocytosis. Therefore, in one embodiment, this invention provides methods for selecting polypeptides or portions that bind antibodies that are internalized within the target cells. The methods preferably include i) contacting one or more of the target cells with one or more of the elements of a phage display library; iv) culturing the target cells under conditions where the elements of the display library can be hospitalized if they bind to an intemable marker; and v) identifying internalizable members of the phage display library if the elements of the phage display library are interned in one or more of the target cells. The methods also include, optionally and additionally preferable, contacting the elements of the phage display library with cells of a subtractive cell line; and then wash the target cells to remove the cells from a subtractive cell line to remove the elements of the display library from phage that do not specifically bind or bind weakly to the target cells. In a preferred embodiment, the phage display library is an antibody phage display library, most preferably an antibody phage display library that displays single chain antibodies (e.g., scFv, scFab, etc.). In a preferred embodiment, the "identification" step comprises recovering the internal phage and repeating the steps of the procedure once more to further select the interchangeable binding portions. In one embodiment, the "recovery" step includes lysing the target cells to release the hospitalized phage; and infecting a bacterial host with the phage interned to produce a phage for a subsequent round of selection. The recovery step may include recovering infectious phage, and / or recovering a nucleic acid encoding an antibody displayed by phage and / or phage selection that expresses a selectable marker (e.g., an antibiotic resistance gene or CDNA). The identification step can include detecting the expression of a reporter gene, detecting the presence, absence or amount of a particular nucleic acid, or phage selection by a selectable marker. In preferred methods, the cells of a subtractive cell line are present at least twice the excess on the target cells. In preferred methods, the target cells form an adherent layer. In preferred methods, the line White cell is grown adhered to a tissue culture plate and coincubated with the subtractive suspension cell line of a single cell culture flask. In particularly preferred methods, contact with a subtractive line is carried out at a temperature (e.g., about 4 ° C) less than the in-culture culture conditions (e.g., about 37 °). In particularly preferred embodiments, the phage expresses a selectable marker and / or a reporter gene. Preferred selectable markers include, but are not limited to genes (or cDNAs) that encode fluorescent protein (s), an antibiotic resistance gene or cDNA, and a chromagenic gene or cDNA (eg, horseradish peroxidase) spicy, ß-lactamase, luciferase, and ß-galactosidase In certain embodiments, target cells may include solid tumor cells, elements of a cDNA expression library, cells overexpressing a cytokine receptor, cells overexpressing a growth factor receptor, metastatic cells, cells of a transformed cell line, cells transformed with a gene or cDNA encoding a receptor surface-specific white, and neoplastic cells derived from the exterior of a solid tumor. In a particularly preferred embodiment, said cells of a subtractive cell line are selected from the same type of tissue as the tissue of the target cells. Suitable subtractive cell line cells include, but are not limited to, fibroblasts, monocytes, stem cells and lymphocytes.

The methods of this invention can also be used to identify internalizable receptors and / or intercept receptor epitopes (regions of the receptor which, when joined, induce the insertion of the binding portion). The methods generally include any of the methods for identifying antibodies or polypeptides that can be internalized as described in the present invention with the additional steps, by which the identified internal antibodies or polypeptides are used to penetrate the original target cells, or different cells. When the incorporable antibodies or polypeptides are joined in this manner, they allow the isolation of the cell carrying the normal receptor and the isolation of the receptor and / or receptor epitope itself. Therefore, in one embodiment, the methods include i) contacting one or more of the target cells with one or more elements of a phage display library; I) optionally, but preferably, contacting elements of the phage display library with cells of a subtractive cell line; iii) optionally, but preferably, washing the target cells to remove said cells from a subtractive cell line and to remove the elements of the phage display library that are not specifically bound or that are weakly bound to said target cells; iv) culturing the cells under conditions wherein the elements of said phage display library can be internalized if they bind to an alternative marker; v) identifying the internal elements of the phage display library if the phage display elements are interned within one or more of said white cells; vi) contacting the same or different target cells with the identified internals of step (v) or the propagated elements thereof, wherein the elements are attached to the surface of the same or different target cells. The method may also encompass the isolation of a component of the same or different target cells to which the elements are attached. In some methods the "identification" step includes retrieving the phage interned and repeating steps from (i) to (v) to further select the internment of the binding portions. The steps of contacting, washing, cultivating and identifying preferably develop as described in the present invention and the target and subtractive cells include the cells described in the present invention. In fact, in another embodiment, this invention provides a multivalent antibody phage display library. The library preferably comprises a variety of phages wherein the phage display, on average, at least two copies of a single chain antibody and the library comprises a variety of species of single chain antibodies. In preferred embodiments, the phage display, on average, at least 3, at least 4, or at least 5 copies of a single chain antibody per phage particle. Particularly preferred libraries comprise, on average, at least 105, preferably at least 106, most preferably at least 107, and most preferably at least 108 of different species of a single chain antibody. In the most preferred mode, antibodies are encoded by nucleic acids that are phage vectors (not phagemids). In certain embodiments, the library will be selected from elements that specifically bind to an internal cell surface receptor (eg, erbB2, EGF receptor, PDGF receptor, VEGF receptor, transfer receptor, etc.). The single chain antibodies are preferably Fv single chain antibodies (scFv) or Fab single chain antibodies (scFab). Filamentous phages are preferably used in the libraries of this invention and the antibodies are preferably expressed as a fusion with a lower coverage protein PIN. The phage can also express a selectable marker (e.g., a gene for resistance to antibiotics or cDNA) and / or a reporter gene or cDNA (for example, green fluorescent protein (GFP) Fflux, β-gal, β-lactamase, etc.). In yet another embodiment, this invention provides a nucleic acid library that encodes one of the phage display antibody libraries described herein. The nucleic acid library comprises at least 105, most preferably at least 106, and most preferably at least 107, of different phage or phagemid vectors. This invention also provides equipment for implementing the methods described herein. The kits preferably comprise one or more containers containing a library of phage display (or a portion thereof) that was described hereinabove. The kit may include nucleic acids encoding the library and / or phage particles expressing single chain antibodies (preferably a multivalent library) and / or cells containing the intact phage or phage nucleic acids.

Definitions As used herein, an "antibody" refers to a protein that consists 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 mu 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 classes of immunoglobulin, IgG, IgM, IgA, IgD and IgE, respectively. It is known that a typical structural unit of immunoglobulin (antibodies) comprises a tetramer. Each tretamer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25kD) and a "heavy" chain (approximately 50-70kD). The N-terminal end of each chain defines a variable region of approximately 100 to 110 or more amino acids mainly responsible for the recognition of antigens. The terms "Variable light chain" (VL) and "variable heavy chain" (VH) 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 several peptidases. Hence, for example, pepsin digests an antibody under the disulfide bonds in the hinge region to produce F (ab) '2, a Fab dimer which in turn is a light chain linked to VH-CH1 by the disulfide bond . The F (ab) '2 can be reduced under light conditions to break the disulfide bond in the hinge region thereby converting the Fab') dimer into a Fab 'monomer. The Fab 'monomer is essentially a 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). Although various antibody fragments are defined in terms of the digestion of an intact antibody, those skilled in the art will appreciate that said Fab 'fragments can be synthesized de novo either chemically or using recombinant DNA methodology. Thus, the term "antibody" as used in the present invention also includes fragments of antibodies 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 light polypeptide chain), most preferably single chain Fv antibodies (scFv or scFv) in which a variable heavy chain and a light chain variable bind to each other (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked VH-VL heterodimer, which can be expressed from a nucleic acid that includes coding sequences for VH- and V- either directly linked or linked by a linker encoding peptides. Huston et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. Although the VH and V chains are each connected as a light polypeptide chain, the VH and V domains are non-covalently associated. The first functional antibody molecules to be expressed on the surface of the filamentous phage were single chain Fv's (scFv). However, alternative expression strategies have also been successful. For example, Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused to a g3 capsid protein and the complementary strand is exported to the periplasm as a soluble molecule. The two chains can be encoded in the same or in different replications; the important point is that the two antibody chains in each Fab molecule are assembled post-translationally and the dimer is internalized in the phage particle by binding one of the chains to g3p (see, for example, US Patent No: 5,733,743 ). The scFv antibodies and a number of other structures that convert the chains of light and heavy polypeptides naturally added, but chemically separated from the V region of an antibody into a molecule that is bent into a structure Three-dimensional substantially similar to the structure of an antigen binding site are known to those skilled in the art (see, for example, US Patent Nos. 5,091, 513, 5,132,405, and 4,956,778). Particularly preferred antibodies include all those which have been displayed on phage I which are preferred antibodies and which should include all those which have been displayed on phages (for example, scFv, Fv, Fab and Fv linked by disulfide (Reiter et.al. ., (1995) Protein Eng. 8: 1323-1331) An "antigen binding site" or "binding portion" refers to the part of an immunoglobulin molecule that participates in antigen binding. Antigen binding is formed by amino acid residues of the N-terminal variable regions ("V") of the heavy chains ("H") and the light chains ("L") .Three highly divergent separations within the V regions of The heavy and light chains are referred to as the "hypervariable regions" that interpose between the most conserved flanking separations known as "frame regions" or "FRs." Therefore, the term "FR" refers to sequences of amino acids qu e are naturally between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are positioned in relation to each other in a three-dimensional space to form a "surface" of binding to the antigen. This surface mediates the recognition and union of white antigen. The three hypervariable regions of each of the heavy and light chains are referred to as the "complementation determining regions" or "CDRs" and are characterized, for example by Kabat et. al., Sequences of proteins of the immunological nterrest 4th edition, U.S. Dept. Health and Human Services, Public Health Services, (Department of Health and Human Services, Public Health Services of the United States) Bethesda, MD (1987). As used herein, the terms "immunological binding" and "immunological binding properties" refer to non-covalent interactions of the type that 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 (Kd) of the interaction; where a lower Kd represents a greater affinity. The immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One of the methods covers the measurement of formation and dissociation of the antigen / antigen binding site complex, where those speeds depend on the concentrations of the complex partner, the affinity of the interaction and the geometrical parameters that also influence the speed in both directions. Therefore, both the "constant in velocity" (kon) and the "constant out of speed" (k0ff) can be determined by calculating the concentrations and the actual association and dissociation velocities. The The koff / kon ratio allows the cancellation of all parameters unrelated to the affinity and is therefore equal to the dissociation constant Kd (see, generally, Davies et al., (1990) Ann. Rev. Biochem., 59: 439-473 The phrase "binds specifically to a protein" or "specifically immunoreactive with", when referring to an antibody, refers to a binding reaction that is determinant of the presence of the protein in the presence of a heterogeneous population of proteins and other biological agents. Thus, under conditions of designated immunological tests, the specified antibodies bind to a particular protein and do not bind in significant amounts to other proteins present in the sample. Specific binding to a protein under such conditions may require an antibody that is selected for its specific character for a particular protein. For example, antibodies F5 or C1, can be raised to the c-erbB-2 protein that binds c-erbB-2 and not to other proteins present in a tissue sample. A variety of immunological test protocols can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid phase immunological ELISA tests 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 the description of test formats immunological conditions that can be used to determine the specific immunoreactivity. The terms "polypeptide", "peptide", or "protein" are used interchangeably herein to designate a linear series of amino acid residues connected together by means of peptide linkages between the alpha-amino carboxy groups of residues adjacent. The amino acid residues are preferably in the natural isomeric ("L") form. However, the residues in the "D" isomeric form can be substituted for any L-amino acid residue, as soon as the desired functional property is retained by the polypeptide. In addition, amino acids, apart from the 20"strd" amino acids, include modified unusual amino acids, which include, but are not limited to, those listed in 37 CFR 3l. 822 (b) (4). Moreover, it should be noted that a dash at the start or end of a sequence of amino acid residues indicates either a peptide linkage to an additional sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group. The term "polypeptide binding" refers to a polypeptide that specifically binds to a target molecule (e.g., a cellular receptor) in a manner analogous to the binding of an antibody to an antigen. Binding polypeptides are distinguished from antibodies by binding polypeptides that are not ultimately derived from immunoglobulin genes or fragments of immunoglobulin genes.

The term "conservative substitution" is used in reference to proteins or peptides to reflect amino acid substitutions that substantially do not alter the activity of the molecule (specific character or binding affinity). Conservative amino acid substitutions typically include the substitution of one amino acid for another amino acid with similar chemical properties (eg, hydrophobic charge). The following six groups each contain amino acids that are typical conservative substitutions for others: 1) Alanine (Ala), Serine (Ser), Threonine (Thr); 2) Aspartic acid (Asp), glutamic acid (Glu); 3) Asparagine (Asn), Glutamine (Gln); 4) Arginine (Arg), Lysine (Lys); 5) Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val); 6) Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides polymers thereof, either in single or double chain form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid are metabolized in a manner similar to nucleotides that occur naturally. Unless otherwise indicated, a The particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) complementary sequences, as well as the sequence that is explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with base-mixed / or deoxyinosin residues (Batzer et al., ( 1991) Nucleic Acid Res. 19: 5081, Ohtsuka et al., (1985) J. Biol. Chem, 260: 2605-2608, Cassol et al., (1992); Rossolini et al., ( 1994) Mol.Cell Probes 8: 91-98). The term nucleic acid is used interchangeably with genes, cDNA mRNA encoded by a gene. The terms "isolated" or "biologically pure" refer to material that is substantially or essentially free of components that normally accompany it as they are in their original state. However, the term "isolated" is not intended to refer to the components present in an electrophoretic gel or other separation means. An isolated component is free of said separation means in a form ready for use in another application or already in use in a new application / medium. A chimeric molecule is a molecule in which two or more molecules that exist separately in their original state join together to form a single molecule that has the desired functionality of all its constituent molecules. Although the chimeric molecule can be prepared by covalently linking two molecules each separately synthesized, those skilled in the art will appreciate that when the chimeric molecule is a fusion protein, the chimera can be prepared de novo as a single "bound" molecule. A fusion protein is a chimeric molecule in which the constituent molecules are all peptides and are linked (fused) together via terminal peptide bonds such that the chimeric molecule is a continuous single chain polypeptide. The various constituents can be linked directly to each other or can be coupled through one or more peptide linkers. An "effector moiety" is a molecule or moiety that typically has a characteristic activity itself that is desired to be delivered to a target cell (e.g., a tumor overexpressing c-erbB-2). Effector molecules include cytotoxins, markers, radionucleotides, ligands, antibodies, drugs, liposomes, and viral coat proteins that produce the virus with the ability to infect a cell that expresses c-erbB-2. A "target" cell refers to a cell or cell type that must be specifically linked by a member of a phage display library or a chimeric molecule of this invention. Preferred target cells are cells for which an internal antibody or binding polypeptide is sought. The white cell is typically characterized by the expression or overexpression of a white molecule that is characteristic of cell type. Thus, for example, a target cell may be a cell, such as a tumor cell, that overexpresses a marker such as c-erbB-2. A "selection portion" refers to a portion (e.g., a molecule) that specifically binds to the target molecule. When the target molecule is a molecule on the surface of a cell and the selection portion is a component of a chimeric molecule, the selection portion specifically binds the chimeric molecule to the cell that carries the target. When the selection portion is a polypeptide it can be referred to as a "selection polypeptide". The terms "internalizable" or "interned" when used in reference to a cell, refer to the transportation of a portion (eg, phage) from the outside to the interior of a cell. The hospitalized portion may be located in an intracellular compartment, for example, a vacuole, a lysosome, the endoplasmic reticulum, the Golgi apparatus, or in the cytosol of the cell itself. An alternative receptor or marker is a molecule present on the outer cell surface that when specifically bound by an antibody or binding protein results in the entry of that antibody or binding protein into the cell. Interceptible receptors or markers include receptors (eg, hormone, cytokine or growth factor receptors) ligands and other cell surface markers joining those resulting in an internment.

