WO2013149237A1 - Targeting intracellular organelle zip codes with functional homing ligands from cell-internalizing phage combinatorial libraries - Google Patents

Abstract

The present invention concerns compositions and methods related to organelle targeting in mammalian cells. In at least certain embodiments, there are bacteriophage that are modified to comprise a cell penetrating moiety, for example as part of a recombinant pVIII major coat protein. In some embodiments, the phage include intracellular homing peptides that target specific organelles, and in certain embodiments the peptides comprise apoptosis-inducing activity and/or cytoskeletal organization modification activity.

Description

DESCRIPTION

TARGETING INTRACELLULAR ORGANELLE ZIP CODES WITH FUNCTIONAL HOMING LIGANDS FROM CELL-INTERNALIZING PHAGE COMBINATORIAL

LIBRARIES The present application claims the priority benefit of United States provisional application number 61/618,008, filed March 30, 2012, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0001] The field of the invention at least generally includes cell biology, molecular biology, biotechnology, and medicine. In at least certain embodiments the field of the invention includes phage display and cell death methods and compositions

BACKGROUND OF THE INVENTION

[0002] For more than a decade, in vivo phage display has allowed identification of novel peptide ligands that home selectively to blood vessels of normal organs or tissues and tumors (Pasqualini et ah, 1996; Arap et ah, 1998). Biochemical approaches such as affinity chromatography or genetic approaches such as similarity mining of online databases have subsequently identified the corresponding organ- and tissue-selective cell surface receptors for such ligands, discoveries which in turn have revealed novel roles for known proteins and identified additional proteins differentially expressed in the vasculature of normal and pathologic tissues (Arap et ah, 2002; Staquicini et ah, 2009; Staquicini et ah, 201 1). These endothelial surface receptors regulate sophisticated vascular functions as either tissue-specific or angiogenesis-related "ZIP codes."

[0003] This combinatorial selection methodology proved also to be versatile when applied to the cell surface; indeed, the biological diversity at the cell membrane level can be probed even when cells have been removed from their usual tissue architecture. For example, by the use of a single step-phase-separation technique (Giordano et ah, 2001), we have ligand- profiled the membrane receptor diversity of the NCI collection of 60 human tumor cell lines and have classified these lines based on expression of their putative tumor cell surface receptor proteome (Kolonin et al, 2006). Moreover, from a screen on white adipose tissue-derived stem cells, we have identified integrin a5bl as a receptor for SPARC (Nie et al, 2008), characterized a novel ternary complex involving netrin-4 that controls the migration and proliferation of neural stem cells (Staquicini et al, 2009), discovered ligand motifs that target the macropinocytotic pathway in leukemia cells (Nishimura et al, 2008), and defined a supramolecular complex, the "leukemia cell invadosome," which is essential for the pericellular proteolysis and migratory capacity of acute myelogenous leukemia cells (Stefanidakis et al, 2009).

[0004] Recently, the inventors have generated prototype drugs for the vascular endothelial growth factor and epidermal growth factor pathways (Giordano et al, 2010; Cardo- Vila et al, 2010), and have developed phage-based hydrogels for three-dimensional tissue culture based on magnetic cell levitation (Souza et al, 2010).

[0005] Whereas phage library screening in vitro and in vivo has consistently uncovered novel protein-protein interactions at the cell surface, the technology has not yet enabled direct and straightforward targeting of intracellular organelles and signal transduction or metabolic pathways. Currently, intracellular ligand-directed delivery can be accomplished by supercharged proteins or cell-penetrating peptides that induce receptor-independent crossing of eukaryotic cell membranes; such peptides are usually arginine-rich cationic sequences (McNaughton et al, 2009; Cunha et al, 2008; Teesalu et al, 2009; Olson et al, 2010; Nguyen et al, 2010). One of the best-characterized of these peptides is penetratin (pen), a motif derived from the third helix of the homeodomain of Drosophila antennapedia protein (Joliot et al, 1991 ; Derossi et al, 1994; Derossi et al, 1996). The mechanism of pen internalization is an energy- dependent and lipid raft-mediated endocytic uptake (Letoha et al, 2005). Pen has previously been used to transport peptides, recombinant proteins, antibodies, small interfering RNA, and micro RNA into cells (Theodore et al, 1995; Chen et al, 1999; Cardo-Vila et al, 2003; Cantelmo et al, 2010; Mi et al, 2000; Apostolopoulos et al, 2006; Jain et al, 2005; Muratovska et al, 2004; Davidson et al, 2004; Fabani et al, 2008). Reports that certain cell- penetrating peptides such as pen or HIV-Tat improve cellular uptake of eukaryotic viruses (such as adenoviruses), and enable delivery of prokaryotic viral particles (such as λ phage) into mammalian cells (Gratton et al, 2003; Eguchi et al, 2001) stimulated us to determine whether combinatorial phage display methodology could be adapted for the direct analysis of intracellular protein-protein interactions and to discover internalizing homing peptides (iHoPe, comprising of a homing peptide and an internalizing peptide sequence) motifs affecting metabolic pathways. Here the inventors introduce a new class of filamentous phage-based reagents that integrate pen (Derossi et ah, 1998) as a fusion protein with the recombinant major coat protein (rpVIII) and thereby enable receptor-independent phage particle entry into mammalian cells. Moreover, either random peptide libraries or specific individual motifs can be simultaneously displayed on the minor coat protein (pill), a feature allowing both intracellular library selection and targeting of organelles. The inventors have termed this new family of reagents "internalizing phage" (iPhage). In the present invention, there is provided an intracellular targeted peptide that would induce programmed cell death as a biological surrogate readout and a general selection approach. The present invention includes internal homing peptides that induce cell death via ribosomal protein L29 (RPL29); therefore, the present invention provides internalizing combinatorial peptide selection. Embodiments of the invention encompass use of the compositions described herein in disease-specific settings.

BRIEF SUMMARY OF THE INVENTION [0006] The present invention is directed to methods and compositions that concern organelle targeting in mammalian cells. In specific embodiments, the compositions are bacteriophage that are modified to express a cell penetrating peptide, for example one that is combined with a major coat protein, such as pVIII. In some embodiments, the bacteriophage also comprise peptides with organelle-specific localization activity. In specific embodiments, the bacteriophage comprise intracellular homing peptides that are linked with, such as covalently attached with, a therapeutic entity and/or diagnostic entity (such diagnostic entity, for example, can be a detectable composition). In specific aspects, the bacteriophage possess two homing peptides, one for specific tissue/cell targeting-receptor mediated internalization and the other for intracellular localization. In some embodiments, the bacteriophage is an adeno-associated virus and phage (AAVP) possessing two homing peptides: one for specific tissue/cell targeting- receptor mediated internalization and the other for intracellular localization, with transgene delivery capabilities. In some embodiments, there are bacteriophage comprising a non-specific cell penetrating peptide; bacteriophage comprising a non-specific cell penetrating peptide and an additional intracellular homing peptide; bacteriophage comprising a non-specific cell penetrating peptide and a diagnostic and/or therapeutic entity (including siRNA, for example); compositions that lack bacteriophage but comprise nonspecific penetrating peptides coupled to intracellular homing peptide and optionally comprising a diagnostic and/or therapeutic entity (including siRNA); compositions that lack bacteriophage but comprise a specific tissue/cell targeting peptide (e.g., RGD-4C, NGR, Crkl-targeting, ILl lRa-targeting, Prohibitin-targeting sequence) coupled to intracellular homing peptide and optionally comprising a diagnostic and/or therapeutic entity (including siRNA); compositions that lack bacteriophage but comprise a specific tissue/cell targeting moiety (e.g., Ab to HER2) coupled to intracellular homing peptide and optionally comprising a diagnostic and/or therapeutic entity (including siRNA).

[0007] Phage display library selection is an unbiased approach to discover and/or to study protein-protein interactions; however, such methodology has not as yet been extended to uncover intracellular targets and pathways within intact live cells, and the present invention provides a solution for this need. In embodiments of the invention, there is a new family of peptide-targeted internalizing-phage (termed "iPhage") libraries and individual clones, which cross eukaryotic cell membranes and home to specific cellular compartments. In at least some embodiments, the display of the antennapedia-derived peptide penetratin on the viral envelope mediates a receptor- independent uptake of bacteriophage particles by mammalian cells. Targeting either nuclei or mitochondria through bifunctional constructs, each containing a pIII- displayed organelle-specific signal localization peptide as well as pVIII-displayed penetratin, was successful. The inventors also screened cells with an iPhage random peptide library to select and isolate internalizing homing peptides (termed "iHoPe", referring to the combination of an "internalizing peptide" and intracellular homing peptide), followed with biochemical characterization of its receptors (which may be referred to as an organelle ZIP Code).

[0008] In certain embodiments of the invention there are peptides that each comprise or consist of a selected targeted motif chemically fused to penetratin, undergo receptor-independent internalization, are functionally active, and recapitulate the observed cellular phenotypes, such as, for example, apoptosis induction and cytoskeletal organization changes. This technology enables ligand-directed combinatorial targeting of cell organelles or intracellular signaling pathways. Many applications for biological or medical discovery include but are not limited to organelle-specific cell biology and pharmacology, for example.

[0009] In some embodiments of the invention, there is provided a new class of filamentous bacteriophage-based viruses that integrate recombinant penetratin (for example) as a fusion protein on a major coat protein (pVIII, for example) thereby enabling receptor- independent viral particle entry and intracellular distribution into mammalian cells. Moreover, either random peptide libraries or specific individual motifs can be simultaneously displayed on the minor coat protein (pill), thus allowing compartment-selection and organelle-targeting. The reagents may be referred to herein as "internalizing phage" (iPhage). The inventors demonstrate subcellular homing of iPhage displaying defined signal peptide sequences to mammalian cell nuclei or mitochondria. Next, the inventors screened cells with iPhage displaying a random peptide library to select and isolate internalizing homing peptides (iHoPe), followed with biochemical identification and validation of their corresponding receptors. Finally, the inventors show that two exemplary iHoPes— each comprising the selected targeted motif chemically fused to penetratin— specifically undergo receptor-independent internalization are functionally active and recapitulate the observed cellular phenotypes mediated by the originally selected iPhage such as apoptosis-induction and cytoskeletal organization changes. Taken together, these results show that this integrated technology platform enables direct ligand-directed combinatorial selection and targeting of cell organelles or intracellular signaling pathways.

[0010] Studies with iPhage libraries have identified peptides which, when delivered intracellularly, modulate cytoskeletal organization and activate apoptosis, indicating that many such functionally relevant peptides can be discovered with this new platform. In some embodiments, one can combine receptor-targeting peptides, which provide tissue selectivity, with intracellular bioactive peptides discovered by iPhage; such constructs are useful to modulate cell function in a tissue- and organ-specific fashion following specific delivery. The approach and molecular tools provided in the invention are useful to target intracellular ZIP codes, interrogate signal transduction pathways, and enable the foundation of a new organelle- targeted cell biology and pharmacology in eukatyotic cells. [0011] Over the past two decades, phage selection in vitro and in vivo has consistently uncovered novel cell-surface biology, in the form of unrecognized functions for known proteins, novel multi-protein complexes, or targetable expression patterns in pathologic settings. In embodiments of the present invention, intracellular processes and pathways can be similarly interrogated with iPhage-based technology. In fact, the inventors found that two of the initially identified peptides have produced striking morphological and functional phenotypes after delivery to the intracellular microenvironment. Embodiments of the invention provide iPhage, a novel family of phage constructs capable of penetrating eukaryotic cells, and thereby extend phage- or AA VP -based technology to the analysis of intracellular pathways. iPhage have identified peptides that, when delivered intracellularly, modulate cytoskeletal organization and activate apoptosis; many such functionally relevant peptides can be discovered with this new platform. It is possible to combine receptor-targeting peptides, which provide tissue selectivity, with intracellular bioactive peptides discovered by iPhage; after specific delivery such constructs are useful to modulate cell function in a tissue- and organ-specific fashion. The new resources introduced here can target intracellular ZIP codes, interrogate signal transduction pathways, and perhaps ultimately enable the foundation of an organelle-targeted cell biology and pharmacology in eukaryotic cells. The products derived by using the iPhage technology would be the identification of bioactive peptides that can alter intracellular signaling leading to apoptosis, cellular proliferation, autophagy, migration or differentiation, as examples. Other exemplary applications include the generation of intracellular zip codes, where peptides will home to specific organelles, these peptides can be coupled with diagnostic or imaging or therapeutic agents, nucleic acid, or any other desirable molecule, for example. It will be appreciated that molecules within the scope of the present invention include virtually any molecule that may be attached to a targeting peptide and administered to a subject for investigative research purposes, diagnostic applications, or for modification of a organelle-specific disease.

[0012] The skilled artisan will appreciate that the scope of the claimed methods of use include any non-disease or disease state. In some embodiments, such diseases may be impacted by intracellular targeted delivery of a therapeutic agent and/or diagnostic agent to a desired tissue and/or cell type. Such disease states include, but are not limited to, organelle- related diseases (e.g., lysosomal storage, mitochondrial, ER misfolding diseases), vascular disease, obesity, and cancer, including non-metastatic cancer, among others. Exemplary lysosomal storage diseases include Adrenoleukodystrophy, Activator Deficiency/GM2 Gangliosidosis, Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, cystinosis, Danon disease, Fabry or Farber disease, fucosidosis, galactosialidosis, Gaucher Disease, GM1 gangliosidosis, Mucoloipidosis, infantile Free Sialic Acid Storage Disease/ISSD, Juvenile Hexosaminidase A Deficiency, Krabbe disease, Lysosomal acid lipase deficiency, Mucopolysaccharidoses disorders, Niemann-Pick Disease, Pompe disease/Glycogen storage disease type II, Pycnodysostosis, Sandhoff disease, Schindler disease, Salla disease, Tay-Sachs disease, and Wolman disease. Exemplary mitochondrial diseases include diabetes mellitus, deafness, diabetes mellitus and deafness, Leber's hereditary optic neuropathy, Wolff-Parkinson- White syndrome, Leigh syndrome, subacute sclerosing encephalopathy, Neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP), Myoneurogenic gastrointestinal encephalopathy (MNGIE), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), and Mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS). Exemplary endoplasmic reticulum stress-related diseases include cardiovascular diseases, such as atherosclerosis, and ischemic diseases.

[0013] The iPhage screenings can be extended to isolate intracellular homing peptide and bioactive peptides not only from human cells, but also from mouse or other eukaryotic cells.

[0014] In embodiments of the invention, peptidesthat target a specific tissue or cell are combined with intracellular bioactive peptides discovered by iPhage such that the composition will provide specific delivery and have the activity to modulate cell function in a tissue- and organ-specific fashion. Compositions of the invention can target intracellular ZIP codes and interrogate signal transduction pathways or selectively deliver agents (including therapeutic, diagnostic and/or imaging agents) to the specific intracellular compartment, and, thus, enable the foundation of an organelle-targeted cell biology and pharmacology in eukaryotic cells. Such agents include but are not limited to drugs, antibodies, polynucleotides, gene therapy vectors and/or fusion proteins.