The term "heterologous nucleic acid" refers to a nucleic acid that is not original to the cell in which it is found or whose final origin is not the cell or cell line in which the "heterologous nucleic acid" is actually found. The idiotype represents the highly variable antigen binding site of an antibody and is itself immunogenic. During the generation of an immune response mediated by antibodies, an individual will develop antibodies to the antigen as well as anti-idiotype antibodies, whose immunogenic binding site (diotype) halves 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) where the phage expresses an external protein (typically heterologous). The outer protein is free to interact (join) other portions with which phages make contact. Each phage displaying an external protein is a "member" of the phage display library. An "antibody library" refers to the phage display library that displays antibodies (binding proteins encoded by one or more antibody genes or cDNA). The antibody library includes the phage population or a collection of vectors that codes for said phage population, or cell (s) that host said phage or vector collection. The library can be monovalent, unfolding in average a single chain antibody per phage particle or multivalently deploying, 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 8, and still more preferably 10 9 different members (i.e., which code for many different antibodies). The term "filamentous phage" refers to a viral particle that has the ability to display a heterogeneous polypeptide on its surface. Although those skilled in the art will appreciate that a variety of bacteriophages can be employed in the present invention, in preferred embodiments, the vector is, or is derived from, a filamentous bacteriophage, such as, for example, fl, fd, Pfl, M13 , etc. The filamentous phage may contain a selectable marker such as tetracycline (for example, "fd-tet"). The various filamentous phage display systems are well known to those skilled in the art (see, for example, Zacher, et al., (1980) Gene 9: 127-140, Smith et al., (1985) Science 228 : 1315-1317 (1985) as well as Parmley and Smith (1988) Gene 73: 305-318.A "viral packaging signal" is a sequence of nucleic acids necessary and sufficient to direct the entry of a nucleic acid into a capsid A pack cell is a cell in which a nucleic acid can be packaged inside a viral coat protein (capsid) .The pack cells can be infected with one or more cells. different virus particles (e.g., a normal or weakened phage and an auxiliary phage) that individually or in combination direct packaging of a nucleic acid in a viral capsid. The term "detectable label" refers to any material that has a detectable physical or chemical property. Said detectable labels have been developed favorably in the field of immunological tests, and in general, any useful label in said methods can be applied to the present invention. Therefore, a trademark is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The labels useful in the present invention include magnetic beads (e.g., Dynabeads ™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radioimmune labels (e.g., 3 H, 125 L, 35 S, 14 C, or 32P), enzymes (eg, LacZ, CAT, horseradish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as genetic markers or in an ELISA test), and colorimetric labels such as Colloidal gold or glass or plastic with color (for example, polystyrene, polypropylene, latex, etc.). Those detectable labels that can be expressed by nucleic acids are referred to as "reporter genes" or "reporter gene products". It will be recognized that fluorescent labels are not limited to organic molecules of a single species, but include molecules inorganic, multiple molecular mixtures of organic and / or inorganic molecules, heteropolymer crystals, and the like. Thus, for example, core-l nanocrystals of CdSe-CdS enclosed in a silica l can be easily derived to be coupled to a biological molecule (Bruchez et al., (1998) Science, 281: 2013-2016). Similarly, highly flurorescent 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). The following abbreviations are used in the present invention: AMP, ampicillin, c-erbB-2 ECD, extracellular domain of c-erbB; CDR, complementarity determining region; ELISA, enzyme-linked immunosorbent test; FACS, fluorescence activated cell sorter; FR, region of the frame; Glu, glucose; HBS, saline solution regulated by PH hepes, 10mM hepes, 150mM NaCl, pH 7.4; IMAC, immobilized metal affinity chromatography; kon, constant of association speed; koff, dissociation speed constant; MPBS, skim milk powder in PBS; MTPBS, skim milk powder in TPBS; PBS, phosphate buffered saline, 25mM NaH2P04, 125mM NaCl, pH 7.0; PCR, polymerase chain reaction; RU, resonance units; scFv or scFv, single chain Fv fragment; TPBS, 0.05% v / v of Tween 20 in PBS; SPR, surface plasma resonance; Vk? variable region of light chain of kappa immunoglobulin; V ?; lambda light chain variable region of Immunoglobulin; VL, immunoglobulin light chain variable region; VH, immunoglobulin heavy chain variable region; wt, wild type.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the method for construction of a larger human scFv phage antibody library. The strategy for the construction of the library included optimizing the individual stages of construction of libraries to increase both the efficiency of the scFv gene packet and to increase both the efficiency of scFv genes assembled by cloning. (A) First, lymphocyte mRNA was used to generate repertoires of VH and V genes by RTPCR that were cloned into different vectors to create VH and VL gene libraries of 8.0 x 108 and 7.2 x 10d members respectively. The cloned V gene libraries provided a stable and unlimited source of VH and VL genes for the scFv package. The DNA encoding the peptide (G4S) 3 was incorporated at the 5 'end of the VL library. This allowed the generation of scFv genes by PCR dividing two DNA fragments. Previously, scFv gene repertoires were assembled from 3 separate DNA fragments consisting of VH, VL, and linker DNA. (B) VH and VL gene repertoires were amplified from the libraries separately and assembled within a repertoire of scFv genes using overlap extension PCR. The initiators that were used to Re-amplifying VH and V gene repertoires were set 200bp upstream of the 5'-end of the VH genes and 200bp downstream of the V genes. These long hanging ends ensured the efficient digestion of restriction enzymes. (C) The repertoire of scFv genes was digested with Ncol and Notl and was cloned into plasmid pHEN1 as fusions with the MI3 gene of layer III protein gene () for phage display. Figures 2A, 2B, and 2C show the scheme depicting the display of antibody phages: phage pattern displaying (2A) a single scFv (2B) single diabody or (2C) multiple scFv. scFv = single chain Fv antibody fragment; VH = Ig heavy chain variable domain; VL = lg light chain variable domain; plll = plll phage coat protein; Ag = antigen bound by scFv. Figure 3 shows the effect of trypsinion on the enrichment of the antigen-specific phage. A mixture of fd phages (5.0 x 10.11 cfu) and scFv phagemids of C6.5 (5.0 x 108 fu) was incubated with SKBR3 cells for 2 hours at 37 ° C. Washes were performed either as described in Table 6 (-) or the cells were trypsinized before cell lysis (+). The phage present in the first wash of the pH regulator of removal (cell surface phage) and the cell lysate (intracellular phage) were titrated in the presence of ampicillin (phagemid C6,5) or tetracycline (phage fd). Figure 4 shows the effect of the incubation period and chloroquine on the recovery of the antigen-specific phage. SKBR3 cells were incubated in the presence (•, •) or in the absence (D.O) of chloroquine (50μM) for 2 hours prior to the addition of the antibotulin phagemid (D, B) or the phagemid scFv C6.5 (O, *) (1.5 x 109 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 trypsinion step and the intracellular phages were titrated. Figure 5 shows the effect of phage concentration on the recovery of the intracellular phage. Various concentrations of phagemid C6.5 scFv, phagemid scFv C6ML3-9, phagemid of diabody C6.5 or phage C6.5 scFv (phage entry title) were incubated with subconfluent SKBR3 cells cultured in 6-well plates during 2 hours at 37 ° C. The cells were treated as described in Figure 4 including the trypsinion step and the intracellular phage and the intracellular phages were titrated (exit phage titer). Figure 6 illustrates strategies for producing anti-ErbB2 phagemids and phages by packaging a eukaryotic reporter gene. Left column: Auxiliary phages are used to infect TG1 containing pHEN-F5-GFP, a phagemid composed of a f1 origin of replicon (fl ori), the scFv anti- Erb2 F5 gene fused to gene III and a reporter gene of eukaryotic GFP manipulated by the CMV promoter. The phage recovered from the culture supernatant displays an average of 1 scFv-plll fusion protein and 99% of them package the GFP reporter gene. Right Column: The anti-ErbB2 F5 scFv gene is cloned into the fd phage genome for expression as a scFv-plll fusion. The phages fd-F5 are used to infect TG1 containing a GFP phagemid reporter vector (pcDNA3-GFP). Purified phages from the culture supernatant display multiple scFv-pIII fusion proteins and approximately 50% package the GFP reporter gene. Figure 7 shows a comparison of the anti-ErbB2 phagemid and the phage that binds to the cells. 105 of ErbB2 were incubated expressing SKBR3 cells with increased concentrations of F5 phagemids (circles) or fd-F5 phages (squares) at 4 ° C for 1 hour. Phages bound to the cell surface were detected with biotinylated anti-M13 and streptavidin-PE. The binding was detected by FACS and the results were expressed as mean fluorescent intensity (MFI). Figure 8A and 8B illustrate the transfer of genes mediated by phagemids in breast cancer cell lines. (Fig. 8A) (1, 2.3) 2.0 x 105 MCF7 (low expression of ErbB2) or (4.5.6) 2.0 x 105 SKBR3 (high ErbB2 expression) of cells cultured in 6-well plates were incubated without (1, 4) without phage, (2.5) 5.0 x 1012 cfu / ml of auxiliary phage packaging GFP or (3,6) 5.0 x 1011 cfu / ml of phagemids F5-GFP for 48 hours. The cells were trypsinized and GFP was detected by FACS. (Fig. 8B) An equal number of MCF7 and SKBR3 cells (1.0 x 105) were cultured together and incubated with 5.0 x 10 11 cfu / ml phagemids F5-GFP for 48 hours. The cells were trypsinized and stained for the expression of ErbB2 using the antibody 4D5 and rhodamine with sheep anti-mouse conjugated Ig to discriminate SKBR3 cells (Region R1) and MCF7 (Region R2). The GFP content of each subpopulation was determined by FACS.

Figures 9A, 9B, 9C and 9D show the concentration dependence and the period of GFP expression mediated by phagemids in SKBR3 cells. Figures 9A and 9B show the concentration dependence of GFP expression mediated by phages and phagemids in SKBR3 cells. 5.0 x 104 cells were cultured in 24-well plates and incubated with increased concentrations of phagemid F5-GFP (frames), phage fd-F5-GFP (diamonds) or phage GFP-helper (circles). After 60 hours, the cells were trypsinized and analyzed by FACS for the expression of GFP. Figures 9C and 9D show the time dependence of GFP expression mediated by phagemids in SKBR3 cells. 5.0 x 104 cells were incubated with 5.1011 cfu / mL of phagemid F5-GFP and analyzed for GFP expression by FACS. For incubation times longer than 48 hours, the phage were added to 2.5 x 104 cells and the culture medium was replaced by fresh medium after 48 hours of incubation. The results are expressed as (9A.9C)% of GFP positive cells and (9B.9D) MF1 of GFP positive cells.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction This invention provides new screening methods for specific polypeptides and / or binding antibodies that are interned by particular target cells. Unlike the techniques methods above, which simply detect binding to an external target in a cell (eg, a receptor), the tests of this invention explicitly identify molecules that bind and are transported into the cell (i.e., within a vacuole and / or endoplasmic reticulum and / or within the cytosol itself). The utility of a number of specific antibodies even those generally known to bind to internalizable receptors (eg, c-erbB-2) has been limited by the frequent lack of internment of the bound antibody or by unacceptably low hospitalization rates. Although said antibodies are useful for delivering portions to the cell surface, it has been found that they are generally unsatisfactory for the delivery of effector molecules which must obtain entry into the cell for their activity. In contrast, the polypeptides and / or binding antibodies identified by the methods of this invention are rapidly internalized within the cell. Thus, are extremely useful for delivering effector portions within the target cell. In addition, once an internal antibody or polypeptide is identified, it can be used to retest one or more cells or cell lines to identify previously unknown internalizable cell targets (eg, receptors). In addition, selecting the hospitalization also selects the biological function. Many receptors (for example, factor receptor growth) use hospitalization as a way to moduland regulthe effect of ligands. For example, ligand binding can result in signal transduction and receptor internment. The decrease in the number of receptors then causes the deregulation of the effect of the additional ligand. The same is true for antibodies that bind to growth factor receptors (Hurwitz et al., (1995) Proc. Nati, Acad. Sci. USA 92: 3353-3357). For example, the "[g] (growth) factors act by binding and activating the intrinsic catalytic activity of their cell surface receptors, thereby initiating a signaling cascade driving the cell response.The growth factor / receptor complexes do not They are static residents of the cell surface membrane but undergo endocytotic trafficking procedures of hospitalization or hospitalization and classification for recycling or degradation, consequently, the growth factors are extermin from the extracellular environment and their receptors suffer a deregulation. , by virtue of their influence on the kinetics of the receptor growth factor / signaling complexes, they are important modulators of the behavioral responses of the cell "(Reddy et al., (1996) Nature Biotech, 14: 1696-1699). In the ErbB2 system, a mechanism by which the ErbB2 binding antibodies inhibit growth is to cause receptor uptake and deregulation (Hurwitz et al., (1995) Proc. Nati. Acad. Sci. USA 92: 3353 -3357). It may also be possible to convert a an internable antibody that binds to a growth factor receptor and elicits growth inhibition within a growth stimulating antibody. For example, the mitogenic properties of EGF have been increased by decreasing the affinity of EGF for the EGF receptor. The lower affinity of EGF causes receptor signaling, but results in reduced hospitalization and deregulation than wild type EGF (presumably of lower affinity) (Reddy et al., (1996) Nature Biotech 14: 1696-1699), Thus, by decreasing the affinity of an internal antibody that inhibits growth, it could become a growth factor. In that way, the identification of internable antibodies can provide compounds / drugs for both growth inhibition and growth stimulation.

II. Methods to identify the internable antibodies v / or receptors. A) Identification of internable polypeptides / antibodies.

In one embodiment, this invention provides methods for identifying antibodies of internable polypeptides. The methods include contacting a "target" cell with one or more elements of a phage display library that displays an antibody or a binding polypeptide. The phage display library is preferably a phage display library and it is believed that this invention provides the first description of a multivalent antibody phage display library. After a suitable incubation period, the cells are washed to remove the externally bound phage (elements of the library) and then the internalized phages are released from those cells for example, by cell lysis. It was a discovery of this invention that the hospitalized phage remains viable (infectious). Therefore, the phage interned in the cell lyscan be recovered and expanded using the lyscontaining the phage interned to infect a bacterial host. The growth of the infected bacteria leads to phage expansion that can be used for a subsequent round of selection. Each round of selection enriches the phage that is hospitalized more efficiently, very specific to the target cell or that has improved the binding characteristics. The phage display library preferably comes in contact with a subtractive cell line, (ie, a subtractive cell line is added to the target cells and the culture medium) to remove the elements of the phage display library that are not specific for the "target" cells. The subtractive cell line is preferably added under conditions in which the elements of the phage display library are not interned (e.g., at a temperature of about 4 ° C to about 20 ° C, most preferably at a temperature of about 4 ° C. ° C) so that the non-specific elements of the library are not interned (kidnapped) before they are subtracted from the subtractive cell line. After subtracting non-specific binding antibodies, the "target" cells are washed to remove the subtractive cell line and to remove the unbound or weakly bound phage. "The target cells are then cultured under conditions where internment is possible. (for example, at a temperature of about 35 ° C to about 39 ° C, most preferably at a temperature of about 37 ° C.) The length of the hospital culture period will determine the rate of hospitalization of the antibodies (deployment elements). of phages) for which the selection is carried out.With shorter hospitalization periods, faster internable antibodies are selected, although with longer hospitalization periods, slower internable antibodies are selected.The period of hospitalization preferably is less than approximately 120 minutes, most preferably less than about 60 minutes, and very preferred possibly even less than about 30 minutes or even less than about 20 minutes. It should be noted that during the period of hospitalization, the target cells are cultured under conditions in which hospitalization may occur. For a number of cell lines this includes culturing the cells in an adherent form on culture plates.

After admission, the fact that the target cells are washed to remove the non-interned ones (for example, the phage bound to the surface) has been allowed to occur. The cells can then be moved to a fresh medium. In the preferred embodiment, where the cells are adherent, the latter are trypsinized to release cells from the extracellular matrix that may contain phage antibodies that bind to the extracellular matrix. Releasing the cells in a solution allows for better washing and movement of the cells to a new flask culture that will be set aside and any phage that may have become stuck in the tissue culture plate. The cells can then be washed with a high volume of PBS and can be lysed to release the hospitalized phage which can then be expanded for example, using to infect E. coli to produce a phage for the next round of selection. It should be noted that there is no need to actually visualize the hospitalized phage. The simple cellular lysis and cellular expansion of the anterior interphase phage is sufficient to recover the elements of unfolding of phages interned.

B) Identification of intemable bodies Once an antibody or polypeptide that has been internalized within a cell has been identified, it is possible to test 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 internable antibody, it is likely that such targets are also desirable targets (for example, elements or portions of internal receivers). In one embodiment, the antibody can be labeled as described below. The cells can come into contact with the antibody (i.e., in vivo or in vitro) and the cells or cell regions to which the antibody binds can be isolated. Alternatively, antibodies can be used, for example, in an affinity matrix (e.g., affinity column) to isolate target cells (e.g., receptor subunits or receptors) to which they bind. Briefly, in one embodiment, affinity chromatography includes immobilization (e.g., on a solid support) of one or more species of the internalizable antibodies identified according to the methods of this invention. The cells, the cell lysate, or homogeneous cells, then come into contact with the immobilized antibody which can bind to its cognate ligand. The remaining material is washed and the bound / isolated cognate ligand can then be released from the antibody for later use. Methods for developing affinity chromatography are well known to those skilled in the art (see, for example, U.S. Patent Nos. 5,710,254.; 5,491, 096; 5,278,061; 5,110,907; 4,985,144; 4,385,991; 3,983.001, etc.). In another embodiment, the antibodies are used to immunoprecipitate the target of the cell lysate. The precipitate then runs on an SDS-PAGE gel that is transferred to Western blot in nitrocellulose. The spot is probed with the precipitating antibody to identify the location of the target. The portion of the spot containing the target can then be sent for N-terminal end protein sequencing. The N-terminal sequence can then be used to identify the target from standard databases, or DNA that can be synthesized for genomic or cDNA libraries. This approach has been used to identify the antigen bound by a phage antibody. Selections from a phage antibody library were made on intact Chlamydia trachomatis (an organism in bacterial form that causes Chlamydia diseases). The selected antibodies were then used as described above to identify the bound antigen.

C) Functional genomics In another embodiment, the tests of this invention can be used to detect libraries and previously identify unknown binding agents. There are two preferred approaches for these proteomic or genomic functional analyzes. In principle, a phage cDNA library is created. MRNA (probably subtracted) is made from the cell line or tissue of interest. The first strand DNA is synthesized and treated with DNAse or fragmented in some other way. This removes the terminal codon towards the 'end and 3' UTR and the 3 'stop codon. A phage library is then produced and selected for entry into cells. Ligands (or ligand domains) that bind to cell surface receptors and interned ones are identified. For example, this approach can be used to identify orphan growth factors that bind to interchangeable growth factor receptors. If a receptor is known, but the ligand is not known, the receptor gene could be transfected into a cell line and the transfected cell line can be used for selection. The selection could also be combined with the supply of a reporter gene. In this case, the phage vector that is used to create the phage library could contain the reporter gene. After selection, one could, for example, isolate immature cells expressing the GFP reporter gene by FACS (instead of lysing all cells to recover the phage interned). This is expected to improve the specific character of selection. For the second approach, second paragraph: the phage library is selected for hospitalization in a white cell line as described above. The selected polyclonal or monoclonal phage is then used to flow distribution cells (eg, COS cells) transfected with a cDNA library. Plasmids from the DNA library are then recovered from cells sorted and amplified in bacteria using standard techniques. The amplified plasmid cDNA library is used to transfect cells (eg, COS) that Again, they are classified using the phage. After several rounds of selection, the sorted cells should contain only plasmids encoding cell surface receptors bound by the internable phage. These can be identified by DNA sequencing, and each plasmid cDNA is tested for phage binding after the plasmid DNA is used to transfect COS cells.

III.- Test components A) Phage display library 1) Monovalent antibody libraries and polypeptide libraries The ability to express polypeptide and antibody fragments on the surface of the viruses infecting the bacterium (Bacteriophage or phage) makes it possible to isolate a single binding polypeptide or antibody fragment from a larger library of clones that are not bound to 1010. To express polypeptide or antibody fragments on the surface of a phage (phage display), a polypeptide or an antibody or fragment gene is inserted into the gene coding for a phage surface protein (pIII) and the pIII fusion protein of the antibody fragment is displayed on the phage surface (McCafferty et al., (1990) Nature, 348: 552-554; Hoogenboom et al. ., (1991) Nucleic Acids Res. 19: 4133-4137). As the fragment antibodies on the phage surface are functional, the phage that the fragments of polypeptides or antibody bound to antigen can be separated from the phage that is not bound by affinity chromatography of the antigen (McCafferty et al., (1990) Nature, 348: 552-554). Depending on the affinity of the antibody fragment, enrichment factors of 20 times-1,000,000 fold are obtained for a single round of affinity selection. By infecting the bacteria with the eluted phage, however, more phages can be cultured and subjected to another round of selection. In this way, an enrichment of 1000 times in a round can be converted into 1, 000,000 in two rounds of selection (McCafferty et al., (1990) Nature, 348: 552-554). Even though the enrichments are low (Marks et al., (1991) J. Mol. Biol .. 222: 581-597), multiple rounds of affinity selection may lead to the isolation of the rare phage. As the selection of phage antibody libraries on enrichment antigen results, most clones bind to the antigen after four rounds of selection. Therefore, only a relatively small number of clones (hundreds of them) need to be analyzed for their binding to the antigen. In a preferred embodiment, analysis for binding including an amber codon between the antibody fragment gene and gene III is simplified. The amber codon makes it possible to exchange easily between the unfolded antibody fragment and the soluble one (native or original) simply by changing the host bacterial chain (Hoogenboom et al., (1991J Nucleic Acids Res. 19: 4133-4137).