[0015] In certain embodiments, an isolated peptide is attached to a molecule, such as an active agent including a therapeutic agent, imaging agent, and/or diagnostic agent. In some embodiments, the attachment is a covalent attachment. In some embodiments, the agent is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, a survival factor, an anti-apoptotic factor, a hormone antagonist, an imaging agent, a nucleic acid (e.g., DNA, RNA, siRNA, miRNA, or antisense RNA), a lipid, or an antigen. It will be appreciated that molecules within the scope of the present invention include virtually any molecule that may be attached to a targeting peptide and administered to a subject. [0016] In some embodiments, the intracellular targeting agent comprises a targeting agent linked with an active agent, such as a diagnostic agent and/or a therapeutic agent. For example, the active agent may comprise a nucleic acid agent, such as DNA, RNA, or a combination thereof. In some embodiments the agent comprises siRNA, miRNA, or antisense RNA.

[0017] In some embodiments, the active agent is or comprises an imaging agent, including a radioisotope, for example. Exemplary radioisotopes include, but are not limited to, "Cu, ^mIn ²¹³Bi, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, ¹¹⁷Sn, ¹⁸⁶Re, ¹⁶⁶Ho and ¹⁸⁸Re. Other examples of imaging agents include one or more of an enzyme, a fluorescent label, a near infrared label, a luminescent label, a bioluminescent label, a magnetic label, and biotin.

[0018] In some embodiments of the invention, the active agent is or comprises an antibody agent. In some embodiments, the antibody agent is or comprises a monoclonal antibody, a polyclonal antibody, an Fc portion, an Fab, an ScFv, and a single domain antibody. [0019] In some embodiments of the invention, there are isolated bacteriophage, comprising a recombinant protein comprising a major coat protein and a cell-penetrating peptide. In specific aspects, the recombinant protein further comprises an intracellular homing peptide. In specific cases, the intracellular homing peptide comprises SEQ ID NO:3 or one of SEQ ID NO: 12-31 and in some cases the intracellular homing peptide is 20 amino acids or less and comprises SEQ ID NO:3 or one of SEQ ID NO: 12-31. In specific embodiments, the major coat protein comprises pVIII. In specific embodiments, the peptides of any one of SEQ ID NO:3 or SEQ ID NO: 12-31 are able to target an entity to one or more particular intracellular locations. In specific cases, SEQ ID NO:3 targets an entity to a mitochondria or endoplasmic reticulum. In certain cases, SEQ ID NO: 12-31 target an entity to the cytoskeleton. [0020] In some embodiments of the invention, there is a method of producing phage having cell penetrating activity and intracellular localization activity, said method comprising the steps of modifying a phage to produce a recombinant protein comprising a major coat protein and a cell-penetrating peptide to produce a modified phage; introducing to a plurality of the modified phage a library of constructs, said constructs comprising randomized peptides inserted within a sequence of the minor pill coat protein to produce test phage; and assaying the test phage for intracellular localization activity. In certain embodiments, the randomized peptides comprise a X₄YX₄ motif, wherein X and Y represent non-identical amino acids.

[0021] In some embodiments of the invention, there is an isolated bacteriophage, comprising a recombinant protein comprising a major coat protein and a cell-penetrating peptide, such as penetratin. In some cases of the bacteriophage embodiments, the major coat protein comprises pVIII. In further aspects of the invention, a bacteriophage of any kind of the invention further comprises an intracellular homing peptide, such as one that selectively binds an organelle. In specific embodiments, the intracellular homing peptide has cell death activity, adhesion activity, and/or migration activity and may or may not comprise SEQ ID NO:3 or SEQ ID NO: 12, or both. The bacteriophage may be configured to comprise a recombinant protein comprising a minor coat protein (such as pill) and an intracellular homing peptide. In some cases of the intracellular homing peptide, the peptide is 50, 40, 30, 20, or 10 amino acids or less and may comprise SEQ ID NO:3 or SEQ ID NO: 12. In certain embodiments of the bacteriophage, the phage further comprises a therapeutic agent, imaging agent, and/or diagnostic agent.

[0022] In some embodiments, there is a method of producing phage having cell penetrating activity and intracellular localization activity, said method comprising the steps of modifying a phage to produce a recombinant protein comprising a major coat protein and a cell- penetrating peptide to produce a modified phage; introducing to a plurality of the modified phage a library of constructs, said constructs comprising randomized peptides (which may be linear or cyclic, for example) inserted within a sequence of the minor pill coat protein to produce test phage; and assaying the test phage for intracellular localization activity.

[0023] In some embodiments, there is an isolated peptide of 50 amino acids or less that selectively binds an intracellular organelle, wherein the isolated peptide comprises a peptide sequence of SEQ ID NO:3 or SEQ ID NO: 12. The isolated peptide may be operatively coupled to a cell penetrating peptide and/or to a specific tissue/cell targeting moiety, and the specific tissue/cell targeting moiety may comprise a peptide or an antibody. The isolated peptide may further comprise a therapeutic agent, an imaging agent, a diagnostic agent, or a combination thereof. The therapeutic agent, imaging agent, or diagnostic agent may be a drug, small molecule, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a cytotoxic agent, a cytocidal agent, a cytostatic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, a hormone antagonist, a nucleic acid (such as one that comprises siR A, shR A, miRNA, or antisense R A ) or an antigen.

[0024] Exemplary anti-angiogenic agents may be selected from the group consisting of thrombospondin, angiostatin5, pigment epithelium-derived factor, angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP- 10, Gro-B, thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM 101, Marimastat, pentosan polysulphate, angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, Docetaxel, polyamines, a proteasome inhibitor, a kinase inhibitor, a signaling peptide, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline. Exemplary pro-apoptosis agents may be selected from the group consisting of etoposide, ceramide sphingomyelin, Bax, Bid, Bik, Bad, caspase-3, caspase-8, caspase-9, fas, fas ligand, fadd, fap-1, tradd, faf, rip, reaper, apoptin, interleukin-2 converting enzyme or annexin V. Exemplary cytokines may be selected from the group consisting of interleukin 1 (IL-1), IL-2, IL- 5, IL-10, IL-1 1, IL-12, IL-18, interferon- (IF- ), IF- , IF-B, tumor necrosis factor- (TNF- ), or GM-CSF (granulocyte macrophage colony stimulating factor).

[0025] In some aspects of the invention, there is a diagnostic agent that is selected from the group consisting of an enzyme, a fluorescent label, a near infrared label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In certain aspects of the invention, there is an imaging agent selected from the group consisting of an enzyme, a fluorescent label, a near infrared label, a luminescent label, a bioluminescent label, a magnetic label, and biotin.

[0026] In certain embodiments, there is employed one or more radioisotopes selected from the group consisting of 64Cu, l l lln 213Bi, 103Pd, 133Xe, 1311, 68Ge, 57Co, 65Zn, 85Sr, 32P, 35S, 90Y, 153Sm, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 1 13Sn, 1 17Sn, 186Re, 166Ho and 188Re. [0027] In some embodiments of the invention, any peptide of the invention is attached to or is part of a molecular complex, such as a virus (including one selected from the group consisting of adenovirus, retrovirus and adeno-associated virus), a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a yeast cell, a mammalian cell or a cell. In some embodiments, a virus is further defined as containing a gene therapy vector. Peptides of the invention may have attached thereto a eukaryotic expression vector, including a gene therapy vector. In some embodiments of the invention, there is a pharmaceutical composition comprising a peptide of the invention.

[0028] In some embodiments of the invention, there is a method of targeting an entity to an organelle in a cell by providing to the cell a composition comprising the entity and a peptide comprising SEQ ID NO:3, wherein the organelle is the mitochondria or endoplasmic reticulum. The composition may further comprise a cell penetrating peptide. The composition may be further defined as comprising a specific tissue/cell targeting moiety. The entity may comprise a therapeutic agent, imaging agent, or diagnostic agent. The composition may be further defined as being a bacteriophage.

[0029] In some embodiments, there is a method of targeting an entity to an organelle in a cell by providing to the cell a composition comprising the entity and a peptide comprising at least one of SEQ ID NOS: 12-31, wherein the organelle is the cytoskeleton. The composition may further comprise a cell penetrating peptide. The composition may be further defined as comprising a specific tissue/cell targeting moiety, and it also may be further defined as being a bacteriophage. In some aspects, the entity comprises a therapeutic agent, imaging agent, or diagnostic agent.

[0030] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing, in which:

[0032] FIG. 1. Display of penetratin on rpVIII mediates receptor-independent cell internalization, (a) Upper: parental f88-4 phage vector contains two capsid genes encoding a wild-type (wt) protein VIII (pVIII, depicted in gray) and a recombinant protein VIII (rpVIII; depicted in green). The recombinant gene VIII contains a foreign DNA insert with a Hindlll and a Pstl cloning site (depicted in red). TetR, tetracycline resistance gene. Lower: representation of the assembled phage particle expressing only the wt major coat protein pVIII (gray); pill, minor coat protein (orange); pVI protein (blue); pVII protein (red), and pIX protein (yellow), (b) Upper: Annealed oligonucleotides encoding the penetratin (pen) peptide were cloned in frame with the recombinant gene VIII. Lower: iPhage particles displaying the pen peptide motif (RQIKIWFQNRRMKWKK; SEQ ID NO: 1) at the amino terminus of the rpVIII (green), (c) Upper: The mutant iPhage genome has nucleotide substitutions in the pen sequence replacing tryptophan (W) by alanine (A) residues (underlined). Lower: Representation of the assembled mutant iPhage displaying mutant pen on rpVIII protein (purple), (d) Immunofluorescence of KS 1767 cells shows internalized viral particles only in cells incubated with iPhage. The nuclear stain DAPI emits blue fluorescence, and internalized phage particles were detected with conjugated antibodies (red fluorescence). Scale bar, 100 μιη. (e) Phage genomic DNA was detected only in KS1767 cells incubated with iPhage particles (Southern blot, upper panel). Total genomic DNA stained with ethidium bromide served as a loading control (lower panel), (f) iPhage particles are internalized and viable in the cytosol and undetected in the membrane fraction of KS1767 cells. Bars represent mean values for TU recovered from the cytosol ± standard error of the mean (SEM), from triplicates. Phage internalization by various types of cultured cells (g) mouse and (h) human. RMA (Rauscher murine leukemia virus antigen) lymphoma, LLC (Lewis lung carcinoma), B 16F 10 (Melanoma), HUVEC (human umbilical vein endothelial cell), leukemia (K562), HEK (human embryonic kidney) cell lines. Internalization assays were run in triplicate; bars represent mean values for phage TU recovered from the cytosol enriched fraction ± SEM, from triplicates.

[0033] FIG. 2. Systematic approach for mammalian organelle targeting, (a) Organelle homing and random peptide iPhage library selection flow diagram, (b) Phage displaying the mitochondrial localization signal of cytochrome c oxidase (MLS-iPhage) in the pill protein is enriched in the mitochondria/ER fraction. Cytosol and nuclear fractions served as internalizing and negative controls, respectively. Bars represent mean values for TU recovered from the mitochondria/ER fraction ± SEM, from triplicates, (c) Confocal fluorescence microscopy analysis of KS 1767 cells exposed to parental phage, iPhage and MLS-iPhage. Intracellular localization was revealed by anti-rabbit Alexa Fluor 488, orange-Mitotracker, and DAPI counterstaining. Scale Bar, 10 μιη. Arrows indicate signal co-localization.

[0034] FIG. 3. Synchronous selection of a random peptide iPhage library is enriched in the mitochondria/ER fraction, (a) Systematic approach of selection in live KS 1767 cells, (b) Phage enrichment after three rounds of selection. Bars represent mean values for phage TU recovered from the mitochondria/ER enriched fraction ± SEM, from triplicates, (c) Bright- field microscopy of KS 1767 cells infected with different peptide-iPhage clones selected from the mitochondrial/ER fraction. Scale bar, 100 μιη. (d, e) Cell viability was measured by WST-1 and MTT assays respectively. Bars represent mean values of cell viability ± SEM, from triplicates. The symbol * indicates significant reduction of cell viability (P < 0.02).

[0035] FIG. 4. The internalizing YKWYYRGAA (SEQ ID NO:3) peptide activates cell death via ribosomal protein L29. (a) Phage binding assay on fractions from YKWYYRGAA (SEQ ID NO:3)affinity chromatography. Fractions were immobilized in a 96-well plate. Insertless iPhage and BSA were used as negative controls. Phage binding assays were run in triplicate; bars represent mean values for phage TU recovered from immobilized proteins ± SEM, from triplicates, (b) RPL29 was identified by mass spectrometry. Amino acids in yellow represent the peptides detected by mass spectrometry, (c) Phage binding to immobilized GST- RPL29 and GST-RPL30 fusion proteins. BSA or GST alone were used as negative controls. Bars represent mean values for phage TU recovered from immobilized proteins ± SEM, from triplicates, (d) Binding of the YKWYYRGAA(SEQ ID NO:3)-phage to RPL29 was inhibited with the corresponding synthetic peptide. Bars represent mean ± SEM, from triplicates, (e) Cell viability is reduced upon exposure of YKWYYRGAA-pen peptide, relative to complete media, penetratin, and YKWYYRGAA (SEQ ID NO:3) peptide (1, 3, 10, and 30 μΜ). The symbol * indicates significant reduction of cell viability (P < 0.001). The MTT assay was performed in triplicate. Bars represent mean ± SEM. (f) Internalization of YKWYYRGAA (SEQ ID NO:3) peptide induces mammalian cell death. FACS of annexin V-FITC-positive cells (apoptotic cells). KS 1767 cells were treated with peptides at 30 μΜ for 6 h at 37°C. Bars represent mean ± SEM, from triplicates, (g) Annexin V-FITC binds to translocated phophatidylserine on the plasma membrane of cells treated with YKWYYRGAA(SEQ ID NO:3)-pen. Control, pen, and YKWYYRGAA(SEQ ID NO:3)-treated cells did not translocate the apoptotic marker. Scale bar, 100 μιη. (h) The internalized YKWYYRGAA (SEQ ID NO:3) peptide activates the caspases in the human cell line KS 1767. Immunoblotting shows cleavage of caspase-7 (Casp 7) and -9 (Casp 9). Actin served as a loading control. Arrows indicate the caspase fragments, (i) Histone- associated DNA fragments are detected only in cells treated with YKWYYRGAA(SEQ ID NO:3)-pen peptide. DNA fragmentation was detected with the Cell Death Detection ELISA plus kit (Roche). Bars represent mean ± SEM. (j) Beclin-1 is upregulated after YKWYYRGAA(SEQ ID NO:3)-pen peptide treatment. Beclin 1, autophagy related protein (atg) 5, and Atg-7 expression were analyzed by Western blot. Actin served as a loading control, (k) YKWYYRGAA(SEQ ID NO:3)-pen induced the translocation of the high mobility group protein Bl (HMGB1) into the extracellular environment. KS1767 cells were treated with different peptides at 30 μΜ or with media as indicated. (1) Normal mitochondria change to vesicular form, and the matrix swells (red arrows). Chromatin condensation and fragmentation (red asterisk) are observed only in KS1767 cells exposed to YKWYYRGAA(SEQ ID NO:3)- pen. Normal mitochondria (white arrows) and nuclei (white asterisks) are observed in control untreated, pen-treated, and YKWYYRGAA(SEQ ID NO:3)-treated cells. Scale bar, 500 nm.