Human antibodies can be produced without prior immunization by displaying very extensive and diverse V gene repertoires in the phage (Marks et al., (1991) J. Mol.Biol 222: 581-597). In the first example, the natural VH and V repertoires present in human peripheral blood lymphocytes were isolated from donors not immunized by PCR. The V gene repertoires were randomly divided together using PCR to create a repertoire of scFv genes that was cloned into a phage vector to create a library of 30 million phage antibodies (Id). From this light "untreated" phage antibody library, fragments of binding antibody 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 autonomous proteins, including thyroglobulin, imunoglobulin, human tumor necrosis factor and CEA (Griffiths et al., (1993) EMBO J. 12: 725-734). It is also possible to isolate antibodies against cell surface antigens by selecting directly on intact cells. For example, antibody fragments against four different erythrocyte cell surface antigens were produced by directly selecting on erythrocytes (Marks et al., (1993), Bio Technology, 10: 779-783). Antibodies were produced against blood group antigens with surface densities as small as 5000 sites / cell. The fragments of antibody were highly specific for the antigen used for selection, and were functional in the immunofluorescence and agglutination tests. Antibodies against the lower density antigens were produced by first selecting the phage antibody library in a type of cell highly related to the deficiency of the antigen of interest. This negative selection removed agglutinants against the higher density antigens and the subsequent selection of the spent phage antibody library on cells expressing the antigen of interest resulted in the isolation of antibodies against that antigen. With a library of this size and diversity, at least one of several binders can be isolated against a protein antigen 70% of the time. Antibody fragments are highly specific for the antigen used for selection and have affinities on the scale of 1: M to 100nM. (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 larger proportion of antigens. The creation of a phage display antibody library of suitable larger dimension is described in detail in Example 1. 2) .- Polyvalent antibody phage display libraries The probability of selecting internatable antibodies from a phage display antibody library increases with increasing valence of the displayed antibody. This approach takes advantage of the biology of the normal cell surface receptor. Frequently, cell surface receptors (eg, growth factor receptors) are activated by binding their cognate ligand through a process of homo- or heterodimerization (or trimerization, or tetramerization, etc.). The association of receptor subunits in this process can be mediated directly (for example, when they are linked by a bivalent ligand) or indirectly causing a change in the configuration at the receptor. It was a discovery of this invention that polyvalent antibodies in a display library (eg, a phage display library) can mimic this process, stimulate endocytosis, be interned and deliver their payload into the cytosol. Therefore, to increase the likelihood of identifying detectable antibodies or recognizing internable epitopes, preferred embodiments of this invention use a polyvalent phage display antibody library. It is considered that multivalent phage display antibodies libraries have not been created prior to this invention. Unlike phage display libraries of multivalently deployed peptides, phage display libraries typically display monomeric fragments of Fv (scFv) or single chain Fab antibodies fused to plll as light copies on the phage surface using a phage display system. phagemids (Marks i went to (1991) J. Mol. Biol., 222: 581-597; Sheets eí al., (1998) Proc. Natis Acad. Sci. USA 95: 6157-6162). As used herein, a polyvalent phage display antibody library refers to a library in which each element (e.g., phage particle) displays on average two or more binding domains, wherein each linking domain includes a variable heavy region and a variable variable. In a very general manner, a multivalent phage display library displays on average two or more plll fusions per phage particle. The deployment of polyvalent phage expressing diabodies (i.e., a protein formed by fusion or conjugation of two single chain antibodies (eg, scFV)) or by deploying, on average, two or more antibodies in each particle can be achieved. phage In contrast, a monovalent library displays, on average, a single chain antibody per viral particle. a) Expression of diabodies Diabodies are scFv dimers where each chain consists of heavy (VH) and light chain (V) variable domains connected using a linker (e.g., a peptide linker) that is too short to result in mating between domains in the same chain. As a consequence, pairing occurs between complementary domains of two different chains, creating a stable non-covalent dimer with two binding sites (Holliger et al., (1993) Proc. Nati. Acad. Sci. 90: 6444-6448). The C6.5 diabody was constructed by cutting the peptide linker between the Ig V and V domains of 15 to 5 amino acids and binds ErbB2 in the SKBR3 cells bivalently with a Kd approximately 40 times less than C6.5 (4.0 x 10 ~ 10 M ) (Adams et al. (1998) Brit. J. Cancer 77: 1405-1412, 1998). In Example 5, which is described herein, the C6.5 diabody genes were subcloned for expression as plll fusions in the phagemid pHEN-1 (Hoogenboom et al. (1991) Nucleic Acid Res. 19: 4133-4137 ). This phagemid was produced by predominantly expressing an affusion of light scFv or diabody-pIII after rescue with an auxiliary phage (Marks et al (1992) J. Biol .. Chem. 267: 16007-16010). The diabody phagemid displays a bivalent antibody fragment resulting from the intermolecular pairing of a scFv-pIII fusion molecule and an original molecule. Using the doctrines that are provided in this, the person skilled in the art can routinely produce other diabodies. Bivalent diabodies that display phage or multiple copies of scFv carried out endocytosis more efficiently than the monomeric scFv of phage display and increased the recovery of infectious phage by preincubation of cells with chloroquine. The results indicate that it is possible to select for antibodies subject to endocytosis, even at the lowest concentrations that could exist for a single phage antibody element in a 109 element library. b) Polyvalent display of single chain antibodies As an alternative to the use of diabodies, antibody phage display libraries are created wherein each viral particle, on average, expresses at least 2, preferably at least 3, most preferably at least 4, and most preferably still 5 copies of a single chain antibody. In principle, each copy of pIII in the phage (and there is controversy if there are 3 or 5 copies of pIII per phage) must express an antibody. However, proteolysis occurs and the number actually displayed is typically less. Therefore, the preferred multivalent antibody libraries are constructed in a phage vector and not in a phagemid vector. This means that the auxiliary phage does not need to be added to be a phage. Auxiliary phage is included in wild type plll of E. coli that competes with the scFv-plll fusion. Therefore, in the phagemid vector, this competence is limited, on average, to only one antibody (or less) per phage. To produce multivalent antibody libraries, the single chain antibodies, typically expressed in the phagemid, are subcloned from the phagemid vector into a phage vector. No helper phage is required and there is no competition between wild-type plll and the scFv fusion with plll fusion. Therefore, on average, the phage display two or more plll fusions. As a consequence, by way of illustration, example 5 describes the subcloning of the C6.5 scFV gene into the fd-Sfi / Not vector. This results in a phage with 3 to 5 copies of the scFv-plll fusion protein.

B) White Cells The target cells of this invention include any cell for which it is desired to identify a polypeptide or antibody which is either neutral or for which it is desired to identify an internally detectable marker (eg, receptor). Cells can include cells from multicellular eukaryotes, unicellular eukaryotes, including plants and fungi, and also prokaryotic cells. Preferred white cells are eukaryotic, most preferably vertebrate cells, and most preferably mammalian cells (e.g., murine cells, bovines, primates including humans, largoments, canines, felines, and so on). The cells can be normal healthy cells or cells characterized by a particular pathology (e.g., tumor cells). The target cells may include any type of cell where it would be useful: 1) to have an antibody that specifically recognizes the type of cell or related types of cells (eg, for cell sorting, cell staining or other diagnostic procedures); 2) having a ligand that is specifically incorporated into the cell type or related types of cells (eg, 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 tissue, e.g., pros or breast). Therefore, an antibody that recognized and annihilated prosthetic cells would be good for pros cancer even if it annihilated normal pros cells (the pros is not an organ essential). White cells may include tissue-specific cells, and cells of a particular developmental stage. The target cells may also include precursor cells, e.g., stem cells from the bone marrow, which would be useful for isolating and perhaps for stimulating differentiation. The target cells can also include cell lines transfected with a gene for a known receptor (e.g., ErbB2) for which it would be useful to have detectable antibodies. Many ErbB2 antibodies are not internable. Instead of being immunized with recombinant protein or selecting a phage library in recombinant protein, the selection in cells transfected with ErbB2 for hospitalization should produce precisely antibodies with the desired characteristics (hospitalization). Finally, a cDNA library could be transfected into a cell line (e.g., COS) from a desired target cell line or tissue and phage antibodies selected for hospitalization. After several rounds of selection, the phage could be used to stain and sort (eg, by FACS) transfected cells. The DNA can be recovered from the cells, producing the sequences of internal receptors as well as the phage antibodies that bind to them.

C) Cells of a subtractive cell line In a preferred embodiment of the tests of this invention, the phage display library is contacted with cells of a line cellular "subtractive". At this stage, attempts are made to deplete or eliminate elements of the phage display library that bind to cells in a non-specific manner or that bind to targets other than the target against which it is desired to obtain a polypeptide or binding antibody. Contact with the cells from a "subtractive" cell line can occur before, during or after the target cells make contact with elements of the phage display library. However, in a preferred embodiment, contact with the cells of a subtractive cell line is simultaneous with the contact of the target cells. Thus, for example, in a preferred embodiment, the white cell line (cultured adherent to a tissue culture plate) is co-incubated with the subtractive (in suspension) cell line in a single cell culture flask. Virtually any cell can act as a subtractive cell. However, in a preferred embodiment, the subtractive cells display all the markers in the target cell except the marker (eg, receptor) which will act as a target for the selection of the desired binding antibodies or binding polypeptides. Therefore, particularly preferred cells are intimately related to the target cell (s), in terms of having common internal cell surface receptors (such as transferin); for example, fibroblasts) If selected over a tumor cell line (e.g., a breast tumor cell line), it could select negatively in a normal breast cell line. However, this may exhaust all antibodies that bind to overexpressed antigens, so once again a parallel path would be to select negatively on fibroblasts. If transfected cells were used, the untransfected cell could be used as the subtractive cell line. When the tumor is of epithelial origin, the preferred subtractive cell will also be epithelial and it is still very preferable that it be from the same tissue or organ. Particularly preferred subtractive cells include, but are not limited to, non-subtractive cell lines, non-transfected cells, mixtures of undifferentiated and non-transfected cells. When selected for entry into tumor cells, the subtractive cell lines are preferably non-tumor cells from the same tissue (e.g., breast tumor cells against normal breast epithelial cells). In addition, for the cDNA expression libraries, the subtractive cell line will be the untransformed cell line that is used for the construction of libraries (eg, COS, CHO, etc.). In a particularly preferred embodiment, the "target" cell is a cell transformed with a gene or cDNA for a specific target receptor. In this case, the subtractive cell line is preferably the untransformed cell line. Then, for example, when the CHO cells are transformed with a vector containing the gene for the EGF receptor, the cells expressing EEGF are used as the white cell line, and the subtractive cell line is the untransformed CHO cells. Using this approach, anti-EGF receptor antibodies were obtained.

Subtractive cells are more effective when they are provided by exceeding target cells. Said excess preferably is at least 2 times to about an excess of 1000 times, most preferably from about 3 times to about an excess of 100 times, and most preferably still from about 5 times to about an excess of 50 times . In one modality, an excess of 5 times is sufficient.

D) Washing steps As indicated above, a variety of washing steps are used in the methods of this invention. In particular, a "weak" wash step can be used to remove the subtractive cells and weak or non-specific binding elements from the phage display library. A second strong wash step is preferably used after the entry of elements of the phage display library. The "strong" washing step has the purpose of removing the phage bound to the surface weakly and strongly. The pH regulators and methods for performing the weak and strong wash steps are well known to those skilled in the art.

For example, weak washings can be performed with standard pH regulators or culture media (eg, phosphate buffered saline (pH regulator) DMEM (culture media), etc.).

E) Culture under hospitalization conditions As explained above, the cells are preferably cultured under "internable" conditions. Harmful culture conditions are conditions wherein the cell, when bound by an element of a phage display library at the recipient's appropriate site (eg, internally), transports the element into the cell. This may include transport within a vesicle, within an endoplasmic reticulum, the Golgi complex, or within the cytosol of the cell itself. The internal conditions are more easily achieved when the cells are cultured under conditions that mimic those of the cell in its original state. Therefore, many cells, e.g., epidermal cells, are preferably cultured in adherent layers attached to a base membrane. Said cells are more effectively bound to polypeptides and binding antibodies when they are cultured as adherent monolayers. Both chloroquine and serum media prevent non-specific hospitalization and increase specific hospitalization (ligands in the serum that induce the hospitalization of the receptor of interest and carry with them non-specific phages found in the vicinity). In addition, for hospitalization to occur, the cells must be cultured at a temperature and at a pH that allows hospitalization. Suitable temperature and pH range from about 35 ° C to about 39 ° C and a pH of 6 to about pH 8, most preferably from about pH 6.5 to about pH 7.5, with the preferred temperature and pH of about 37 ° C and pH of 7.5, respectively. In a preferred embodiment, the cells are preincubated in serum culture medium for approximately two hours before adding the competing phages and (subtraction) cells.

F) Identification of the hospitalized phage The elements of the hospitalized phage display library can be identified directly or indirectly. Direct identification can be achieved simply by visualizing the phage within the cell, for example, by confocal or immunofluorescent microscopy. Phage internment can be identified by its ability to deliver a reporter gene that is expressed within the cell. The reporter gene may be one that produces a detectable signal (eg, a fluorescent signal (eg, green fluorescent protein, or lux, etc.) or colorimetric signal (eg, HRP, β-galactosidase) or by itself may be a selectable marker (e.g., an antibiotic resistance gene) The use of both ß-galactosidase and GFP as reporter genes is described herein Alternatively, the phage display element may carry a marker ( for example, a brand) and the cells that contain the hospitalized phage can be detected simply by the detection of the brand (for example, in a flow cytometer.) Direct methods are developed that are preferably used for the identification of receptors or cells that join after the selections. It should be noted that the approaches of Cellular Classification (FAC) will work with the identification of both the phage bound to the surface and the boarding school. However, an additional level of specificity can be achieved if the cells are first classified to locate the presence of the hospitalized phage prior to lysis. Direct methods are also used during the analysis phase to demonstrate that the selected phage is in fact hospitalized. Alternatively, the elements of the phage display library can be identified indirectly. In indirect detection methods, the element (s) of the phage display library do not need to be detected while they are present inside the cell. It is enough that they have simply been interned. For example, indirect identification can be achieved by isolating and expanding the phages that were interned within the cells as described above. Indirect identification is particularly well suited where the identified elements of the phage display library are to be used in subsequent rounds of selection or to isolate bacterial host monoclonal phage genomes for the subsequent characterization of monoclonal phages (ie, for the analysis of selection results).

G) Isolation and expansion of the hospital phage It was a discovery of this invention that the elements of the phage display library that had been interned within White cells (e.g., mammalian tumor cells) remain viable and can be recovered and expanded into a "selected" library for subsequent rounds of selection and / or isolation, as well as characterization of particular elements. As used herein, the term "recovery" is intended to include the recovery of the infectious phage and / or recovery of the phage antibody gene and / or recovery of a heterologous nucleic acid by accompanying the antibody gene. The hospitalized phage can be isolated and expanded using standard methods. Typically, 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., TG1 from E. Coli.The phage-containing bacteria then cultured in accordance with standard methods (see, for example, Sambrook supra, Marks et al. (1991) J. Mol. Biol. 222: 581-597).

IV.- Preparation and modification of neutral antibodies As described below, once a neutral antibody is identified, additional copies can be prepared using either synthetic synthetic means or through the use of recombinant expression systems. In addition, other "related" bendable antibodies can be identified by selecting for antibodies that bind to the same epitope and / or by modification of the identified internal antibody to produce libraries of modified antibodies and then re-select antibodies at the in-house of the library.

A) Synthesis of antibodies 1) Chemical synthesis The neutral antibodies, once identified by the methods of this invention, can be synthesized chemically using well-known methods of peptide synthesis. The solid phase synthesis in which the C-terminal amino acid of the sequence is bound to an insoluble support followed by the sequential addition of the amino acids remaining in the sequence, is a preferred method for the chemical synthesis of single chain antibodies. The techniques for solid phase synthesis are described by Barany and Merrifield, Solid Phase Peptide Synthesis; 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol.2: Special Methods in Peptide Synthesis. Panel A., Merrifield et.al., (1963) J. Am. Chem. Soc, 85: 2149-2156, and Stewart et al., (1984) Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford 2. - Recombinant Expression of Internable Antibodies In a preferred embodiment, neutral antibodies, once identified by the methods of this invention, are prepared using standard techniques well known to those skilled in the art. The Nucleic acid sequences encoding the internable antibodies are determined (for example by Sanger sequencing). Using the sequence information, the nucleic acids can be chemically synthesized according to a number of standard methods known to those in the art. Oligonucleotide synthesis is preferably carried out in solid phase commercially available in oligonucleotide synthesis machines (Needham-VanDevanter et al., (1984) Nucleic Acid Res. 12: 6159-6168) or by manually synthesizing using the solid phase of the oligonucleotide. phosphoramidite triester method described by Beaucage et al., (Beaucage et al., (1981) Tetrahedron Letts.22 (20): 1859-1862). Alternatively, the nucleic acids encoding the antibody can be amplified and / or cloned according to standard methods. Molecular cloning techniques to achieve these ends are well known in the art. A wide variety of methods of cloning and in vitro amplification suitable for the construction of recombinant nucleic acids. Examples of these techniques and sufficient instructions to direct those skilled in the art through cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152, Academic Press Inc., San Diego, CA (Berger); Sambrook ei. al., (1989) Molecular Cloning- A Laboratory Manual (2nd ed.) Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY., (Sambrook) and Current Protocols in Molecular Biology F.M. Ausubel eí. to the., eds., Current Protocols, a partnership between Greene Publishing Associates, Inc. and John Wiley & Sons Inc., (1994 Supplement) (Ausubel). Methods for producing recombinant immunoglobulin are also well known in the art. See, Cabilly, EU Patent. 4,816,567; and Queen ei al., (1989) Proc. Natl.Acad. Sci. USA 86: 10029-10033. In addition, detailed protocols for the isolation and cloning of the antibody are provided herein in the examples, and in Schier et al., (1996 J. J. Mol. Biol. 263: 551-567.

B) Identification of other antibodies that bind to the same "internable" epitope. Once one or more internable antibodies are identified by the screening methods of this invention, other "related" internal antibodies can be identified by selection for antibodies that react with the different internable antibodies, either in the epitope bound by the antibodies or with an elevated ideotypic against the internable antibodies. 1) Reactivity with anti-idiotypic antibodies. The idiotype represents the highly variable antigen binding site of an antibody and is itself immunogenic. During the generation of an immune response mediated by antibody, an individual will develop antigen antibodies as well as anti-idiotype antibodies, whose immunogenic binding site (idiotype) mimics the antigen.