[0036] FIG. 5. iPhage internalization was not inhibited with penetratin (pen) peptide in different mouse and human cell types. Cells were preincubated with pen peptide (10, 30, 100 μΜ), and incubated with phage particles for 16 h at 37°C. Bars represent mean values for phage transducing units (TU) recovered from the cytosol fraction ± SEM, from triplicates. Central bars are results of experiments with the iPhage.

[0037] FIG. 6. iPhage particles were distributed evenly in the cytosol. The KS1767 cells were incubated with phage, iPhage and mutant iPhage for 24 h at 37°C. Intracellular distribution of phage particles within the cytosol were detected by using anti-phage (green fluorescence), and anti-organelle (red fluorescence) antibodies. Subcellular markers used: antiearly endosome antigen- 1 (EEA1), anti-mannose 6 phosphate receptor [M6PR, (late endosome)], anti-58K (Golgi apparatus), and anti-ERAB [hydroxysteroid (17-beta) dehydrogenase 10; mitochondria]. DAPI counterstain nucleus (blue fluorescence). Scale bar, 10 μιη.

[0038] FIG. 7. Confocal fluorescence microscopy analysis of KS 1767 cells exposed to parental phage, iPhage and iMLS-phage. Intracellular localization was revealed by anti-rabbit Alexa Fluor 488, Orange-Mitotracker (200 nM/ 25 min), and DAPI counterstain. Scale bar, 10 μιη. Arrows indicate signal co-localization. [0039] FIG. 8. Colocalization index analysis of MLS-iPhage versus controls.

Images were analyzed as a complete z-stack series with the Olympus FV10-ASW V31 co- localization software. The fluorograms compared the colocalization of the Phage-FITC (Channel 2) on the x-axis with the mitotracker-RFP (Channel 3) on the Y-axis. Confocal images of MLS- iPhage (a), iPhage (c), phage (e) and fluorograms respectively (b, d, f). The co-localization index for this data indicates 10- and 1000-fold more co-localization in the MLS-iPhage compared to iPhage and Phage respectively.

[0040] FIG. 9. Targeting frequency of YKWYYRGAA (SEQ ID NO:3)/or MLS- iPhage clone versus iPhage particles in the mitochondria fraction. The YKWYYRGAA(SEQ ID NO:3)-iPhage (a) and MLS-iPhage (b) clones were enriched in the mitochondria/ER fraction when they were mixed to equal or 10,000-fold more iPhage particles. KS 1767 cells were superimposed with different admixtures of phage particles overni ght at 37°C [i.e., 10⁵ TU of iPhage + 10⁹ TU of MLS-iPhage (1 : 10,000), 10⁷ TU of iPhage + 10⁷ TU of MLS-iPhage (1 : 1), 10⁹ TU of iPhage + 10⁵ TU of MLS-iPhage (10,000: 1). Mitochondria/ER fractions were isolated for phage recovery by K91 bacteria infection. The pie charts represent the percentage (%) frequency of sequences randomly selected from single bacteria colonies. [0041] FIG. 10. The MLS and YKWYYRGAA (SEQ ID NO:3) iPhage clones were enriched in the mitochondria/ER fraction after 24 h post-incubation, (a) Plasma membrane (left bars), nucleus (right bars) and mitochondria/ER fractions (center bars) were isolated either immediately or 24 h post-incubation with phage particles, (b) Cytosol fraction was used as a control of phage viability and internalization for immediate and 24 h post-incubation respectively. Bars represent mean values for TU recovered from the subcellular fractions ± SEM, from triplicates.

[0042] FIG. 11. Cell viability was reduced upon exposure of YKWYYRGAA(SEQ ID NO:3)-pen, relative to controls (1, 3, 10 and 30 μΜ). Cell death was not detected in non- conjugated admixtures of pen plus YKWYYRGAA peptides. The MTT assay was performed in triplicate. Bars represent mean ± SEM.

[0043] FIG. 12. Induction of cell death in different murine and human cell types upon exposure to YKWYYRGAA(SEQ ID NO:3)-pen peptide. Cells were plated on a 96-well plate and were exposed to each peptide (1, 3, 10, 30 and 100 μΜ) for 6 hours at 37°C. The MTT assay was performed in triplicate. Bars represent mean ± SEM.

[0044] FIG. 13. Electron microscopy analysis of KS1767 cells untreated (media) and treated with peptides at different time points. The cells treated only with the YKWYYRGAA(SEQ ID NO:3)-pen peptide (30 μΜ) suffered ultrastructural alterations as early 30 min; whereas the nucleus (N) is still intact and present a defined nucleoli, the plasma membrane and the cytoplasm were extensively dismantled (red arrows) compared to cells treated with peptide controls (30 μΜ) or media. DNA fragmentation and total organelle disruption was observed at 4h. Scale bar 2 μιη.

[0045] FIG. 14. Ultrastructural analysis of intracellular compartments by transmission electron microscopy. Normal-vesicular (black arrow) to swollen mitochondria (red arrow) structures were only observed (0.5 - 4h) in KS 1767 cells treated with YKWYYRGAA(SEQ ID NO:3)-pen peptide (30 μΜ). Normal mitochondria were observed in KS 1767 cells treated with control peptides (30 μΜ) and media. Scale bar 500 nm.

[0046] FIG. 15. Receptor purification of the peptide-ligand LGRFYAASG (SEQ ID NO: 12). A, phage binding assay on fractions from LGRFYAASG (SEQ ID NO: 12) affinity chromatography. Fractions were immobilized in a 96-well plate. Insertless iPhage and BSA were used as negative controls. B, protein candidates eluted from fraction 46 were analyzed by mass spectrometry. Peptides sequences from LIM and SH protein, F-actin capping protein and annexin A2. C, bioinformatic protein interaction analysis of molecules eluted from the LGRFYAASG (SEQ ID NO: 12) peptide column. We used the STRING protein network prediction program to determine the protein-protein interactions.