Anti-idiotypic antibodies can be raised against the variable regions of internally detectable antibodies identified in the screening systems of this invention using standard methods well known to those skilled in the art. Briefly, anti-idiotype antibodies can be made by injecting internalable antibodies of this invention, or fragments thereof (eg, CDRs) into an animal in that manner by choosing antiserum against various antigenic determinants in the antibody, including determinants in the region idiotypic Methods for the production of anti-analyte antibodies are well known in the art. High molecular weight antigens (greater than about 5000 Daltons) can be injected directly into animals, where the low molecular weight compounds (less than about 5000 Daltons), are preferably coupled to a high molecular weight immunogenic carrier, generally a protein, to render them immunogenic. Antibodies produced in response to immunization may be used as serum, as ascites fluid, as an immunoglobulin (Ig) fraction, an IgG fraction, or as an affinity-purified monospecific material. Polyclonal anti-idiotype antibodies can be prepared by immunizing an animal with the antibodies of this invention prepared as described above. In general, it is desirable to immunize an animal whose species and allotype combines with the animal from which the antibody was derived (e.g., phage display library). This reduces the minimal production of antibodies directed against non-idiotypic determinants. The antiserum thus obtained is generally absorbed extensively against normal serum from the same species from which the phage display library was derived, thus eliminating antibodies directed against non-idiotypic determinants. Absorption can be achieved by passing antisera on a gel formed by interlacing serum proteins or normal (non-immune) crosslinking with glutaraldehyde. Antibodies with specific anti-idiotypic character will pass directly through the gel, while those that have specific character to be non-idiotypic determinants will bind to the gel. Non-immune serum proteins immobilizing on an insoluble polysaccharide support (e.g., sepharose) also provides a matrix suitable for absorption. Monoclonal anti-idiotypic antibodies can be produced using the method of Kohier et al., (1975) Nature 256: 495. In particular, monoclonal anti-idiotype antibodies can be prepared using hybridoma technology which comprises fusing spleen cells (1) from an immunized mouse with the conjugated hapten antigen or vehicle of interest (ie, the antibodies of this invention or subsequences thereof). ) or (2) a myeloma cell line that has been selected for resistance to a drug (e.g., 8-azaguanine). In general, it is desirable to use a myeloma cell line that does not secrete an immunoglobulin. Various determined lines are known in the art. A preferred cell line is P3X63 Ag8.653. This cell line is on deposit in the American Type Culture Collection culture collection as CRL-1580. The fusion should be carried out in the presence of polyethylene glycol according to established methods (see, for example, Monoclonal Antibodies, R. Kennett, J. McKeam &K. B echtol, eds. NY, Plenum Press, 1980, and Current Topics in Microbiology &Immunology, Vol.81, F. Melchers, M. Potters &NL Warner eds., NY, Springer-Verlag, 1978. The resulting mixture of fused and unfused cells are plated on selective media hypoxanthin-aminopterin-thymidine (HAT). Under these conditions, only hybrid cells will be grown.When sufficient cell growth has occurred, (typically 10-14 days after the fusion), the culture medium is harvested and then selected for to obtain the presence of idiotypic anti-analyte antibodies, by any of the various methods including the RIA solid phase and the enzyme-linked immunosorbent assay Cells from the culture wells containing the The desired specific character is expanded and re-cloned. Cells from those cultures that remain positive for the antibody of interest generally pass as ascites tumors in susceptible, histocompatible, mice primed with pristane. The ascitic fluid is harvested by piercing the peritoneal cavity, which was re-tested for the antibody, and purified as described above. If a line of non-secreting myeloma is used in the fusion, the affinity purification of the monoclonal antibody is generally not necessary since the antibody is already homogeneous with respect to its antigen-binding characteristics. All this is necessary to isolate it from contaminating proteins in ascites, that is, to produce an immunoglobulin fraction. Alternatively, the hybrid cell lines of interest can be cultured in serum free tissue culture and the antibody can be harvested from the culture medium. In general, it is a less desirable method to obtain large amounts of antibodies because the yield is low. It is also possible to pass the cells intravenously in mice and harvest the antibodies from the serum. This method is generally not preferred because the small amount of serum can be obtained by bleeding and due to the need for extensive purification from other components of the serum. However, some hybridomas will not grow as ascites and therefore one of these alternative methods should be used to obtain antibodies. 2.- Cross reactivity with the epitope F5 to C1. In place of the anti-idiotypic antibody, other neutral antibodies can be identified by reactivity with the identified "prototypic" antibodies, against the epitope (s) used in the original selection. The competition between "prototypical" internable antibodies and new candidates in an epitope mapping format establishes that antibodies compete for the same epitope.

C) Methods of phage display to select other "related" bendable antibodies. 1) Chain intermixing methods To create higher affinity antibodies, the repertoires of mutant scFv genes, based on the sequence of a binding of an identified internal antibody, are created and expressed on the surface of the phage. The scFvs are of higher affinity are selected on the antigen as described above and in the examples. One approach to creating repertoires of modified single chain antibody genes (scFv) has been to replace the original VH OV gene with a repertoire of V- genes to create new partners (chain intermix) (Claxon et al., (1991J Nature, 352: 624-628.) Using chain intermixing and phage display, the affinity fragment of human scFv antibody to which the phenyloxazolone hapten (phOx) binds was increased from 300 nM to 1 nM (300 times) (Marks et al., (1992) Bio / technology 10: 779-783). Therefore, for example, to alter the affinity of an internal antibody, a repertoire of mutant scFv genes containing the VH gene of the internableable antibody and a repertoire of the VL gene (single chain intermix) can be created. The scFv gene repertoire can be cloned into the phage display vector pHEN-1 (Hoogenboom et al., (1991) Nucleic Acids Res., 19: 4133-4137) and after transformation a transformant library is obtained. Similarly, for the heavy chain intermixing, the internal antibody VH CDR1 and / or CDR2, and / or CDR3 and the single chain are cloned into a vector containing a repertoire of human VH genes to create mutants of antibody libraries of phage For detailed descriptions of chain intermixing to increase antibody affinity, see Schier et al., (1996,) J. Mol.Biol .., 255: 28-43, 1996. 2) Site-directed mutagenesis to improve binding affinity. The majority of amino acid side chains that contact antigens are located in the complementarity determining regions (CDRs), three in the VH (CDR1, CDR2 and CDR3) and three in the VL (CDR1, CDR2 and CDR3) (Clothia et al., (1987) J. Mol.Biol., 196: 901-917; Clothia et al., (1986) Science, 233: 755-8; Nhan et al. ., (1991) J. Mol. Biol., 217: 133-151). These residues contribute to the majority of binding energy responsible for the antibody affinity for the antigen. In other molecules, the mutation amino acids that make contact with the ligand have been shown to be the effective means to increase the affinity of a protein molecule for its binding partner (Lowman et al., (1993) J. Mol. Biol. .., 234: 564-578; Wells (1990) Biochemistry, 29: 8509-8516). Site-directed mutagenesis of CDRs and selection against c-erbB-2 can be used to generate C6 antibodies that have improved binding affinity and / or hospitalization of a known neutral antibody. 3) Random CDR classification to produce higher affinity human scFv. In an extension of simple site-directed mutagenesis, libraries of mutant antibodies can be created where the partial or complete CDRs are randomly selected (V CDR1 and CDR2 and VH CDR1, CDR2 and CDR3). In one embodiment, each CDR is randomly selected in a separate library, using the internalizable antibody known as a template or template. The CDR sequences of the highest affinity mutants from each CDR library are combined to obtain a further increase in affinity. A similar approach has been used to increase the affinity of human growth hormone (hGH) for the growth hormone receptor over 1500 times from 3.4 x 1010 to 9.0 x 1013M (Lowman et al., (1993) J. Mol. Biol .., 234: 564-578). V CDR3 frequently occupies the center of the binding pocket, and therefore mutations in this region are likely to result in an increase in affinity (Claxon et al., (1995) Science, 267: 383-386). In one embodiment, four V CDR3 residues are randomly selected at one time using the NNS nucleotides (see, eg, Schier et al., (1996) Gene, 169: 147-155; Schier and Marks (1996) Human Antibodies and Hybridomas. 7: 97-105, 1996; and Schier et al., (1996 J. Mol. Biol., 263: 551-567, 1996). 4) Creation of homodimers. To create antibodies (scFv ') 2 two scFvs can be internalized, either through a linker (for example, a carbon linker, a peptide) or through the disulfide linkage, between, for example, two cysteines. Thus, for example, to create disulfide-linked scFv, a cysteine residue is introduced by site-directed mutagenesis between the myc tg and a hexahistidine tag in the carboxy-termini of the antibodies described hereinbefore. The introduction of the correct sequence can be verified by DNA sequencing. If the construct is in pUC119, the leader directs expressed pelBs to the periplasm and to the scFv cloning sites that exist to introduce (Ncol and Notl) or scFv mutant of F5 or C1. The expressed scFv is labeled tag myc at the C-terminus, followed by two 2 glycines, one cysteine, and then 6 histidines to facilitate purification by IMAC. After the disulfide bond formation between two cysteine residues, the two scFvs are separated from each other by approximately 26 amino acids (two myc tags of 11 amino acids and 4 glycines). A scFv can be expressed from this construct, purified by IMAC, and analyzed by gel filtration. To produce dimers (scFv ') 2, cysteine is reduced by incubation with 1 mM3-mercaptoethanol, and half of the scFv blocked by the addition of DTNB. The blocked and unblocked scFvs are incubated together to form (scFv ') 2 and the resulting material can be analyzed by gel filtration. The affinity of monomers F5 and C1 scFv 'and those of dimers F5 and C1 (scFv') 2 is determined by core BIA. In a particularly preferred embodiment, the dimer (scFv ') 2 is created by joining the scFv' fragments through a linker most preferably through a peptide linker can be achieved by a wide variety of means well known to those skilled in the art. technique. For example, a preferred approach is described by Holliger et al., (1993) Proc. Nati Acad. Sci. USA, 90: 6444-6448 (see, in addition WO 94/13804).

) Antibody affinity for the target antigen. As explained above, selection for increased activity includes measurement of antibody affinity for the target antigen (e.g., c-erbB2). The methods for making such measurements are described in detail in the co-pending application USSN 08 / 665,202. Briefly, for example, the Kd of F5, C1 F5-or C1, or an antibody derived from, the binding kinetics c-erbB-2 is determined in a BIA core, a biosensor that relies on surface plasma resonance. For this technique, the antigen is coupled to a derived sensor chip capable of detecting changes in the mass. When the antibody is passed over the chip sensor, the antibody binds to the antigen which results in an increase in the mass that is quantifiable. The measurement of the association rate as well as the function of the antibody concentration can be used to calculate the association rate constant (kon) - After the association phase, the pH regulator is passed over the chip and determined the dissociation rate of the antibody (k0ff). Kon is typically measured on the scale from 1.0 x 102 to 5.0 x 106 and koff, on the scale of 1.0 x 10"1 to 1.0 x 10" 6. The equilibrium constant ka is often calculated as k0ff / kon and therefore is typically measured on the scale 10"5 to 10" 12. The affinities that are measured in this way correlate well with affinities that are measured in the solution by fluorescence tempering titration.

V) Genotes and vectors. In another embodiment, this invention provides libraries and vectors for practicing the methods described herein. The libraries are preferably polyvalent libraries, which include diabody libraries and most preferably include multivalent single chain antibody libraries (e.g., scFv), (e.g., expressed by phage). The libraries can take a number of forms. Therefore, in one embodiment, the library is a collection of cells that contain elements of the phage display library, although in another embodiment, the library consists of a collection of isolated phages, and still the The library consists of a nucleic acid library that codes for a polyvalent phage display library. The nucleic acids can be phagemid vectors encoding the antibodies and are ready for subcloning within a phage vector or the nucleic acids can be a collection of phagemids that already carry nucleic acids encoding the subcloned antibody.

SAW. Equipment for selecting internalable antibodies In another embodiment, this invention provides equipment for practicing the methods described herein. The kits preferably include elements from a phage display library (e.g., as phage particles, as vectors, or as phage-containing cells). The test equipment may additionally include any of the other components described herein for the practice of the tests of this invention. Such materials preferably include, but are not limited to, auxiliary phage, to one or more mammalian cell lines, ph regulators, antibiotics, tags, and the like. In addition, the equipment may optionally include instructional materials that contain the instructions (i.e., protocols) that describe the selection methods described herein. Although instructional materials typically comprise written or printed materials, they are not limited to such materials. Any medium capable of storing said instructions and communicating them to an end user is contemplated by this invention. Such means include, but are not limited to, electronic storage means (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Said means may include addresses of Internet sites that provide said instructional materials. Said means may include addresses of Internet sites that provide said instructional materials.

EXAMPLES The following examples are offered to illustrate, but not to limit the claimed invention.

EXAMPLE 1 Creation of a non-immune human Fab phage antibody library containing 109-1011 elements The manipulation of phage display libraries of 107 elements revealed two main limitations: 1) Fabs expression levels were too low to produce a material suitable for characterization, and 2) the library was relatively unstable. These limitations are the result of creating the library in a phage vector, and the use of cre-lox recombination systems. Therefore it was decided that the The best approach for this project was to create a very large scFv library using a phage vector. The target was to produce a library at least 100 times larger than our scFv library of 3.0 x 107 previous elements. The approach that was taken was to clone the VH and VL libraries in separate replicas, combine them into a repertoire of scFv genes starting by overlapping extension, and clone the pHEN1 gene repertoire into the phage display vector. The human peripheral blood lymphocyte and spleen RNA were initialized with heavy chain constant region IgM, the kappa and lambda single chain constant region primers and the first cDNA chain synthesized. The first strand cDNA was used as a template for the PCR amplification of the VH gene repertoires Vkk and V ?. The repertoires of VH genes were cloned into the vector pUC119Sfi-Not as fragments of Ncol-Not1, to create a library of PCR elements. The library was diverse by digital PCR printing. A single-stranded linker DNA was split over the VL gene repertoires using PCR and the repertoire was cloned as an Xhol-Notl fragment into the pHENIXscFv vector to create a library of 7.2 x 106 elements. The repertoires of gene V and VL were amplified from their respective vectors and divided together using PCR to create a repertoire of scFv genes. The scFv gene repertoire was cloned as an Ncol-Notl fragment into the vector to create a scFv phage antibody library of 7.0 x 109 elements. The library was diverse as determined by digital BstNL imaging. To verify the quality of the library, the phage was prepared and selected on 14 different protein antigens. The results are shown in table 1. ScFv antibodies were obtained against all the antigens used for selection, with between 3 and 15 unique scFv isolated by TABLE 1 Results of the selections of the antibody library.

For each antigen (column 1), the number and percentage of selected positive clones (column 2) and the number of different isolated antibodies are indicated (column 3) Percentage (number) of Number of different Protein antigen used for selection positive clones ELISA isolated antibodies ECG FGF receptor 69 (18/26) 15 Type I BMP receptor ECD 50 (12/24) 12 Type I fortune teller ECD 66 (16/24) 7 Type II fortune-teller receiver ECD 66 (16/24) 4 Erb-B2 ECD 91 (31/34) 14 VEGF 50 (48/96) 6 BoNT / A 28 (26/92 ) 14 C-fragment of BoNT-A 95 (87/92) 10 BoNT / B 10 (9/92) 5 BoNT / C 12 (11/92) 5 BoNT / E 9 (8/92) 3 Bungarotoxin 67 (64 / 96) 15 Cytochrome b5 55 (53/96) 5 Chlamydia trachomatis EB 66 (63/96) 7 of antigen (average 8.7) (Table 1). This compares favorably to the results obtained. This compares favorably to the results obtained from the smaller scFv libraries (from 1 to a few linkers obtained against only 70% of the antigens used for selection). The affinities of 4 anti-ErbB-2 scFv and 4 anti-Botulino scFv were measured using surface plasma resonance in BIA core and were found to range from 4.0 x 10'9M to 2.2 x 10"10M for the anti-ErbB2 scFv and 2.6 x 10"8M to 7.15 x 10" 8M for the anti-Botulinum scFv (Table 2) .ScfV was highly specific for the antigen used for selection (Fig. 2) .The library could be successfully selected in complex mixtures of antigens.

TABLE 2 Affinities and binding kinetics of fragment C of anti-BoNT A and anti-Erb-B2 scFv. Rate of association (kon) and dissociation (koff) constants for purified scFv were measured using surface plasma resonance (BIA core) and Kd calculated as (k0ff / k0n) - For example, the selection in elementary bodies Chlamyda trachomatis (the organism that causes the Chlamyda disease) yielded seven that specifically recognized chlamydia (Table 1). The scFv could be used successfully in a number of immunological tests including ELISA, immunofluorescence, Western blot, epitope mapping and immunoprecipitation. The number of binding antibodies for each antigen, and the affinities of the scFv are comparable to the results obtained from the best phage antibody libraries (Table 3). Therefore, the library was established as a source of human antibody panels against any antigen with affinities at least equivalent to the secondary murine response.

TABLE 3 Comparison of protein binding antibodies selected from non-immune phage display antibody libraries. * For type of library, we obtained repertoires of N = V-gene genes obtained from V genes that were re-placed in vivo, V, SS = semi-synthetic genes were constructed from V gene segments and synthetic oligonucleotides coding for VHCDR3. ND = indeterminate.

These experiments demonstrated the creation of a high complexity human scFv phage antibody library from which a high affinity human scFv panel can be generated against any purified antigen. This library is ideal for testing the cell surface to identify novel cell surface markers.

EXAMPLE 2 Infection of scFv in cells by receptor-mediated endocytosis and subsequent recovery The 7.0 x 109 phage antibody library of equis scFv elements that was previously described was selected in malignant breast tumor cell lines MB231 and ZR-75-1, both with or without negative selections on the normal breast cell line HBL100. Similar results were obtained as described in the previous section. scFv was isolated and could not be distinguished from malignant or non-malignant cell lines. To increase the specificity of the selections, it was concluded that phage-binding cell surface receptors could be recovered in cells that could be taken into cells by receptor-mediated endocytosis and could be recovered from cells using said cells. Therefore, it was concluded: 1) that the phage could be hospitalized by receptor-mediated endocytosis and 2) that the phage could be recovered in the infectious state within the cells prior to lysosomal degradation. The ability to select interphase phage antibodies would have two main benefits: 1) the identification of antibodies that bind to receptors capable of hospitalization and 2) an added level of specificity in the selection procedure. The identification of antibodies that are interned would be extremely useful for many therapeutic approaches of white, where hospitalization is essential (for example, as immunotoxins, selected liposomes, selected gene therapy vectors, and others).

A) Phage-mediated hospitalization of phage F5 or C1 To determine the proof of principle, phage C6.5 and diabody phage C6.5 were used (see co-pending application USSN 08 / 665,202). It has previously been shown that C6.5 scFv is hospitalized, but at a slow rate, and that the C6.5 diabody is somehow better hospitalized (probably because it causes the dimerization of the receptor). The C6.5 phage, the C6.5 diabody phage or an irrelevant anti-botulino phage were incubated with SKBR3 cells (ErbB2 expressing breast tumor cell line) that is at 37 ° C or 4 ° C and a phage not Boarding removed by sequential washing with PBS and low pH glycine regulator. The cells were waterproofed and biotinylated with the addition of anti-M13-antibody followed by streptavidin Texas Red. Cells and were subsequently examined using a confocal microscope. Both the C6.5 phage and the C6.5 diabody phage were observed within the cytoplasm). Approximately, 1% of the cells had entered the C6.5 phage and 20% had entered the diabody phage C6.5. There was no hospitalization of the anti-Botulino phage. To determine if the infectious phage could be specifically taken and recovered from the interior of the cells, phage C6.5 and phage of diabody C6.5 were incubated with SKBR3 cells at 37 ° C. It was not removed no binding phage washing with PBS and phage binding to the cell surface was eluted by washing twice with low pH glycine. The cells were then glissed and each fraction (first and second glycine and the cytoplasmic fraction) was used to infect E. coli TG1. Twenty times (C6.5) or 30 times (of diabody C6.5) more phages were bound to the cell surface compared to the anti-Boulino phage (a wash with glycine) (Table 4). After the second wash with glycine, the infectious cell surface phage titre decreased, indicating that the wash was effective to remove the surface binding phage (Table 4). After cell lysis, the titer increased more than 10-fold (C6.5 phage) or 50-fold (C6.5 diabody phage) from the second glycine wash. It was considered that this title represents the phage recovered from inside the cell. The recovery of the phage inside the cell was 100 times higher than for ErbB2 binding to C6.5 than for the anti-Botulino phage and 200 times higher for the diabody phage C6.5 (Table 4).