[0047] FIG. 16. Annexin A2 is the receptor for the internalizing LGRFYAASG (SEQ ID NO: 12) peptide. A, iPhage displaying the LGRFYAASG (SEQ ID NO: 12) peptide binds specifically to annexin A2 (ANXA2). LASP-1, and CAPZAl were receptor candidates identified by mass spectrometry. GST and BSA served as negative controls. B, LGRFYAASG(SEQ ID NO: 12)-iPhage binds with higher specificity to ANXA2 in comparison to other annexin family members. Phage binding assays were performed as described in A. C, concentration-dependent inhibition of LGRFYAASG(SEQ ID NO: 12)-iPhage to ANXA2 by the cognate synthetic peptide. iPhage displaying LGRFYAASG (SEQ ID NO: 12) were incubated in wells coated with ANXA2 in the presence of increasing concentrations of LGRFYAASG (SEQ ID NO: 12) or control peptide. D, alanine scanning of LGRFYAASG (SEQ ID NO: 12) displayed on the iPhage revealed that arginine (R) and phenylalanine (F) are critical residues for binding to ANXA2. All Phage binding assays mentioned above were run in triplicate; bars represent mean values for phage TU recovered from immobilized proteins ± SEM, from triplicates. [0048] FIG. 17. Internalization of LGRFYAASG (SEQ ID NO: 12) peptide impairs cell adhesion. A, reduced cell F-actin protrusions in KS 1767 cells treated with LGRFYAASG(SEQ ID NO: 12)-pen peptide (red arrows), compared to normal F-actin cytoskeleton structures (white arrows). Cell were treated with 30 mM of each peptide and incubated overnight. Confocal images of fixed cells stained with phalloidin-FITC (Green fluorescence) and DAPI (Blue fluorescence). Scale bar 25 mm. B, concentration-dependent inhibition of cell adhesion of KS1767 cells exposed to LGRFYAASG(SEQ ID NO: 12)-pen. BSA and complete media served as negative and positive controls, respectively. Cells were pre- incubated with peptides; after 30 min at 37°C, the cells were added to plates coated with fibronectin or vitronectin. Bars represent mean ± SEM of triplicate experiments. [0049] FIG. 18. LGRFYAASG(SEQ ID NO: 12)-pen impairs cellular migration in scratch (wound)-covering assay. A, KS 1767 cells exposed to LGRFYAASG(SEQ ID NO: 12)- pen do not close over the abraded area, in comparison to controls after 16 ours. Cells were exposed to each peptide (30 μΜ), and control received only complete media. Scale Bar, 1 mm. B, dose-dependent peptide inhibition of wound closure assay using control (first bars); pen (second bars); LGRFYAASG(SEQ ID NO: 12)-pen (third bars); and LGRFYAASG (SEQ ID NO: 12; fourth bars). KS1767 cells were exposed at different peptide concentrations for 16 h at 37°C. C, KS 1767 cells proliferate normally at different peptide concentrations (1, 3, and 30 mM) for 96 hours. Bars represent mean ± SEM, from triplicates.

[0050] FIG. 19. A Scheme of lung metastasis model. B16F 10 cells were preloaded with different peptides (30 mM) and intravenously administrated. After 21 days, lungs were surgically removed and analyzed for lung mircometastasis.

[0051] FIG. 20. Internalizing LGRFYAASG (SEQ ID NO: 12) synthetic peptide disruptions lung metastasis. A, lung weights are shown as mean ± SEM (*, P < 0.01). B, representative lung photos, scale bar 5 mm. C, metastatic foci were counted on lungs of wt and null mice, (*, P < 0.0001). D, Formalin fixed lungs were sectioned and H&E-stained. Black arrows indicate lung metastatic foci. Scale bar 100 mm.

[0052] FIG. 21. A, screening strategy of the cytosol using the peptide iPhage display library. B, Selection of an iPhage display peptide library from cytosol fraction produce serial enrichment.

[0053] FIG. 22. Eluted control (fraction 22) and targeted (fraction 46) fractions were analyzed by SDS-PAGE, and proteins were visualized by staining with Coomassie blue. Arrows indicate proteins extracted for mass spectrometry analysis.

[0054] FIG. 23. Adhesion assays with various types of cells. Inhibition of cell adhesion exposed to LGRFYAASG(SEQ ID NO: 12)-penetratin. BSA and complete media served as negative and positive controls, respectively. Cells were pre-incubated with peptides; after 30 minutes at 37°C, the cells were added to plates coated with fibronectin (black bars) or vitronectin (white bars). LGRFYAASG (SEQ ID NO: 12) without penetratin conjugation; pen, penetratin peptide only. Bars represent mean ± SEM of triplicate experiments. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

I. Definitions

[0055] As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claim(s), in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more of an item.

[0056] An "intracellular homing peptide" is a term that encompasses various peptides that may be used to enhance the intracellular localization of a moiety to a particular location in an animal, including organs, tissues, particular cell types, diseased tissues or tumors. Intracellular homing peptides may include peptides, peptide mimetics, and fragments thereof. In certain embodiments, an intracellular homing peptide will enhance the localization of a substance to desired intracellular location. Selective localization intracellularly may be determined, for example, by methods disclosed herein, and in specific embodiments the intracellular homing peptide sequence is incorporated into a protein that is displayed on the outer surface of a phage.

[0057] A "cell-penetrating peptide" in certain embodiments comprises a peptide, as well as variants and fragments thereof, that facilitates receptor-independent uptake of bacteriophage particles by mammalian cells. An example of a cell-penetrating peptide is penetratin. [0058] A "phage display library" refers to a collection of phage that have been genetically engineered to allow identification of intracellular homing peptides. One of skill in the art recognizes that any phage display library may be employed so long as the library can detect protein-protein interactions, including peptide libraries and antibody fragment libraries. In particular embodiments, DNA sequences encoding the putative intracellular homing peptides are inserted in frame into a gene encoding a phage capsule protein. In other preferred embodiments, the putative targeting peptide sequences are in part random mixtures of all twenty amino acids and in part non-random. In certain preferred embodiments the putative targeting peptides of the phage display library exhibit one or more cysteine residues at fixed locations within the targeting peptide sequence. Cysteines may be used, for example, to create a cyclic peptide, which may provide some structure to portions of the peptide. In some embodiments, the intracellular homing peptides comprise a X₄YX₄ motif.

[0059] A "receptor" for a targeting peptide includes but is not limited to any molecule or molecular complex that binds to a targeting peptide. Non-limiting examples of receptors include peptides, proteins, glycoproteins, lipoproteins, epitopes, lipids, carbohydrates, multi-molecular structures, DNA, RNA, microRNA, ncRNA, and specific conformation of one or more molecules. In preferred embodiments, a "receptor" is a naturally occurring molecule or complex of molecules that is present on the lumenal surface of cells forming blood vessels within a target organ, tissue or cell type. [0060] A "targeting moiety" is a term that encompasses various types of affinity reagents that may be used to enhance the localization or binding of a substance to a particular location in an animal, including organs, tissues, particular cell types, diseased tissues or tumors. Targeting moieties may include peptides, peptide mimetics, polypeptides, antibodies, antibodylike molecules, nucleic acids, aptamers, and fragments thereof. In certain embodiments, a targeting moiety will enhance the localization of a substance to organelles, for example. Selective binding of a targeting moiety of the present invention, e.g., an targeting peptide or antibody, as well as variants and fragments thereof is when the targeting moiety binds a target and does not significantly bind to unrelated proteins. A targeting moiety is still considered to selectively bind even if it also binds to other proteins that are not substantially homologous with the target so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that target moiety binding to the target is still selective despite some degree of cross-reactivity. Typically, the degree of cross- reactivity can be determined and differentiated from binding to the target.

[0061] A "targeting peptide" is a peptide comprising a contiguous sequence of amino acids, which is characterized by selective localization to an organ, tissue or cell type, which includes specific binding with an extracellar protein or molecule that is specfically expressed or produced in a specific tissue or cell type(s). Selective localization may be determined, for example, by methods disclosed below, wherein the putative targeting peptide sequence is incorporated into a protein that is displayed on the outer surface of a phage. [0062] A "subject" refers generally to a mammal. In certain preferred embodiments, the subject is a mouse or rabbit. In even more preferred embodiments, the subject is a human.

II. General Embodiments of the Invention

[0063] Phage display selection allows the study of functional protein-protein interactions at the cell surface but targeting intracellular organelles remains challenging. In embodiments of the present invention, there are internalizing-phage (iPhage) libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and home to specific organelles, an approach enabling peptide selection and intracellular receptor identification. First, the inventors demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of iPhage into cells. Next, the inventors show that an iPhage construct displaying an established mitochondria- specific localization signal target that organelle. Finally, the inventors constructed an iPhage random peptide library to select internalizing homing peptide (iHoPe) motifs to different cellular compartments; one exemplary iHoPe chemically fused to penetratin recapitulated receptor- independent internalization, mitochondria targeting, and cell death promotion. This combinatorial platform technology allows direct organelle targeting and intracellular ligand- receptor discovery with applications ranging at least from fundamental cell biology to drug development.