TABLE 4 Title of phage bound to cell surface and hospitalized phage. The phage 5.0 x 1011 (anti-Botulinum or anti-ErbB2) were incubated with approximately 1. 0 x 105 SKBR3 cells expressing ErbB2 at 37 ° C. The cells were washed 10 times with PBS and the surface bound phage was eluted with two low pH glycine washes. The cells were then washed once with PBS and the cells were lysed to release the phage interned. The phage titer was then determined for each of the glycine washes and for the cell fraction lysed with E. coli TG1 infection.

All the results are considered, indicating that: 1) phage-binding cell surface receptors can be taken up by cells and the infectious phage can be recovered from the cytoplasm. The amount of intake is significantly greater than the intake of phage that has no binding, and the difference of 100 to 200 times is adequate within the scale that could allow the enrichment of a library. What is unknown about the results is whether the phage antibodies are mediated by receptor or mediated by admission or if they are simply taken after binding by membrane replacement.

B) .- Selection and characterization of antibodies that can be internalized from a phage antibody library. The results described above encouraged to attempt the selection of the phage antibody library described above to identify new phage antibodies that were interned The phage antibodies were rescued from the library and selected in SKBR3 cells. For selection, the phages were incubated with cells at 37 ° C, the phage containing no binding was removed by washing cells with PBS and the phage bound to the cell surface antigens were removed by sequential washings with low pH glycine. The cells were then lysed to release the internal phage and the lysate was used to infect E. coli TG1 to prepare the phage for the next round of selection. Three rounds of selection were made. One hundred clones from each round of selection were analyzed for binding to SKBR3 cells and the extracellular domain ErbB2 by ELISA. It was concluded that they were likely to obtain linkers to ErbB2 since SKBR3 cells are known to express high levels and ErbB2 is a receptor that is known to be hospitalized. After each round of selection, the phage titer recovered from the cytoplasm increased (Table 5). After the third round, 45% of the clones were positive bound to SKBR3 cell and 17% bound to ErbB2 (Table 5).

TABLE 5 Results of the selection of a phage antibody library for hospitalization. For each round of selection, the phage titer in lysed cells, the number of lysed cells and the phage number per cell is indicated. After the third round, the individual clones were analyzed for binding to SKBR3 cells by ELISA and for ErbB2 ECD by ELISA.

To calculate the number of unique linkers, the scFv gene from the ELISA positive clones was amplified with PCR and was digitized by digestion with BstNL. Two unique restriction patterns were identified. The scFv genes were sequenced and two unique ErbB2s were identified bound to scFv. Similar analysis of ELISA SKBR3 positive clones that did not bind to ErbB2 were identified as only 11 additional scFv. To verify that the phage antibodies were specific for SKBR3 cells, the phage was prepared from each unique clone and analyzed for binding to SKBR3 cells (high expression of ErbB2) as well as other 2 epithelial epithelial tumor cell lines ( SKBR3), 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 C6.5 (positive control), 3TF5 and 3GH7 antibodies. The last two clones were isolated of the library with ErbB2 binding to 3TF5 and and the antigen bound by 3GH7 was unknown. All 3 phage antibodies intensely stained SKBR3 cells (the selected cell line and high ErbB2 expressor.) C6.5 phage weakly stained MCF7 cells (low ErbB2 express.) The anti-ErbB2 clone of 3TF5 the library stains MCF7 cells more intensely than C6.5, as does 3GH7.SKBR3, SK-OV-3, MCF7 and HST578 cells were studied using original purified 3TF5 and 3GH7 scFv.For these studies, scFv genes were cloned into a vector that fused a hexameridin label at the C-terminal scFv, then scFv was expressed, cultured from the bacterial periplasm and purified by immobilized metal affinity chromatography.The two scFv stained intensely SKBR3 cells, and did not stain the normal breast cell line HST578. minimal staining of the cell line of expression of low ErbB2 and MCF7, as well as the intermediate staining of SK-OV-3 cells (moderate ErbB2 expression) .In general, the intensity of staining is less than when it is appreciated with a phage. This should be expected since the secondary antibody for phage staining recognizes the major coat protein (2500 copies / phage) which results in tremendous signal amplification. The anti-ErbB2 phage of 3TF5 antibody was also studied to determine if it had indeed been hospitalized. This antibody was selected for the initial study from its hospitalization that could be compared to ErbB2 bound to C6.5 5.0 x 1011 3TF5 or to phage C6.5 was incubated with SKBR3 cells at 37 ° C or at 4 ° C. After washing with PBS, phage 3TS5 stained cells more intensely than phage C6.5. After washing with low pH glycine, confocal microscopy revealed that phage 3TF5 was internalized by a greater than 95% number of cells, whereas C6.5 was interned only by a small percentage of cells. Incubation of any antibody at 4 ° C led to no hospitalization. The purified original 3TF5 scFv was analyzed in a similar manner and was also efficiently internalized by SKBR3 cells. It should be noted that scFv 3TF5 existed only as a monomer without appreciable dimerization or aggregation as determined by gel filtration. These experiments demonstrate that phage antibodies can be interned by cells and recovered from the cytoplasm. The phage that binds to an internable cell surface receptor can be enriched more than 100 times over the non-binding phage. This level of enrichment is greater than that achieved by selecting on the cell surface. This approach has been applied to the selection of libraries and isolated phage antibodies that bind and are interned by SKBR-3 cells. Several of these antibodies bind to ErbB2, but are interned more efficiently than antibodies, such as C6.5 that were generated by selecting on the pure antigen. Many other antibodies have been isolated that bind specifically to SKBR-3 and other breast tumor cell lines and are efficiently interned. These antibodies should prove useful for the detection of tumors and for identifying potentially novel recipients of potentially cancerous cells.

EXAMPLE 3 Increase the affinity of antibody fragments with the desired binding characteristics by creating antibody libraries of mutant phage and selecting in the appropriate breast tumor cell line Phage display has the potential to produce antibodies with affinities that can not be produced using conventional hybridoma technology. Human ultra-high affinity antibody fragments could result in excellent tumor penetration, prolonged tumor retention and rapid release from the circulation, leading to a high specificity. Accordingly, a series of experiments was conducted to develop methodologies for generating fragments of human antibodies of high affinity. The experiments were developed to answer the following questions: 1) What is the most effective way to select and detect low affinity phage antibodies sparse among a background group of lower affinity binders; 2) Which is the most effective means to remove the bound phage of the antigen, to ensure the selection of the highest affinity phage antibodies; 3) What are the most efficient techniques to elaborate mutant phage antibody libraries (random mutagenesis or site-directed mutagenesis); 4) What is the region of the antibody molecule that must be selected for mutagenesis that more efficiently increases the affinity of the antibody fragment. To answer these questions, we studied human scFv C6.5, which binds to the extracellular domain (ECD) of ErbB2 tumor antigen (32) with a Kd of 1.6 X 108 M and Koff of 6.3 X 10"3 s "1 (Schier et al. (1995) Immunotechnology, 1: 63-71). The isolation and characterization of C6.5 is briefly described below and in detail in the co-pending application USSN 98 / 665,202). Despite the excellent ratios of tumor / normal tissue in vivo, the quantitative supply of C6.5 was not suitable for curing tumors in animals using radioimmunotherapy (Schier et al. (1995) Immunotechnology, 1: 63-71). To improve the quantitative supply of antibody to tumor, the affinity of C6.5 was increased. The techniques that allowed the selection of phage antibodies based on affinity, instead of differential growth in E. coli or host strain toxicity ((Schier et al., (1996) J. Mol. Bio. 255) were developed. : 28-43, (Schier et al. (1996) Gene; (Schier et al. (1996) Human antibodies and hybridomas 7: 97-105). Subsequently, the locations in the scFv gene were determined to mutate with the target to achieve the greatest increases in affinity. (Schier et al. (1996) J. Mol. Biol. 255: 28-43); (Schier et al. (1996) Gene; (Schier et al. (1996) J. Mol. Biol. 263: 551-567.) Random mutagenesis does not produced such a high affinity increase as site-directed mutagenesis of complementarity determining regions (CDRs) containing the amino acids with contact antigen. The results of diversifying the CDRs indicated that: 1) the greatest increase in affinity was achieved by mutating the CDRs located in the center of the binding pocket (VL and VHCDR3); 2) half of the CDR residues have a structural role in the scFv and when they are mutated they return as wild type; and 3) these structural residues can be identified prior to the construction of the library by modeling on a homologous atomic crystal structure. These observations led to the development of a generic strategy to increase the affinity of antibodies in which mutations are randomly introduced sequentially into VL and VHCDR3, with preservation of postulated residues to have a structural role through homology modeling (Schier et al. (1996) J. Mol. Biol. 263: 551-567). Using this approach, the affinity of C6.5 was increased 1200 times to a Kd of 1.3 X 10-11 M (/). Bio-distribution studies revealed a close correlation between affinity and the dose injected as a percentage of scFv / gram of tumor (% ID / g) at 24 hours (Adams et al. (1998) Cancer Res. 58: 485- 490). The highest degree of tumor retention was observed with 125l-C6ML3-9 (1.42% ID / g, Kd = 1.0 X 10"9M). Less significant tumor retention was achieved with 125I-C6.5 (0.80% ID / g, Kd = 1.6 X 10"8) and C6G98A (0.19% ID / g, Kd = 3.2 X 10" 7M). The tumoral / normal organ relationships also reflected the differences in affinity, for example, the blood-blood relationships of 17.2, 13.3, 3.5 and 2.6, and the tumor-to-liver ratios of 26.2, 19.8, 4.0 and 3.1 for C6ML3-9, C6.5 and C6G98A respectively at 24 hours. The highest affinity studies of scFv are pending. The results demonstrate the ability to increase the affinity of the antibody to values that can not be achieved from the hybridoma technology and confirm the importance of affinity in the selection of tumors.

EXAMPLE 4 Clinical development of breast cancer therapies based on C6.5 Two approaches to develop breast cancer therapies based on C6.5 have been collaboratively followed. Molecules based on C6.5 are manipulated for radioimmunotherapy. To increase the quantitative tumor supply and retention of the antibody fragment, dimeric scFv "diabodies" were created by trimming the linker between the VH and VL domains of 15 to 5 amino acids. As a consequence, mating occurs between the complementary domains of two different chains, creating a dimer non-covalently bound, stable, with two binding sites. In vitro, the diabodies produced from the V6 genes of C6.5 have a significantly higher apparent affinity and a longer retention on the surface of SK-OV-3 cells compared to scFv of C6.5 (T 1 / >5hr.v. 5 min.) (Adams Ket al. (1998) Brit J. Cancer). The Bio-distribution studies of the C6.5 diabody revealed 6.5% ID / g tumor at 24 hours compared to only 1% ID / g for C6.5 scFv. When the diabody retentions were examined for 72 hours and the cumulative area was determined under curve values (AUC), the resultant tumor: organ ratios were greater than those reported for other monovalent or divalent scFv molecules. The therapeutic potential of these molecules is examined in radioimmunotherapy studies in nude mice. As the in vivo characterization of molecules based on C6.5 was not formally one of the technical targets, the use of C6.5 affinity mutants and C6-based diabodies is continued. 5 to study the relationship between antibody affinity, size, valence and tumor specific selection as part of NIH R01 1 CA 65559-01 A1. In another collaboration molecules based on C6.5 are being used to select doxorubicin containing furtive liposomes for breast cancer expressing ErbB2 (Kirpotin et al) 1997) Biochemistry. 36: 66-75). To facilitate chemical coupling of the scFv to the liposomes, the C6.5 gene was subcloned into an E. coli expression vector resulting in the addition of a cysteine-free residue at the C-terminus of the scFv. The scFv of C6.5cys was conjugated to the liposomes and the uptake was determined in vitro using SKBR3 cells. The total uptake was 3.4 mmol of phospholipid / 106 cells at 6 hours, with 70% of the intake uptake. The intake is comparable to that achieved using the Fab 'anti-HER2 of 4D5 from Genentech. There was no intake of liposomes not conjugated The results indicate that C6.5 binds to the epitope of ErbB2 which results in hospitalization at a rate comparable to that of the best antibody for hospitalization produced from hybridomas (4D5). In vivo therapy studies in scid mice indicate that the liposomes directed to C6.5 caused a higher degree of tumor regression and a higher cure rate than that of the untargeted liposomes or a combination of non-directed liposomes and systemic anti-convulsive for 4D5.

Conclusions The experiments described in the present invention establish that a large gene library (7.0x109 elements) of phage antibodies has been created which can provide panels of human antibodies against purified antigens with affinities comparable to the affinities of the antibodies produced by the immunization of murine The receivers cell surface antibodies that bind to phage antibodies can be interned by cells and recovered in an infectious state from inside the cell. Methodologies were developed that allow the enrichment of phage antibodies for hospitalization with respect to non-internant antibodies in more than 100 times. These methodologies were then applied in SKBR-3 cells to select new antibodies against scFv that bind to the recipients for hospitalization. Several of these antibodies bind to ErbB2, but are interned more efficiently than scFv based on C6.5. Many other antibodies bind to unknown recipients for hospitalization. All these scFv bind specifically to SKBR-3 cells or related tumor cell lines. The results indicate that this method of selection is a powerful method to generate antibodies that can distinguish one cell type (malignant) from the other type (non-malignant). Furthermore, it has been shown that it is not only possible to make the selection with respect to union, but also to make the selection in terms of function (hospitalization). In the short term, the isolated antibodies will be further characterized with respect to their specific character, and in the case of scFv that binds to ErbB2, the affinity. In the long term, these reagents will be used to: 1) study the effect of affinity and valence on the speed of hospitalization; and 2) identify the bound antigens using immunoprecipitation techniques. It is likely that the results will lead to the identification of novel receptors on the surface of the tumor cells to effect hospitalization, which will be useful therapeutic targets. If this method proves to be useful, it is planned to be applied to primary tumor cells and DCIS. It is also intended to evaluate 3TF5 (scFv that binds to ErbB2 which is internally faster than C6.5) to select the liposome as target. It is possible that it is more effective than C6.5. In addition, the experiments demonstrate that the methodologies increase in vitro the affinity for antibody to values not previously achieved in vivo. These methodologies have been applied to generate novel scFvs that bind to ErbB2.

EXAMPLE 5 Selection of antibodies for hospitalization from phage libraries In this example, a human scFv (C6.5) that binds to ErbB2 was studied to determine the feasibility of directly selecting the antibodies for hospitalization from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv deployed in monovalent form in a phagemid, it was shown that anti-ErbB2 phage antibodies can be subjected to receptor-mediated endocytosis. Using affinity mutants and dimeric C6.5 diabodies deployed either as single copies in a phagemid or as multiple copies in a phage, the role of affinity, valence, and display format over phage endocytosis is defined and identified the factors that lead to the greatest enrichment for hospitalization. Phages displaying bivalent diabodies or multiple copies of scFv were ingested by endocytosis more efficiently than phages displaying monomeric scFv and the recovery of infectious phage was increased by pre-incubating the cells with chloroquine. The measurement of phage recovery from inside the cytosol as a function of the applied phage titer indicates that it is possible to select antibodies that can be ingested by endocytosis, even at the low concentrations that could exist for an individual element of phage antibody in a library of 109.

A) Materials and methods 1) Cells The SKBR3 cell line of breast tumor was obtained from the ATCC and cultured in RPMI medium supplemented with 10% FCS (Hyclone) in an atmosphere with 5% C02 at 37 ° C. 2) Antibodies and phage antibody preparations The phage vector C6.5 scFv was constructed by subcloning the C6.5 gene as a S1 / I / I fragment from scFv C6.5 pHEN1 (Schier et al. 1995) Immunotechnology 1: 63-71) in the vector fd / S / 7 l /? I heard of phage (a gift from Andrew Griffiths, MRC Cambridge, UK). The phagemid diabody vector of C6.5 was constructed by subcloning the diabody gene of C6.5 (Adams et al. (1998) Brit. J. Cancer 77: 1405-1412, 1998) as an Ncol / Notl fragment in pHEN1. (Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137). The phagemid of anti-botulism scFv (clone 3D12) (Amersdorfer et al. (1997) Infection and Immunity 65: 3743-3752), the phagemid scFv of C6.5 (Schier et al. (1995) Immunotechnology) have already been described. 1: 63-71) and the phagemid scFv of C6ML3-9 scFv (Schier et al. (1996) J. Mol. Biol. 263: 551-567) in pHEN1. The phages were prepared (Sambrook et al., 1990) Molecular cloning-a laboratory manual., New York: Cold Spring Harbor Laboratory) from appropriate vectors and were titred in E. coli TG1 as previously described (Marks et al (1991) J. Mol. Biol. 222: 581-597) using ampicillin resistance (100 μg / ml) for the titration of constructions in pHEN1 and tetracycline (50 μg / ml) for the titration of the constructions in fd. The C6.5 scFv, the C6.5 diabody and the soluble anti-botulism scFv were expressed from the vector pUC119mycHIS (Schier et al. (1995) Immunotechnology 1: 63-71) and purified by affinity chromatography. immobilized metal as described elsewhere (Id.)). 3) Detection of original antibody fragments and interphase phage antibodies. SKBR3 cells were grown on cover slips in 6-well culture plates (Falcon) up to 50% confluence. The culture medium was renewed 2 hours before the addition of 5.1011 cfu / ml of phage preparation (the phage preparation representing a maximum of 1-10 of the culture medium volume) or 20 μg / ml of scFv or purified diabody. in phosphate buffered saline, pH 7.4 (PBS). After 2 hours of incubation at 37 ° C, the wells were quickly washed 6 times with ice-cold PBS and 3 times for 10 minutes each with 4 ml of buffer for removal (50 mM glycine pH 2.8, 0.5 M NaCl, 2M urea, 2% polyvinylpyrrolidone) at room temperature. After 2 additional washes with PBS, the cells were fixed in 4% paraformaldehyde (10 minutes at room temperature), washed with PBS, permeabilized with acetone at -20 ° C (30 seconds) and washed again with PBS. The coverslips were saturated with PBS-1% BSA (20 minutes at room temperature). Phage particles were detected with M13 anti-immunoglobulins treated with biotin (5 Prime-3 Prime, Inc., diluted 300-fold) (20 minutes at room temperature) and Texas-conjugated streptavidin-red (Amersham, diluted 300-fold) (20 minutes at room temperature). Soluble scFv and diabodies containing a C-terminal myc peptide tag were detected with mouse mAb 9E10 (Santa Cruz Biotech, diluted 10-fold) (45 minutes at room temperature), biotin-treated anti-mouse immunoglobulins ( Amersham, diluted 100 times) and conjugated streptavidin-red Texas. The confocal optical sections were taken using a confocal microscope with a BioRad MRC 1024 laser scan. Alternatively, the coverslips were analyzed with a Zeiss Axioskop UV fluorescence microscope. 4) Retrieval and titration of phage bound to the cell or hospital surface. Subconfluent SKBR3 cells were grown in 6-well plates. The culture medium was renewed 2 hours before the experiment. The cells were incubated for varying times with different concentrations of phage preparation at 37 ° C. After washing with PBS and with a regulatory solution for removal, performed exactly as described above for the detection of original antibody fragments and of internalized phage antibodies, the cells were again washed twice with PBS and lysed with 1 ml of 100 mM triethylamine (TEA). The washings with buffer for removal and the lysate with TEA were neutralized with V2 volume of 1M Tris-HCl, pH 7.4. For some experiments, the cells were treated with trypsin after three washes with buffer for removal, collected in a 15 ml Falcon tube, washed twice with PBS and then lysed with TEA. In the experiments performed in the presence of chloroquine, the SKBR3 cells were pre-incubated for two hours in the presence of total medium containing 50 μM of chloroquine before adding the phage. The corresponding control samples were prepared at the same time in the absence of chloroquine. For all experiments, the phage were titered in E. coli TG1 as described above.

B) Results 1) The model system used to study the entry of antibody against phage The scFv C6.5 of anti-ErbB2 was obtained by selecting a phage antibody library for human scFv on the extracellular domain of recombinant ErbB2 (13) . The scFv C6.5 binds to ErbB2 with a Kd = 1.6 x 10"8 M and is a stable monomeric scFv in solution that does not show any tendency to dimerize or to aggregate spontaneously (Schier et al. (1995) Immunotechnology 1: 63 -71) To determine the impact of the affinity on hospitalization, we studied a scFv (C6ML3-9) which differs from C6.5 by three amino acids (Schier et al (1996) J. Mol. Biol. 263: 551-567). The scFv C6ML3-9 is also a stable monomer in solution and binds to the same epitope to which scFv C6.5 binds but with a Kd (1.0 x 10"9 M) 16 times smaller (Schier et al. (1996) J Mol. Biol. 263: 551-567; Adams et al. (1998) Cancer Res. 58: 485-490.) Because homodimerization typically appears to be a requirement for antibody entry, the C6 diabody was also studied. Dimeric (Adams et al. (1998) Brit. J. Cancer., 77: 1405-1412, 1998.) Diabodies are scFv dimers in which each chain consists of variable domains of heavy (VH) and light chain ( VL) connected using a peptide linker which is too short to allow pairing between domains on the same chain.As a result, mating occurs between complementary domains of two different chains, creating a stable non-covalent dimer with two binding sites (Holliger et al. (1993) Proc. Nati, Acad. Sci. 90: 6444-6448) The diabody C6.5 is struyó by shortening the peptide linker between the VH and VL domains of Ig from 15 to 5 amino acids and binds ErbB2 in SKBR3 cells bivalently with a Kd approximately 40 times less than that of C6.5 (4.0 x 10"10 M ) (Adams et al. (1998) Brit. J. Cancer. 77: 1405-1412, 1998). The original C6.5 scFv and the C6.5 diabody were expressed and purified from E. coli and analyzed for endocytosis in SKBR3 breast tumor cells expressing ErbB2 by Immunofluorescent confocal microscopy. As expected, the C6.5 scFv is not significantly internalized whereas the dimeric C6.5 diabody can be detected in the cytoplasm of all the observed cells. For subsequent experiments, the genes for scFv C6.5 and C6ML3-9 and diabody C6.5 were subcloned into the phagemid pHEN-1 to express them as plll fusions (Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137). This should produce phagemid that predominantly expresses an individual scFv or a diablo-fusion plll complex after rescue with helper phage (Marks et al (1992) J. Biol. Chem. 267: 16007-16010) (Figures 2A and 2B ). The diabody phagemid displays a bivalent antibody fragment resulting from the intermolecular pairing of a scFv-fusion plll molecule and an original scFv molecule (Figure 2B). The gene for scFv C6.5 was also subcloned into the phage vector fd-Sfi / Not. This results in phages each with 3 to 5 copies of the scFv-fusion protein plll (Figure 2C). The SKBR3 line of human breast cancer cells was used as a target cell line for endocytosis. Its density of ErbB2 on the surface is approximately 1.0 x 106 per cell (Hynes et al (1989) J. Cell. Biochem 39: 167-173). 2) Phagemids C6.5 are ingested by endocytosis by human cells Phagemids with scFv C6.5 were incubated for 2 hours with SKBR3 cells cultured on coverslips at 37 ° C to allow the active hospitalization. The cells were thoroughly washed with PBS to remove the non-specific binding and washed another three times with buffer solution with high salt content and low pH (removal) to remove the phage bound specifically to the cell surface receptors. Inpatient phagemids were detected with an M13 antiserum treated with biotin that recognizes the pVIII protein of the main phage coat. A phagemid anti-botulism toxin was used as a negative control. The staining was analyzed using immunofluorescent microscopy. Approximately 1% of the cells incubated with the phagemid scFv C6.5 showed strong intracellular staining consistent with the endosomal localization, although no staining was observed for the anti-botulism phagemid. In addition, no staining is observed if the incubation is carried out for 2 hours at 4 ° C instead of 37 ° C (data not shown). Staining performed after washing with PBS, but before the washings with a regulating solution for removal, they showed staining of the membranes of all the cells, which indicates that mult washes with the regulatory solution for removal are necessary to eliminate the phagemids bound to the surface. The results also indicate that only a fraction of the phages bound to the cell is ingested by endocytosis. 3) Increased affinity and bivalence lead to increased phage endocytosis. The internment of phagemids scFv C6.5, scFv C6ML3-9 and diabody C6.5 and phage scFv C6.5 was compared using immunofluorescence. Both the phagemids of scFv C6ML3-9 and of diabody C6.5, as well as the phage of scFv C6.5 produced increased intensity of the immunofluorescence observed at the cell surface compared to that of the phagemid of scFv C6.5. For the phagemid of scFv C6ML3-9, approximately 10% of the cells showed intracellular fluorescence after 2 hours of incubation. This value was increased to approximately 30% of the cells for the diabody phagemid of dimeric C6.5 and up to 100% of the cells for the multivalent C6.5 scFv phage. 4) Infectious phage can be recovered from inside the cell and its titer correlates with the level of absorption observed using immunofluorescence. To determine if the infectious phage antibody particles can be recovered from inside the cell, they were incubated 5.0 x 105 SKBR-3 cells for 2 hours at 37 ° C with 3.0 x 10 11 cfu of different phagemids or phages. Six washes with PBS were used to remove the unbound phage specifically and the specifically bound phage was removed from the cell surface by three washes consecutive with a regulatory solution for removal (washes I, II and III respectively, Table 6). The cells were then lysed with 1 ml of a 100 mM solution of triethylamine (TEA) (representing the intracellular phage). The three removal washes and the cell lysate were neutralized and their phage titers determined by E. coli TG1 infection. The recovered phage titles are reported in table 6.

TABLE 6 Titration of phage bound to membrane and intracellular phage Title of the phage titer on the cell surface (x 10"5) phage Antibody against phage 1st wash 2nd. 3rd wash intracellular lavage (x 10"5) Phagemid anti-botulism 280 36 2.8 15 Phagemid of scFv C6.5 600 96 7.6 52 Phagemid of scFv 2500 140 32 270 C6ML3-9 Diabody phagemid of 1800 120 13 450 C6.5 Phage of scFv C6.5 2300 620 56 2200 3.0 x 1011 cfu of phagemid of monovalent C6.5 scFv, of monovalent scFv C6ML3-9 with 16-fold higher affinity, of bivalent C6.5 diabody phagemid or multivalent C6.5 fd phage, were incubated with SKBR3 cells subconfluents for 2 hours at 37 ° C. The cells were washed 6 times with PBS, 3 times with buffer for removal and then lysed to recover the intracellular phage. The various fractions were neutralized and the phage was titrated. The number is reported total cfu for each of the fractions. Non-specific anti-botulism phagemid was used to determine non-specific recovery. A considerable background binding for the anti-botulism phage was observed in the first wash of removal even after 6 washes with PBS (2.8 x 107 cfu, Table 6). This value probably represents the phage not specifically bound to the cell surface, as well as the phage trapped in the extracellular matrix. The amount of phage bound to the surface was increased only 2.1 times above this background for the phagemid of scFv C6.5 (tables 6 and 7). With the increased affinity and avidity of the expanded C6.5 antibody fragment, the titer of the phagemid or phage bound to the surface was increased (Table 6). The phage titer in consecutive washings for removal decreased by approximately 10 times with each wash. These additional washings for removal led to a minor increase in the eluted specific phage titer compared to the background binding of the anti-botulism phage (2.7 times for the phagemid of scFv C6.5 up to 20 fold for the C6 scFv phage. 5, table 7). The only exception was the title of the diabody phagemid of C6.5, in which the relation of fact decreased from 6.4 to 4.6 times. This is probably due to the fact that in the diabody the VH and v domains comprising a single binding site are not covalently bound to one another by the peptide linker. This increases the likelihood that an astringent eluent (such as glycine) can dissociate VH from V and derogate the binding to the antigen.

TABLE 7 Specific Enrichment of Anti-ErbB2 phage compared to anti-botulism phage Relation of anti-ErbB2 phage titer / anti-botulism * Relation of phage titer Antibody against phage Surface cephalide Superf | C? E ce | u¡ar, ntrace | u | ar intracellular / surface (1st wash) (3rd wash) cellular Phagemid of scFv C6.5 2.14 2.7 3.5 6.8 Phagemid of scFv C6ML3-9 8.9 1 1.4 18 8.4 Diabody phagemid of 6.4 4.6 30 35 C6.5 Phage of scFv C6.5 8.2 20 146 39 * The anti-ErbB2 phage titers are divided between the anti-botulism phage titers (Table 6) to obtain an enrichment relationship for the specific vs. non-specific binding or for hospitalization. ** The intracellular phage titer is divided between the phage titer bound to the cell surface (Table 6) to obtain the ratio of internal phage vs phage bound to the surface.

Three removal washes were required to ensure that the titer of the phage recovered after lysis of the cells was greater than the titer of the last clearance wash (Table 6). It is considered that after three washings of removal, the majority of phage eluted represented by the infectious particles comes from the interior of the cells and not from the cell surface. In fact, because the titer of the cell lysate observed with the non-specific anti-botulism phage was considerable (1.5 x 106) and greater than the observed in the last wash of removal, it is probable that a good part of the phage remains trapped inside the extracellular matrix and relatively inaccessible to the washings with regulatory solution for removal. In addition, some amount of anti-botulism phage could have been ingested, by endocytosis, by the cells in non-specific form, but this is probably a small amount given the immunofluorescence results. The phage titer in the TEA fraction increased with the increasing affinity and avidity of C6.5, with the highest titers observed for the diabody phagemid of dimeric C6.5 and for the multivalent C6.5 scFv phage ( box 6). The values represent an increase of 30 times (diabody phagemid of C6.5) and 146 times (phage of scFv C6.5) in the titer compared to that of the anti-botulism phage (Table 6). It is believed that the increase in the phage titer in the cell lysate compared to the last clearance wash is due to phage ingested by endocytosis. In fact, some of these phages could come from the cell surface or from the intracellular matrix. Although this may be true for a fraction of the phages from the cell lysate, the immunofluorescence results indicate that at least some of the phage are ingested by endocytosis. An indicator of the relative fraction of phages, ingested by endocytosis, for the C6.5 molecules is to compare the amount of phage remaining on the cell surface before lysing the cells (last wash for removal) recovering said amount after cell lysis . This relationship shows only a minor increase for the phagemid of scFv of C6.5 or of scFv of C6ML3-9 (6.8 and 8.4 times respectively) in comparison with the phagemid of anti-botulism (5.4) (table 7). In contrast, the ratios for the diabody phagemid of C6.5 and for the multivalent C6.5 scFv phage are increased to a greater degree (35 and 39 respectively) compared to the anti-botulism phagemid.

) Increasing the enrichment ratios of phage ingested specifically by endocytosis The above results indicate that phage antibodies can be subjected to receptor-mediated endocytosis and remain infectious in a cell lysate. The selection of inpatient phage from a phage library requires the optimization of the method to increase the enrichment of phages interned specifically with respect to the non-interned phages. Two parameters can be improved: (1) reduction of non-specific or non-hospital phage recovery and (2) preservation of the infection capacity of the hospitalized phage. To examine these parameters, the phagemid of wild type C6.5 scFv was studied. This molecule was chosen because it is clearly ingested by endocytosis based on confocal microscopy, although the molecule underwent the lowest degree of specific endocytosis. The phagemid of scFv of C6.5 also represents the most commonly used format for the deployment of antibody libraries against non-immune phage (copy individual of plll in a phagemid vector) and has an affinity (16 nM) more typical of the Kd of scFv from such libraries than the affinity of mature C6ML3-9 scFv (Sheets et al. (1998) Proc. Nati Acad Sci USA 95: 6157-6162; Vaughan et al. (1996) Nature Biotech 14: 309-314). a) Reducing the non-hospital phage background To reduce the background of non-specific phage recovery, the effect of trypsin treatment on cells before lysis with TEA was studied. This should remove the phage trapped in the extracellular matrix. The trypsin treatment also dissociates the cells from the cell culture flask, allowing transfer to a new vessel and removal of any phage bound to the cell culture flask. For these experiments, phagemid of C6.5 scFv (5.0 x 10 8 cfu resistant to ampicillin) was mixed with a 1000-fold excess of wild type fd phage (5.0 x 10 11 cfu tetracycline resistant). After incubating the phagemid with SKBR-3 cells for two hours at 37 ° C, the cells were washed with PBS and three times with buffer for removal. The cells were then lysed directly with TEA or treated with trypsin, washed twice with PBS and then lysed with TEA. The phagemid of the first clearance wash and the cell lysate were titrated by infection of E coli TG1 and plated on ampicillin and tetracycline plates. The fd phage titer and the phagemid of scFv of C6.5 recovered from the cell surface were comparable for the two experimental groups (figure 3). The relationship of phage fd / phagemid of scFv of C6.5 in the fractions of the cell surface (160/1 and 250/1) gives an enrichment of 4 to 6 times that is achieved by specific binding to the cell surface from the initial relationship of 1000 times. Without trypsin treatment, the fd / phagemid phage ratio of scFv of C6.5 in the cell lysate is increased only 6.1 fold; in contrast, the ratio increases 209 times with trypsin treatment (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). b) Improving the recovery of infectious hospital phage To increase the recovery of infectious hospital phage, it was studied whether the prevention of lysosomal acidification through the use of chloroquine would protect or not the phages, ingested by endocytosis, of the endosomal degradation (Barry et al. (1996) Nat. Med. 2: 299-305). SKBR3 cells were incubated with chloroguin and either C6.5 scFv phagemid or anti-botulism phagemid. The cell phones were used at different times to determine the number of intracellular phagemids. The phagemids of scFv of C6.5 were presented at 20 minutes and the amount of phagemid was comparable with or without the addition of chloroquine. In later times, approximately twice as much infectious phagemid was recovered using chloroquine. In contrast, much smaller amounts of anti-botulism phage were present and chloroquine had no effect on the titer, which suggests that the phagemid results from non-specific binding to the surface instead of non-specific endocytosis in the endosomes. In general, the results indicate that the prevention of endosomal acidification increases the amount of infectious phage recovered for incubations lasting more than 20 minutes. 6) Recovery of phage hospitalized at low concentrations of phage Only very large antibody libraries against phages containing more than 5.0 x 109 elements are capable of generating panels of high affinity antibodies against all antibodies (10, 23, 24). Because phages can only be concentrated to approximately 1013 cfu / ml, a typical phage preparation from a large library will contain only 104 copies of each element. In this way, the selection of libraries for endocytosis will only work if the phages can be recovered when they are applied to the cells at titers as low as 104. Thus, the recovery of infectious phages within SKBR3 cells was determined as a function of the titer of applied phage SKBR3 cells were incubated with phagemids of scFv of C6.5, scFv of C6ML3-9 or diabody of C6.5 or with phage of scFv of C6.5 for 2 hours at 37 ° C. The cells were washed three times with buffer for removal, treated with trypsin and washed twice with PBS. The cells were lysed and the intracellular phage was titrated on E. coli TG1. Phage recovery is increased with increasing phage titer for all the phages that were studied (figure 5). For the antibodies displayed in monovalent form, the phagemids could not be recovered from the interior of the cell with entry titers less than 3.0 x 105 (scFv of C6.5) up to 3.0 x 106 (scFv of C6ML3-9). This threshold value decreased for bivalent and multivalent deployment (3.0 x 104 for the diabody phagemid of C6.5 and for the phage of scFv of C6.5).

C) Discussion It was demonstrated for the first time that phages displaying an antibody against anti-receptor can be ingested by endocytosis in a specific manner by means of cells that express the receptor and that can be recovered from the cytosol in an infectious manner. The results show that it is possible to directly select antibodies that are intemalized from rather large non-immune phage libraries and identify the factors that will lead to successful selections. Phage display antibody fragments specifically anti-ErbB2 ingested by endocytosis SKBR3 cells expressing ErbB2, can be visualized within the cytosol and can be recovered in an infectious form from within the cells. When the fragments of antibody against monovalent scFv are displayed in monovalent form in a phagemid system, the recovery of the phages interned was only 3.5 to 18 times above the background. The deployment of bivalent diabody or the multivalent deployment of scFv in a phage vector increased the recovery of phage interned up to 30 to 146 times above the background. This result is consistent with studies of C6.5 scFv Original monomeric and dimeric C6.5 diabody as well as studies of other monoclonal anti-ErbB2 antibodies, where dimeric IgG but not monomeric Fab is dimerizes and activates the receptor and is endocytosed (Yarden (1990) Proc Natl Acad Sci USA 87: 2569-2573; Hurwitz ef al (1995) Proc Natl Acad Sci USA 92:... 3353-3357....) . Indeed it is likely that endocytosis of C6.5 scFv phagemid and C6ML3-9 reflect the small percentage of phage display two or more scFv (Marks ef al (1992) J. Biol Chem. 267:.. 16007 -16010). The importance of valence to mediate either high avidity binding or receptor cross-linking and subsequent endocytosis is confirmed by the other report demonstrating specific phage endocytosis. Phage display about 300 copies of an Arg-Gly-Asp high affinity peptide that binds to integrin on pVIII were ingested efficiently by endocytosis by mammalian cells (Hart ef al (1994) J. Biol Chem 269...: 12468-12474). Phage recovery after endocytosis also increases the specificity of cell selections compared to phage recovery from the cell surface. These enrichment ratios for specific surface binding versus non-specific varied from 2 to 30 times. These values are comparable to the enrichment of approximately 10 times reported by others for a single cell surface selection round (Pereira et al. (1997) J. Immunol., Meth. 203: 11-24); Watters I went to. (1997) Immunotechno / ogy 3: 21-29). In contrast, the enrichment ratios for specific versus non-specific endocytosis ranged from 3.5 to 146 fold. Based on these results, the selection of antibodies for hospitalization from phage antibody libraries could be more successful with any of the homodimeric diabodies in a phagemid factor or a multivalent scFv using a phage vector. Although such libraries have not been published, there are no technical barriers that prevent their construction. The multivalent libraries will present the antibody fragment in the form that most likely crosses the receptor and can be subjected to endocytosis. Antibodies from such libraries will need to be bivalent in order to mediate endocytosis. In alternative form, the monomeric receptor ligands can activate the receptors and be subjected to endocytosis, either causing a conformational change in the receptor that favors the dimeric form or simultaneously uniting two receptors. The monomeric scFv that binds to the receptor in a similar manner could also be ingested by endocytosis. Therefore the selection of monovalent scFv libraries in a phagemid vector could result in the selection of ligand mimics that activate the receptors and that are ingested as monomers by endocytosis. Such scFvs could be especially useful for the construction of fusion molecules for the delivery of drugs, toxins or DNA in the cytoplasm. Such antibodies which mediate the admission of the receptor can cause deregulation of the receptor and inhibition of growth (Hurwits et al., (1995) Proc. Nati, Acad. Sci. USA 92: 3353-3357; Hudziak et al. (1989 ) Mol Cell Cell Biol. 9: 1165-1172 Stancovski et al. (1991) Proc. Nati Acad Sci USA 88: 8691-8698 Lewis et al. (1993) Cancer Immunol. Immunother. 37: 255- 263), the selection for antibodies that can be ingested by endocytosis can also identify antibodies that directly inhibit or modulate cell growth.