[0064] An exemplary method of identifying intracellular homing peptides includes administering to a cell system or a subject a library of vectors that have been genetically engineered to comprise sequences encoding a cell-penetrating peptide, for example, on a major coat protein, and an intracellular homing peptide, for example, a phage expressing a particular intracellular homing peptide sequence is considered to be selectively intracellularly localized to a an intracellular region or organelle if it exhibits greater binding in that respective region or organelle compared to a control. Preferably, selective intracellular localization of a targeting peptide should result in a two-fold or higher enrichment of the phage in the target localization, compared to a control. Selective localization resulting in at least a three-fold, four-fold, five- fold, six- fold, seven- fold, eight- fold, nine-fold, ten-fold or higher enrichment in the target region or organelle compared to a control is more preferred. A phage expressing a intracellular homing peptide sequence that exhibits selective localization preferably shows an increased enrichment in the target organelle compared to a control when phage recovered from the target organelle are reinjected into a second host or cell system for another round of screening. Further enrichment may be exhibited following a third round of screening. Another alternative means to determine selective localization is that phage expressing the putative intracellular homing peptide preferably exhibit a two-fold, more preferably a three-fold or higher enrichment in the target organelle or location compared to control phage that express a non-specific peptide or that have not been genetically engineered to express any putative intracellular homing peptides. Another means to determine selective localization is that binding to the target organelle or protein of phage expressing the intracellular homing peptide is at least partially blocked by the coadministration of a synthetic peptide containing the target peptide sequence. "Targeting peptide" and "homing peptide" are used synonymously herein. "Targeting peptide" and "homing peptide" may be used synonymously herein.

III. Proteins and Peptides

[0065] In certain embodiments, the present invention concerns novel compositions comprising at least one protein or peptide. As used herein, a protein or peptide generally refers, but is not limited to, a protein of greater than about 200 amino acids, up to a full length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide" and "peptide" are used interchangeably herein.

[0066] In certain embodiments the size of at least one protein or peptide may comprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 1 10, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater amino acid residues.

[0067] As used herein, an "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative or any amino acid mimic known in the art. In certain embodiments, the residues of the protein or peptide are sequential, without any non-amino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence may comprise one or more non-amino acid moiety. In particular embodiments, the sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.

[0068] Accordingly, the term "protein or peptide" encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid, including but not limited to Aad, 2-Aminoadipic acid; EtAsn, N-Ethylasparagine; Baad, 3- Aminoadipic acid, Hyl, Hydroxylysine; Bala, β-alanine, β-Amino-propionic acid; AHyl, allo-Hydroxylysine; Abu, 2-Aminobutyric acid; 3Hyp, 3-Hydroxyproline; 4Abu, 4- Aminobutyric acid, piperidinic acid; 4Hyp, 4-Hydroxyproline; Acp, 6-Aminocaproic acid, Ide, Isodesmosine; Ahe, 2-Aminoheptanoic acid; Alle, allo-Isoleucine; Aib, 2-Aminoisobutyric acid; MeGly, N-Methylglycine, sarcosine; Baib, 3-Aminoisobutyric acid; Melle, N-Methylisoleucine; Apm, 2-Aminopimelic acid; MeLys, 6-N-Methyllysine; Dbu, 2,4-Diaminobutyric acid; MeVal, N-Methylvaline; Des, Desmosine; Nva, Norvaline; Dpm, 2,2'-Diaminopimelic acid; Nle, Norleucine; Dpr, 2,3-Diaminopropionic acid; Orn, Ornithine; and EtGly, N-Ethylglycine.

[0069] Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. The nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's GenBank® and GenPept databases (on the world wide web at ncbi.nlm.nih.gov). The coding regions for known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.

[0070] Peptide mimetics

[0071] Another embodiment for the preparation of polypeptides is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al, 1993, incorporated herein by reference. The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used to engineer second generation molecules having many of the natural properties of the targeting peptides disclosed herein, but with altered and even improved characteristics.

[0072] Fusion proteins

[0073] Other embodiments concern fusion proteins. These molecules generally have all or a substantial portion of a targeting peptide (e.g., an APA targeting peptide, or Crkl, or prohibitin, or RGD, or NGR, etc.), linked at the N- or C-terminus, to all or a portion of a second polypeptide or protein. For example, fusions may employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions. In preferred embodiments, the fusion proteins of the instant invention comprise a targeting peptide linked to a therapeutic protein or peptide. Examples of proteins or peptides that may be incorporated into a fusion protein include cytostatic proteins, cytocidal proteins, pro-apoptosis agents, anti-angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab fragments antibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins, cell adhesion proteins and binding proteins. These examples are not meant to be limiting and it is contemplated that within the scope of the present invention virtually any protein or peptide could be incorporated into a fusion protein comprising a targeting peptide. Methods of generating fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by chemical attachment using bifunctional cross-linking reagents, by de novo synthesis of the complete fusion protein, or by attachment of a DNA sequence encoding the targeting peptide to a DNA sequence encoding the second peptide or protein, followed by expression of the intact fusion protein.

[0074] Protein purification

[0075] In certain embodiments a protein or peptide may be isolated or purified. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the homogenization and crude fractionation of the cells, tissue or organ to polypeptide and non-polypeptide fractions. The protein or polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. An example of receptor protein purification by affinity chromatography is disclosed in U.S. Patent 5,206,347, the entire text of which is incorporated herein by reference. A particularly efficient method of purifying peptides is fast performance liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC). [0076] A purified protein or peptide is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. An isolated or purified protein or peptide, therefore, also refers to a protein or peptide free from the environment in which it may naturally occur. Generally, "purified" will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition. [0077] Various methods for quantifying the degree of purification of the protein or peptide are known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a "-fold purification number." The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the expressed protein or peptide exhibits a detectable activity. [0078] Various techniques suitable for use in protein purification are well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like, or by heat denaturation, followed by: centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[0079] There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

[0080] Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind. This is a receptor-ligand type of interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g., altered pH, ionic strength, temperature, etc.). The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.

[0081] Synthetic Peptides

[0082] Because of their relatively small size, the targeting peptides of the invention can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, 1984; Tarn et ah, 1983; Merrifield, 1986; Barany and Merrifield, 1979, each incorporated herein by reference. Short peptide sequences, usually from about 6 up to about 35 to 50 amino acids, can be readily synthesized by such methods. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultivated under conditions suitable for expression.

[0083] Antibodies

[0084] In certain embodiments, it may be desirable to make antibodies against APA, APA targeting peptides or antibody idiotopes. The appropriate targeting peptide, APA protein or portions thereof, may be coupled, bonded, bound, conjugated, or chemically-linked to one or more agents via linkers, polylinkers, or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions are familiar to those of skill in the art and may be suitable for administration to humans, i.e., pharmaceutically acceptable. Preferred agents are the carriers are keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). [0085] Antibodies can be used to therapeutically or diagnostically by inhibiting APA, targeting APA, or detecting APA. These antibodies may be used in various diagnostic or therapeutic applications, described herein below.

[0086] As used herein, the term "antibody" is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE or antibody like molecule. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Harlow and Lane, 1988; incorporated herein by reference).

[0087] Monoclonal antibodies (MAbs) are recognized to have certain advantages (e.g., reproducibility and large-scale production). The invention thus provides for monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies may be preferred. However, "humanized" antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.

[0088] The methods for generating monoclonal antibodies (MAbs) and polyclonal antibodies are well known in the art. MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. It is also contemplated that a molecular cloning approach may be used to generate monoclonals. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or by expression of full- length gene or of gene fragments in E. coli.