EXAMPLE 5 Transfection of the cells The gene for scFv of F5 was removed from pHEN1-F5 by digestion of the phagemid DNA with the restriction enzymes Sfil and Notl. A phage vector based on FdDOGI (see previous reference), but modified to insert a Sfil site in the leader sequence of gene III, was digested with Sdil and Notl and the gene for digested F5 was ligated into the DNA of the Fd vector of digested phage Recombinant transformants were identified. E. coli containing the recombinant phage F5 was grown in culture to produce the phage F5-Fd (see Maniatis for phage preparation). F5 phages were then used to infect E. coli harboring a phagemid which contains a mammalian promoter (CMV) followed either by the β-galactosidase gene (pcDNA3.1 / HisB / LacZ, Invitrogen) or the gene for the protein fluorescent green (pN2EGFP, Clonetech plasmid) and a sequence of eukaryotic polyadenylation. Bacteria were grown overnight in the presence of 15 ug / ml tetracycline and either 100 ug / ml ampicillin (bacteria containing pcDNA3.1 / HisB / LacZ) or 30 ug / ml kanamycin (bacteria that contain pN2EGFP). Phages prepared from the supernatant of a mixture of F5-Fd shells contain either a reporter gene (approximately 50% phage) in a single-stranded format or the phage F5-Fd genome (approximately 50 % of phages). Incubation of 5,105 positive SKBR3 cells for ErbB2 with 107 pfu of the phage mixture (filtered twice through a 0.45 nm filter to sterility) allowed expression of the reporter gene in 1% of the cells. The cells incubated with a control phage 10 times more negative, ie the reporter gene that is packaged in wild-type Fd, did not present expression of the reporter genes. In an experiment in which a mixed population of cells with high content of ErbB2 (SKBR) and with low content of ErbB2 (MCF7) (Lewis et al., (1993) Cancer Immunol. Immunother 37: 255-263) was incubated with the phages F5-Fd-EGFP during two days, the expression of the reporter gene was obtained only in the erbB2-positive cells, the cells being differentiated by their level of ErbB2 by FACS.

EXAMPLE 6 Provision of gene directed to mammalian cells by filamentous bacteriophage This example demonstrates that prokaryotic viruses can be genetically engineered to infect eukaryotic cells that result in the expression of a portion of the bacteriophage genome. Phages that can bind to mammalian cells expressing the ErbB2 receptor for growth factor and which are subjected to receptor-mediated endocytosis, were isolated by selection of a phage antibody library in breast cancer tumor cells and by recovery of infectious phages from inside the cell. As determined by immunofluorescence, F5 phages were ingested efficiently by endocytosis in SKBR3 cells expressing 100% ErbB2. To achieve the expression of a portion of the phage genome, the F5 phages were genetically engineered to package the reporter gene of the green fluorescent protein (GFP) controlled by the CMV promoter. When these phages are applied to the cells, they are subjected to ErbB2-mediated endocytosis which leads to the expression of GFP. GFP expression occurred only in cells that express ErbB2 in excess, is dose dependent, reaching 4% of the cells after 60 hours and was detected with phage titers as low as 2.0 x 107 cfu / ml (500 phages / cell). The results show that bacterial viruses which display the appropriate antibody can bind to the mammalian receptors and use the endocytic pathway to infect the eukaryotic cells which results in the viral expression of the gene. This represents a novel method for discovering target selection molecules that can deliver a gene intracellularly in the correct trafficking path for gene expression by directly selecting the phage antibodies. This should significantly facilitate the identification of appropriate targets and molecules to be targeted for gene therapy or other applications in which cytosol delivery is required. This method can also be adapted to directly select, rather than just select, phage antibodies for directed gene expression. The results also demonstrate the potential of phage antibodies as a vehicle for targeted delivery of the gene in vivo or in vitro.

A) Introduction The widespread application of gene therapy requires that a therapeutic gene can be targeted to the appropriate cell or tissue type with high efficiency (Michael and Curiel (1994) Gene Ther. 1: 223-232). It has been reported to target retroviral vectors by inserting ligands for the receptor or single chain Fv (scFv) antibody fragments into the viral coat protein (Kasahara et al (1994) Science 266: 1373-1376). It has been possible to select as target the adenoviral vectors through the use of fusion molecules "adapters" which consist of an antibody fragment which binds to the adenoviral protuberance and to a molecule that is directed to the cell such as a ligand for the receptor or an antibody (Douglas et al. (1996) Nat. Biotechnol. 14: 1574- 1578; Watkins et al. (1997) Gene Ther. 4 (10): 1004-1012). It has also been reported to target non-viral vectors using ligands or antibodies to the cell surface receptor (Fominaya and Wells (1996) J. Biol. Chem. 271 (18): 10560-10568; Michael and Curiel (1994). Gene Ther.1: 223-232). All of these methods depend on the use of white molecules which bind to the cell surface receptor which results in the delivery of the gene delivery vehicle with the subsequent supply of DNA to the nucleus. The identification of the appropriate target molecules has been largely developed by individually selecting the antibodies or ligands for the receptor. In the case of scFv antibody fragments this typically requires the construction of the scFv from the V genes of a hybridoma, the construction of a vehicle for targeted gene delivery, and the in vitro evaluation of the ability to select the target . More recently, it has been shown that it is possible to directly select peptides and antibody fragments that bind to cell surface receptors from filamentous phage libraries (Andersen et al. (1996) Proc. Nati. Acad. Sci. USA 93 (5): 1820-1824; Barry et al. (1996) Nat. Med. 2: 299-305; Caí and Garen (1995) Proc. Nati. Acad. Sci. USA 92 (24): 6537-6541; de Kruif e al. (1993) Proc. Nati. Acad. Sci.

USA 92 (6): 3938-3942; Marks e to al. (1993) Bio / Technology 11 (10): 1145-1149). This has led to a marked increase in the number of potential molecules for target selection. The ability of bacteriophages to undergo receptor-mediated endocytosis (Barry et al. (1996) Nat. Med. 2: 299-305; Hart et al. (1994) J. Biol. Chem. 269: 12468-12474) indicates that phage libraries can be selected not only for cell binding but also for entry into mammalian cells. If the single-stranded phage genome can be transcribed and translated, then it should prove that it is possible to separate or select phages that bind to the receptor in a way that leads to endocytosis and delivery of the phage genome on the trafficking route correct to lead to expression. It has previously been shown that the phage can enter mammalian cells after chemical alteration of the cell membrane which leads to the expression of the reporter gene (Okayama and Berg (1985) Mol. Cell. Biol. 5 (5): 1136-1142; Yokoyama-Kobayashi and Kato (1993) Biochem. Biophys. Res. Commun. 193 (2): 935-939). More recently, Larocca went to. demonstrated that indirect delivery of bacteriophage-mediated gene can be presented by targeting biotin-treated phage via streptavidin and fibroblast growth factor (FGF) treated with biotin to mammalian cells expressing the FGF receptor (Larocca et al. (1998) Hum). Gene Ther 9: 2393-2399). In this report, it is demonstrated that the filamentous phages that display the anti-ErbB2 F5 scFv as a genetic fusion with the protein plll of the minor coat of phage, they can directly infect mammalian cells that express ErbB2 which leads to the expression of a reporter gene contained in the phage genome. This offers a new way of discovering molecules for target selection for intracellular drug delivery or for gene therapy by directly selecting antibodies against phage to identify those that can be subjected to endocytosis and that can deliver a gene in intracellular form in the correct traffic route for the expression of the gene. This should significantly facilitate the identification of appropriate targets and molecules to select the target for gene therapy or other applications in which cytosol delivery is required. We also analyze how this method could be used to directly select anti-phage anti-genes for targeted gene expression. Finally, the potential use of phage antibodies by themselves for vectors for targeted delivery of the gene in vivo or in vitro is discussed.

B) Materials and methods 1) scfV of F5 anti-ErbB2 An anti-ErbB2 scFv (F5) was obtained in the vector pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res. 19 (15): 4133-4137 ) (pHEN-F5) by selection of an antibody library against non-immune phage (Sheets et al. (1998) Proc. Nati. Acad. Sci. USA 95 (11): 6157-6162) in cells SKBR3 expressing ErbB2 followed by the selection for binding in the Extracellular domain (ECD) of ErbB2 recombinate. The original F5 scFv binds to the ErbB2 ECD with a Kd = 1.6 x 10"7 M as determined by surface plasmon resonance in a BLAcore apparatus as previously described (Schier et al. (1996) J. Mol. S / 'or / .255 (1): 28-43) 5 2) Phage and phagemid vectors pcDNA3-GFP (6.1 kbp) was obtained by subcloning the Hind lll / Not l fragment from pN2EGFP (4.7 Kpb) (Clontech) in the base structure pcDNA3- HisB / LacZ (Invitrogen) HindIII / Not I. A phage vector was constructed fd-F5 subcloning the S // l / Not I scFv insert from pHEN-1 into the Sfi l / Not I sites of fd-Sfi l / Not I (constructed from fd-tet-DOG (Clakson et al. 1991) Nature 352 (6336): 624-628) by shifting the Apal cloning site in leader gene III to Sfil The phagemid vector pHEN-F5-GFP (6.8 Kpb) was obtained by subcloning the blunted Ase \ IAfl II fragment of pN2EGFP in the EcoR I site blunted by pHEN-F5. The orientation of the insert was analyzed by restriction digestion with Not I. 3) Culture and transfection of the cell line SKBR3 and MCF7 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) (Hyclone). SKF3 cells were transfected into 50% confluence cultured in 6-well plates with 1 μg of DNA per well using Lipofectamine (GIBCO BRL) following the manufacturer's instructions. The double DNA was prepared chain (sdDNA) of pN2EGFP by lysis in alkaline medium using the Maxiprep Qiagen Kit (Qiagen Inc.). The single chain DNA (ssDNA) was extracted from 500 μl of phagemid preparation (see below) by two phenol extractions followed by ethanol precipitation. The DNA was quantified by spectrophotometry with 1.0 A260 nm equal to 40 μg / ml for ssDNA or 50 μg / ml for dsDNA. To detect GFP, the cells were detached using a trypsin-EDTA mixture (GIBCO BRL) and analyzed in a FACScan (Becton Dickinson) apparatus. 4) Preparation of phage and phagemid Phagemids of pHEN-F5, pHEN-F5-GFP, pcDNA3-GFP or pN2EGFP from E. coli TG1 were prepared by superinfection with the auxiliary phage VCS-M13 (Stratagene) as described previously (Marks et al. (1991) J. Mol. Biol. 222 (3): 581-597). The Fd-F5 phages were prepared from E. coli TG1 as previously described (McCafferty et al (1990) Nature 348 (6301): 552-554). The phages F5-GFP and F5-LacZ were prepared by superinfection of E. coli TG1 containing pc-DNA3-GFP with the phage fd-F5. Viral particles were purified from the culture supernatant by 2 precipitations with polyethylene glycol (Sambrook et al., 1990), Molecular cloning- a laboratory manual, Cold Spring Harbor Laboratory, New York) were resuspended in phosphate buffered saline, pH 7.4 (PBS), filtered through a 45 μm filter and stored at 4 ° C. . Alternatively, the preparations were sent to an additional step of ultracentrifugation with CsCl (Smith and Scott (1993) Meth, Enzymol, 217: 228-257). The ratio of auxiliary phage DNA packaged against phagemid DNA was determined by titering the phage (Sambroock et al., Supra) for resistance to ampicillin and kanamycin (for pHEN-F5 rescued by helper phage) or for resistance ampicillin and tetracycline (for pcDNA3-GFP rescued by phage fd-F5).

) FACS analysis for phage The cells were treated with trypsin, washed with PBS containing 1% FBS (regulatory solution for FACS) and resuspended at a concentration of 10 5 cells / ml in the same buffer. The staining procedure was performed on ice using reagents diluted in FACS buffer. Aliquots of 100 μl cells were distributed in a conical 96-well plate (Nunc), centrifuged at 300 g and the cell pellets were resuspended in 100 μl of serial dilutions of the phage or phagemid preparation and incubated for 1 hour. The cells were centrifuged and washed twice, the cell pellets were resuspended in 100 μl of anti-M13 antibody (5 Prime, 3 Prime Inc.) (diluted 1/400) and incubated for 45 minutes. The cells were washed as indicated above, resuspended in 100 μl streptavidin-phycoerythrin (Jackson Inc.) (diluted 1/400) and incubated for 20 minutes. After a final wash, the cells were analyzed by FACS. 6) Immunofluorescence The cells were grown on cover slips up to 50% confluence in 6-well plates. The 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 minutes with glycine buffer (50 mM glycine, pH 2.8, 500 mM NaCl) were neutralized with PBS and fixed with PBS-4% paraformaldehyde for 5 minutes at room temperature. The cells were permeabilized with cold acetone for 30 seconds, saturated with PBS-1% BSA and incubated with anti-M13 antibody (diluted 1/300 in the saturation solution) followed by streptavidin-Texas red (Amersham) ( diluted 1/300 in the saturation solution). The coverslips were analyzed with an Axioskop fluorescent microscope (Zeiss). 7) Bacteriophage-mediated cell infection Phage preparations with CsCl were diluted at least 10-fold in the cell culture medium, filtered through a 0.45 μm filter and added to cells with 30% up to 80% of confluence. After incubation, the cells were treated with trypsin, washed with regulatory solution for FACS and analyzed for GFP expression by FACS. In the experiments in which MCF7 and SKBR3 cells were co-cultured, the expression of ErbB2 was quantified by FACS using the 4D5 of mouse anti-ErbB2 mAb which binds to the ErbB2 ECD (10 μg / ml) (1 hour), sheep anti-mouse immunoglobulins treated with biotin (Amersham) and streptavidin-phycoerythrin.

C) Results 1) Entry of monovalent and multivalent F5 phage particles that bind to ErbB2 by cells expressing ErbB2 The anti-ErbB2 scFv-F5 was isolated from a scFv library displayed on the surface of bacteriophages as fusions to plll (Sheets et al. (1998) Proc. Nati. Acad. Sci. USA 95 (11): 6157-6162) by selection in SKBR3 cells of breast tumor expressing ErbB2 and by recovery of infectious phage from inside of the cell (M. Poul et al., report in preparation). The selection strategy was used to select the scFv that could be subjected to endocytosis after binding to the receptor. When the phagemid vector pHEN-F5 is rescued with the auxiliary phage VCS-M13, the resulting virus particles (phagemid-F5) display an average of 1 copy of scFv-fusion protein and 3 to 4 copies of the protein plll of the wild type of the lower coat from the auxiliary phage (Marks et al. (1992) J. Biol. Chem. 267 (23): 16007-16010). As a result, the phagemid binds monovalently. To improve the binding of the virus particles to the cells expressing ErbB2, multivalent antibodies against phage were created by subcloning the F5 scFv DNA into the fd-Sfi / Not phage vector so that it fuses with the pIII protein. The virus particles, to which it refers as phage fg-F5, they display 4 to 5 copies of scFv-fusion protein pIII (Id.). To determine whether antibodies against phage F5 can be internalized or not by mammalian cells, SKBR3 cells expressing ErbB2 in excess were incubated for 16 hours with phage fd-F5 (109 colony forming units / ml, cfu / ml) , with phagemid F5 (1011 cfu / ml), or with phagemids displaying a scFv-irrelevant anti-botulism plll fusion protein (1012 cfu / ml) (Amersdorfer et al., 1997) as a negative control. The cell surface was stripped of phage antibodies using a glycine buffer solution of low pH. In an outstanding manner, all the cells showed strong intracellular staining when they were incubated with phage fd-F5 or with phagemid of F5 but not when they were incubated with the anti-botulism phagemid. This demonstrates the dependence of the phage entry on the specific character of the scFv fused to pIII. 2) Preparation of phages and phagemids that bind to ErbB2 that pack a reporter gene for expression in eukaryotic cells Two strategies were used to investigate whether phage F5 could or could not supply a reporter gene to mammalian cells leading to the expression. To prepare monovalent phages containing a reporter gene, the gene for the green fluorescent protein (GFP) controlled by the CMV promoter was cloned into the phagemid vector pHEN-F5, whereby the vector pHEN-F5-GFP is generated ( figure 6, left panel). It got infected Escherichia coli TG1 containing pHEN-F5-GFP (resistant to ampicillin) with the auxiliary phage (resistant to kanamycin) and high titers of phagemids F5-GFP (5.0 x 1010 cfu ampicillin resistant / ml of culture supernatant) were obtained . It was determined that the ratio of phagemid DNA packaged against helper phage DNA (cfu resistant to ampicillin vs cfu kanamycin resistant) is 100: 1. To prepare multivalent phages containing a reporter gene, phages fd-F5-GFP were generated by infecting E. coli TG1 carrying the phagemid pcDNA3-GFP (resistant to ampicillin) with phage fd-F5 (resistant to tetracycline), using in this way to phage fd-F5 as an auxiliary phage. The phage fd-F5-GFP titer was approximately 5.0 x 108 cfu ampicillin resistant / ml culture supernatant. Low phage titers are obtained when fd is used as an auxiliary phage because it lacks a plasmidic replicon origin that leads to interference from the phagemid f1 origin (Cleary and Ray (1980) Proc. Nati. Acad. Sci USA 77 (8): 4638-4642). The ratio of reporter gene DNA packed against phage DNA (cfu resistant to ampicillin vs. cfu resistant to tetracycline) was 1: 1. The lowest ratio of reporter gene / helper genome when fd is used as an auxiliary phage is due to the presence of a fully functional packaging signal in the fd genome compared to the mutated packaging signal in VCS-M13 (Vieira and Messing (1987) Meth. Enzymol. 153: 3-11). Both phage and phagemid preparations were evaluated for binding to SKBR3 cells (Figure 7). Although both preparations bound to the SKBR3 cells, the binding did not it could be detected with an amount as low as 108 cfu / ml cfu / ml phage fd-F5-GFP (160 fentomolar) compared to 1010 cfu / ml phagemids F5-GFP (15 picomolar). Therefore the multivalent binding leads to an increase in the apparent binding constant of the virus particles. 3) Phagemid and phage-mediated gene transfer directed into breast cancer cells expressing ErbB2 To determine whether phagemids that bind to ErbB2 are capable of targeted gene delivery, 2.0 x 10 5 SKBR3 cells were incubated (a cell line of breast tumor expressing high levels of ErbB2) or 2.0 x 10 5 MCF7 cells (a breast tumor cell line expressing low levels of ErbB2) with 5.0 x 10 11 cfu / ml phagemids F5-GFp at 37 ° C. The cells were analyzed for GFP expression by FACS after 48 hours (Figure 8A). 1.37% of the SKBR3 cells expressed to GFP after incubation with the phagemids F5-GFP (Figure 8A6). The expression of GFP was identical without considering the orientation of the packaging signal f1 (data not shown), which indicates that the transcription / translation proceeds via the synthesis of the complementary strand of DNA. GFP expression was not detected in SKBR3 cells incubated without phage or with auxiliary phage packaging the reporter gene (Figures 8A4 and 8A5). Neither was the expression obscured in MCF7 cells incubated without phage, helper phage or pHEN-F5_GFP, indicating the requirement for ErbB2 expression for the targeted delivery of genes (FIGS. 8 A1, 8 A2 and 4 A3). Due that it is likely that the transfer applications involve the selection of specific cell targets in a heterogeneous population of cells, the same experiments were performed in a co-culture of SKBR3 and MCF7 cells (Figure 8B). The cells were stained for the expression of ErbB2 to discriminate MCF7 cells from SKBR3 cells and the GFP expression of each subpopulation was analyzed by FACS. Only SKBR3 cells (1.91%) expressed GFP. Similar results were found using F5-FGP phages instead of phagemids F5-GFP (data not shown). These data confirm that the delivery of genes mediated by phage fd-F5-GFP and phage F5-GFP is restricted to cells that express excess ErbB2 and can be directed to such cells in the presence of cells that do not express it. 4) Characterization of phage-mediated gene transfer To determine the dose-response characteristics of phage-mediated gene transfer, SKBR3 cells were incubated for 60 hours with increasing amounts of phage fd-F5-GFP or phagemid F5-GFP and the percentage of GFP-positive cells was determined (Figures 9A and 9B). The minimum phage concentration required for the detection of a significant number of GFP-positive cells (figure 9A) was approximately 4.0 x 107 cfu / ml for phage fd-F5-GFP (0.13%) and 1.0 x 1010 cfu / ml for phagemid F5-GFP (0.12%). The values are correlated closely within the binding curves (Figure 7) and indicate that multivalent phages are 100 to 1000 times more efficient than phagemids in terms of gene expression. A significant number of positive cells with up to 4.0 x 1013 cfu / ml of helper phage packaging the reporter gene were not observed. For both the phage and the phagemid, the percentage of GFP-positive cells increased with the phage concentration without evidence of a plateau. The maximum values achieved were 2% of cells for phage fd-F5-GFP and 4% for phagemids F5-GFP and appear to be limited by the applied phage titer (1.5 x 109 cfu / ml and 4.0 x 1012 cfu / ml respectively). The amount of GFP expressed per cell (estimated by mean fluorescent intensity (MFI), (Figure 9B) was also increased with phage concentration, with a small number of cells expressing phage titers as low as 2.0 x 107 cfu / ml (phage fd-F5-GFP) up to 1.0 x 1010 cfu / ml (phagemid F5-GFP) To compare the performance of phage gene expression with traditional transfection methods, single-stranded DNA was transfected ( ssDNA) or double-chain (dsDNA) in SKBR3 cells using lipofectamine The efficiency of genes mediated by phagemid, per μg of ssDNA, (approximately 1%) was comparable to the transfection of ssDNA with lipofectamine (0.98%) and dsDNA (1.27%) (Table 8) The efficiency was approximately 500 times higher for phage-mediated transfection, with 2.25 ng of ssDNA resulting in the transfection of 0.87% of the cells.

TABLE 8 Efficiency in transfections in SKBR3 cells Transfection method Reporter plasmid ... ,. % of cells positive to GFP r plasmid reporter r 15 ug 3.84 Mediated by phagemid F5 pHEN-Fd-GFP 3.1 μg 1.44 0.78 μg 0.64 5 ng 169 Mediated by phage fd-F5 pcDNA3-GFP 2.25 ng 0.87 1.25 ng 0.57 100 μg 0.12 Mediate by auxiliary phage pN2GFP 20 μg 0.07 5 ug 0.06 Lipofectamine pN2GFP i μg 1.27 i μg 0.98 * Cells were analyzed 48 hours after transfection for GFP expression using FACS. The results are expressed in% of GFP-positive cells. ** For phage, the amount of reporter plasmid was calculated from the size of the plasmid and the number of ampicillin resistant colonies (pHEN-F5-GFP or pcDNA3-GFP) or kanamycin (pN2GFP). Cells transfected with mimic contained an average of 0.05% of GFP-positive cells. To determine the time course of gene expression, 5.0 x 1011 cfu / ml of phagemid F5-GFP were incubated with SKBR3 cells. After 48 hours, the culture medium was replaced with fresh medium. Cells expressing GFP can be detected within 24 hours after applying the phage and the percentage of positive cells increases linearly with the increase in time to a maximum 4.5% in 120 hours (Figure 9C). The GFP content of the positive cells, as estimated by the MFI, is increased up to 96 hours (Figure 9D). After 96 hours, the number of GFP-positive cells continues to increase, but the MFI decreases, probably because the molecules are distributed to the daughter cells during cell division.

C) Discussion It has been shown that filamentous phages displaying an anti-ErbB2 scFv antibody fragment as a genetic fusion with the plll protein of the minor coat can be directed directly to mammalian cells expressing the specific character of the scFv. Because the phages can be subjected to receptor-mediated endocytosis and enter an intracellular trafficking path which ultimately leads to the expression of the reporter gene. This is an outstanding discovery that procaryotic viruses can be genetically redesigned to infect eukaryotic cells that result in the expression of a portion of the bacteriophage genome. Gene expression was detected with an amount as small as 2.0 x 107 cfu phage and increased with increasing phage titers up to 4% of the cells. Multivalent display reduced the threshold value for detectable gene expression by approximately 500 fold compared to the monovalent display, most likely due to an increase in functional affinity and an increased rate of receptor-mediated endocytosis from the cross-linking of the receiver. However, the maximum percentage of transfected cells was higher for the monovalent (phagemid) display due to the significantly higher generated phage titer. The lowest titer of the multivalent phage is due to the interference of the f1 origin of the replicon in the phagemid reporter with the origin of the anti-cough replicon against phage fd (Cleary and Ray (1980) Proc. Nati. Acad. Sci. USA 77 (8 ): 4638-4642). It is likely that targeted infection of mammalian cells using phages that bind to receptors that can be ingested by endocytosis is a general phenomenon. For example, fusing an anti-transferrin receptor scFv to gene III of pHEN-GFP results in the expression of GFP in 10% of MCF7 cells, in 4% of SKBR3 cells, in 1% of cells LNCap and in 1% of the primary melanone cells. Similarly, the delivery of the gene for GFP directed towards cells expressing the FGF receptor using phage treated with biotin and a streptavidin-FGF fusion molecule (Larocca et al. (1988) Hum. Gene Ther. 2393-2399). However, direct gene fusion of the molecule for target selection via gene III could be more efficient than using adapter molecules. Thus, although the maximum percentage of transfected cells was not reported using FGF-adapter molecule, it is estimated that this is only 0.03% of rat L6 myoblasts that express FGF based on the number of infected cells, the time from infection to the measurement of gene expression and the number of cells expressing GFP. Although observed a higher frequency of expression (0.5%) in COS-1 cells, this results from the presence of large T antigen and the SV40-mediated DNA replicon and this can not be generalized to most cells. The method described represents a novel method for discovering ligands for targeted intracellular delivery of genes or drugs. First, phage antibody or peptide libraries for endocytosis are selected by mammalian cells (Barry et al (1996) Nat. Med. 2: 299-305) or for binding to the purified antigen, to cells, tissues or organs. After subcloning the genes for scFv selected in the vector pHEN-GFP, phage produced from individual colonies can be screened directly for gene expression. This is possible because the expression can be detected with quantities as low as 1.0 x 1010 cfu of phagemids. This allows not only the direct identification of scFv ingested by endocytosis but also the identification of the subset of antibodies against the receptor that can be subjected to the correct traffic for the expression of the gene. If multivalent deployment is needed for efficient endocytosis, the genes for scFv can be subcloned into fd-Sfi-Not which are then used to rescue the phagemid reporter. The use of the scFv-fd phage also allows targeting a large number of different reporter genes in various expression vectors since many commercially available mammalian vectors contain replicon origins. As such, the phage directed with antibody could prove to be a useful transfection reagent, especially for cells difficult to transfect by standard techniques. It could also be shown that it is possible to use this method to directly select, rather than just select, the antibodies for targeted gene delivery. For example, mammalian cells are incubated with a phage antibody library containing the gene for GFP, and are then classified based on the expression of GFP using FACS. The phage antibody DNA could 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 amounts of recoverable phage DNA are inadequate, inclusion of the gene for neomycin in the vector pHEN-GFP could allow the selection of mammalian cells expressing GFP using G418 (Larocca et al., Supra). Finally, this system is promising as a vehicle for in vitro or in vivo gene targeting therapy. The main limitations are infection efficiency, pharmacokinetics and immunogenicity. With respect to infection efficiency, the values achieved by the phage addressed in this report (8.0 x 104 / ml of phage preparation) are not unequal to the values reported for the targeted retrovirus (103-105 / ml of virus) ( Kasahara et al. (1994) Science 266: 1373-1376; Somia et al. (1995) Proc. Nati. Acad. Sci. USA 92 (16): 7570-7574) but lower than those reported for strategies to select as white to adenoviruses (Douglas et al. (1996) Nat. Biotechnol. 14: 1574-1578; Watkins et al. (1997) Gene Ther. 4 (10): 1004-1012). However, the factors that limit the highest infection efficiencies are likely to differ between systems. Thus, although the percentage of cells infected by retroviruses is significantly higher than that observed for bacteriophages, the infection is limited by the problems encountered in producing large numbers of viruses that can enter the cell. Because all cells ingest the targeted phage, gene expression is limited by one or several post-ingestion events (eg, phage degradation to release DNA, endosomal escape, target selection or transcription) . It is likely that more detailed studies of the fate of the phage and its DNA suggest where the blocks are that allow the genetic manipulation of the phages to increase the efficiency of infection. For example, endosomal escape could be increased by co-administration of adenovirus with defects in the replicon (Curiel et al (1991) Proc. Nati, Acad. Sci. USA 88 (19): 8850-8854) or by incorporating peptides for endosomal escape (Wagner et al. (1992) Proc. Nati. Acad. Sci. USA 89 (17): 7934-7938) or proteins (Fominaya and Wells (1996) J. Biol. Chem. 271 (18): 10560- 10568) in the pVIII protein of the main phage coat. Alternatively, the infection efficiency can be increased combinatorially by creating scFv-directed libraries of the pVIII mutants and selecting for increased gene expression. With respect to pharmacokinetics, although it has not been studied extensively, it is likely that the biodistribution of the phage is limited to the intravascular space. This would not affect in vitro phage gene therapy, but could limit in vivo uses to those that target vascular tissue. This however leaves numerous applications including those in which neovascularization plays a role, such as in cancer. With respect to immunogenicity, the phage is likely to be immunogenic, thus limiting the number of times the phage could be administered in vivo. Alternatively, it could be shown that it is possible to develop the pVIII protein from the main envelope to reduce or eliminate the immunogenic character by, for example, selecting negatively a pVIII library in immune serum (Jenne et al., 1998) J. Immunol. 161 (6): 3161-3168). It is understood that the examples and embodiments described in the present invention are for illustrative purposes only and that various modifications or changes will be suggested in light thereof to those skilled in the art and that these should be included within the scope and anticipation of this. application and within the scope of the appended claims. All publications, patents and patent applications cited in the present invention are therefore incorporated for reference in their entirety for all purposes.

Claims (50)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for selecting polypeptide or antibody binding portions that are incorporated into target cells, said method comprising: i) contacting one or more of said target cells with one or more elements of a phage display library; ii) contacting the elements of said phage display library with cells of a subtractive cell line; iii) washing said target cells to remove said cells from a subtractive cell line and removing the elements of said phage display library that do not bind specifically or weakly bind said target cells; iv) culturing said target cells under conditions wherein the elements of said phage display library can be incorporated if they bind to an incorporable marker; and v) identifying the incorporated elements of said phage display library are incorporated in one or more of said target cells.

2. The method according to claim 1, further characterized in that said phage display library is a library of antibody phage display.

3. The method according to claim 2, further characterized in that said antibody phage display library displays Fv regions of single chain antibodies.

4. The method according to claim 1, further characterized in that said identification comprises recovering the incorporated phage and repeating the steps between (i) to (v) to further select the incorporable connection portions.

5. The method according to claim 4, further characterized in that said recovery comprises: (a) lysing said target cells to release the incorporated phage; and (b) infecting a bacterial host with said incorporated phage to produce the phage for a subsequent round of selection.

6. The method according to claim 4, further characterized in that said recovery comprises recovering nucleic acids encoding the antibody displayed by phage.

7. The method according to claim 1, further characterized in that said intensification comprises detecting the expression of a reporter gene or a selectable marker.

8. The method according to claim 1, further characterized in that said cells of a subtractive cell line are present at least twice the excess of said target cells.

9. - The method according to claim 1, further characterized in that said target cells form an adherent layer in said method.

10. The method according to claim 1, further characterized in that step (ii) is developed at a lower temperature than step (iv).

11. The method according to claim 1, further characterized in that step (i) is developed at about 4 ° C and step (iv) is developed at about 37 ° C.

12. The method according to claim 1, further characterized in that said phage expresses a selectable marker.

13. The method according to claim 12, further characterized in that said selectable marker is selected from the group consisting of a fluorescent protein, an antibiotic resistance gene and a chromagenic gene.

14. The library according to claim 13, further characterized in that said chromogenic gene is selected from the group consisting of horseradish peroxidase, b-lactamase, luciferase and b-galactosidase.

15. The method according to claim 1, further characterized in that said target cells are selected from the group consisting of solid tumor cells, elements of a cDNA expression library, cells overexpressing a cytokine receptor, cells that overexpress a growth factor receptor, metastatic cells, cells of a transformed cell line, cells transformed with a gene or cDNA that codes for a target specific surface receptor, and neoplastic cells derived from the outside of a solid tumor.

16. The method according to claim 1, further characterized in that said cells of a subtractive cell line are selected from the same type of tissue as those of the target cells.

17. The method according to claim 1, further characterized in that said cells of a subtractive cell line are selected from the group consisting of fibroblasts, monocytes, stem cells, and lymphocytes.

18. A method for identifying an incorporable receptor, said method comprises: i) contacting one or more of said target cells with one or more elements of a phage display library; ii) contacting the elements of said phage display library with cells of a subtractive cell line; iii) washing said target cells to remove said cells from a subtractive cell line and removing the elements of said phage display library that do not bind specifically or weakly bind said target cells; iv) culturing said target cells under conditions wherein the elements of said phage display library can be incorporated if they bind to an inexpressible marker; v) identify the incorporated elements of said phage display library are incorporated in one or more of said target cells and vi) contacting the same or different target cells with the identified incorporated elements of step (v) or the propagated elements thereof, wherein said elements are attached to the surface of said target cells same or different.

19. The method according to claim 18, further characterized in that it comprises isolating a component of the same or different target cells to which said elements are attached.

20. The method according to claim 18, further characterized in that said phage display library is a library of antibody phage display.

21. The method according to claim 20, further characterized in that said antibody phage display library displays Fv versions of single chain antibodies.

22. The method according to claim 18, further characterized in that said identification comprises recovering the incorporated phage and repeating steps (i) to (v) to further select the incorporable connection portions.

23. The method according to claim 22, further characterized in that said recovery comprises: (a) lysing said target cells to release the incorporated phage; and (b) infecting a bacterial host with said incorporated phage to produce the phage for a subsequent round of selection.

24. - The method according to claim 18, further characterized in that said cells of a subtractive cell line are present at least twice the excess of said target cells.

25. The method according to claim 18, further characterized in that said target cells form an adherent layer in said method.

26. The method according to claim 18, further characterized in that step (ii) is developed at a lower temperature than step (iv).

27. The method according to claim 18, further characterized in that step (ii) is developed at approximately 4 ° C and step (iv) is developed at approximately 37 ° C.

28. The method according to claim 18, further characterized in that said cells of a subtractive cell line are selected from the same type of tissue as those of the target cells.

29. The method according to claim 1, further characterized in that said target cells are selected from the group consisting of solid tumor cells, elements of a cDNA expression library, cells overexpressing a cytokine receptor, cells overexpressing a growth factor receptor, metastatic cells, cells of a transformed cell line, cells transformed with a gene or cDNA encoding a target specific surface receptor, and neoplastic cells derived from the exterior of a solid tumor.

30. - A multivalent antibody phage display library, said library comprises a diversity of phages wherein said phage display, on average, at least two copies of a single chain antibody and said library comprises a variety of species of chain antibodies simple.

31. The library according to claim 30, further characterized in that said phage display, on average, at least four copies of a single chain antibody.

32. The library according to claim 30, further characterized in that said library comprises at least 105 different species of single chain antibodies.

33. The library according to claim 30, further characterized in that said antibodies are encoded by a nucleic acid that is a phage vector.

34. The library according to claim 30, further characterized in that said library is selected from elements that specifically bind to an incorporable cell surface receptor.

35. The library according to claim 34, further characterized in that said cell surface receptor is selected from the group consisting of erB2, EGF receptor, PDGF receptor, VEGF receptor, and transferrin receptor.

36. - The library according to claim 30, further characterized in that said single chain antibodies are single chain Fv or single chain FAB antibodies.

37. The library according to claim 30 further characterized in that said phage is a filamentous phage.

38.- The library according to claim 30, further characterized in that said antibodies are expressed as a fusion with a lower layer protein Plll.

39. The library according to claim 30, further characterized in that said phage expresses a selectable marker.

40. The library according to claim 30, further characterized in that said selectable label is selected from the group consisting of a fluorescent protein, an antibiotic resistance gene, and a chromagenic gene.

41. The library according to claim 40, further characterized in that said chromagenic gene is selected from the group consisting of horseradish peroxidase, blactamase, luciferaza and b-galactosidase.

42.- A library of nucleic acids encoding an antibody library, said library comprises a variety of phage vectors, wherein said library encodes a variety of single chain antibodies.

43. - The reivinidcation 42, further characterized in that said library comprises at least 105 different phage vectors.

44. The library according to claim 42, further characterized in that said single chain antibodies are a single chain Fv (scFv) or a single chain Fab (scFab).

45. The library according to claim 42, further characterized in that said phage vectors further comprise a selectable marker.

46. The library according to claim 42, further characterized in that said library is selected for the elements that encode antibodies that specifically bind to an incorporable cell surface receptor.

47. The library according to claim 46, further characterized in that said receptor is selected from the group consisting of erB2, EGF receptor, PDGF receptor, VEGF receptor, and transferrin receptor.

48. A device comprising one or more containers comprising one or more containers containing a polyvalent phage display antibody library.

49. The equipment according to claim 48, further characterized in that said phage display library contains a variety of phage vectors encoding single chain antibodies in fusion with a viral coat protein.

50. - The equipment according to claim 48, further characterized in that said phage display library contains a diversity of phage particles deployed, on average, in at least two copies of an antibody per phage particle.