[0089] In certain embodiments, compositions comprising a mixture of 2, 3, 4, 5, 6, or more antibodies may be used to treat various pathologic conditions associated with angiogenesis. The antibodies may be polyclonal or monoclonal antibodies selected for either binding of APA, inhibition of APA, or both. These antibodies may also be conjugated to vairous therepeutic or diagnostic agents. [0090] Antibody Conjugates

[0091] Certain embodiments of the invention provide antibodies to APA peptides, proteins, polypeptides or antibody idiotopes thereof that are linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. A reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.

[0092] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. "Detectable labels" are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired. An example of such a detectable label is gold nanoparticles. Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti- cellular agent, and may be termed "immunotoxins".

[0093] Antibody refers to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Harlow and Lane, 1988; incorporated herein by reference).

IV. Therapeutic or Diagnostic Embodiments [0094] Targeting moieties identified using these methods may be coupled or attached to various substances, including therapeutic or diagnostic agents, for the selective delivery of the conjugate to a desired organelle or intracellular location. For example, targeted delivery of chemotherapeutic agents and proapoptotic peptides to receptors located in tumor angiogenic vasculature result in a marked increase in therapeutic efficacy and a decrease in systemic toxicity in tumor bearing mouse models (Arap et ah, 1998; Ellerby et ah, 1999). [0095] Cytokines and chemokines

[0096] In certain embodiments, it may be desirable to couple specific bioactive agents to one or more targeting moieties for targeted delivery to an organ, tissue or cell type. Such agents include, but are not limited to, cytokines, chemokines, pro-apoptosis factors and anti-angiogenic factors. The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators.

[0097] Examples of such cytokines are lymphokines, monokines, growth factors and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; pro insulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-. alpha, and -.beta.; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-.beta.; platelet-growth factor; transforming growth factors (TGFs) such as TGF-. alpha, and TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -.β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-l.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12; IL- 13, IL-14, IL-15, IL-16, IL-17, IL-18, LIF, G-CSF, GM-CSF, M- CSF, EPO, kit-ligand or FLT- 3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. [0098] Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine gene in combination with, for example, a cytokine gene, to enhance the recruitment of other immune system components to the site of treatment. Chemokines include, but are not limited to, RANTES, MCAF, MIP1 -alpha, MIPl-Beta, and IP- 10. The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines.

[0099] Imaging agents and radioisotopes

[0100] In certain embodiments, the targeting moieties of the present invention may be attached to imaging agents of use for imaging and diagnosis of various diseased organs, tissues or cell types. Many appropriate imaging agents are known in the art, as are methods for their attachment to proteins or peptides (see, e.g., U.S. Patents 5,021,236 and 4,472,509, both incorporated herein by reference). Certain attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the protein or peptide (U.S. Patent 4,472,509). Proteins or peptides also may be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.

[0101] Non-limiting examples of paramagnetic ions of potential use as imaging agents include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper

(II) , neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium

(III) , dysprosium (III), holmium (III) and erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III). [0102] Radioisotopes of potential use as imaging or therapeutic agents include astatine211, 14carbon, 51chromium, 36chlorine, 57cobalt, 58cobalt, copper67, 152Eu, gallium67, 3hydrogen, iodinel23, iodinel25, iodinel31, indiuml l l, 59iron, 32phosphorus, rheniuml 86, rheniuml 88, 75selenium, 35sulphur, technicium99m and yttrium90. 1251 is often being preferred for use in certain embodiments, and technicium99m and indiuml l l are also often preferred due to their low energy and suitability for long range detection.

[0103] Radioactively labeled proteins or peptides of the present invention may be produced according to well-known methods in the art. For instance, they can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Proteins or peptides according to the invention may be labeled with technetium-99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the peptide to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNC12, a buffer solution such as sodium-potassium phthalate solution, and the peptide. Intermediary functional groups that are often used to bind radioisotopes that exist as metallic ions to peptides are diethylenetriaminepenta-acetic acid (DTPA) and ethylene diaminetetra-acetic acid (EDTA). Also contemplated for use are fluorescent labels, including rhodamine, fluorescein isothiocyanate and renographin. [0104] In certain embodiments, the claimed proteins or peptides may be linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. Preferred secondary binding ligands are biotin and avidin or streptavidin compounds. The use of such labels is well known to those of skill in the art in light and is described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149 and 4,366,241; each incorporated herein by reference.

[0105] In still further embodiments, a targeting moiety may be operatively coupled to a nanoparticle. Nanoparticles include, but are not limited to colloidal gold and silver nanoparticles. Metal nanoparticles exhibit colors in the visible spectral region. It is believed that these colors are the result of excitation of surface plasmon resonances in the metal particles and are extremely sensitive to particles' sizes, shapes, and aggregation state; dielectric properties of the surrounding medium; adsorption of ions on the surface of the particles (For examples see U.S. Patent Application 20040023415, which is incorporated herein by reference). [0106] Cross-linkers

[0107] Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies. Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially. The bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino, sulfhydryl, guanidino, indole, carboxyl specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied. A majority of heterobifunctional cross- linking reagents contains a primary amine-reactive group and a thiol-reactive group. [0108] Exemplary methods for cross-linking ligands to liposomes are described in

U.S. Patents 5,603,872 and 5,401,511, each specifically incorporated herein by reference in its entirety. Various ligands can be covalently bound to liposomal surfaces through the cross- linking of amine residues. Liposomes, in particular, multilamellar vesicles (MLV) or unilamellar vesicles such as microemulsified liposomes (MEL) and large unilamellar liposomes (LUVET), each containing phosphatidylethanolamine (PE), have been prepared by established procedures. The inclusion of PE in the liposome provides an active functional residue, a primary amine, on the liposomal surface for cross-linking purposes. Ligands such as epidermal growth factor (EGF) have been successfully linked with PE-liposomes. Ligands are bound covalently to discrete sites on the liposome surfaces. The number and surface density of these sites are dictated by the liposome formulation and the liposome type. The liposomal surfaces may also have sites for non-covalent association. To form covalent conjugates of ligands and liposomes, cross-linking reagents have been studied for effectiveness and biocompatibility. Cross-linking reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and a water soluble carbodiimide, preferably l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Through the complex chemistry of cross-linking, linkage of the amine residues of the recognizing substance and liposomes is established.

[0109] In another example, heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described (U.S. Patent 5,889, 155, specifically incorporated herein by reference in its entirety). The cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example, of aldehydes to free thiols. The cross-linking reagent can be modified to cross-link various functional groups.

V. Nucleic Acids

[0110] Nucleic acids according to the present invention may encode an intracellular homing peptide or a cell-penetrating peptide, or one nucleic acid may comprise sequences that encode both. The nucleic acid may be derived from genomic DNA, complementary DNA (cDNA) or synthetic DNA.

[011 1] A "nucleic acid" as used herein includes single-stranded and double- stranded molecules, as well as DNA, RNA, chemically modified nucleic acids and nucleic acid analogs. It is contemplated that a nucleic acid within the scope of the present invention may be of almost any size, determined in part by the length of the encoded protein or peptide.

[0112] It is contemplated that targeting peptides may be encoded by any nucleic acid sequence that encodes the appropriate amino acid sequence. The design and production of nucleic acids encoding a desired amino acid sequence is well known to those of skill in the art, using standardized codon tables. In preferred embodiments, the codons selected for encoding each amino acid may be modified to optimize expression of the nucleic acid in the host cell of interest.

[0113] Targeted Delivery of Gene Therapy Vectors

[0114] There are a number of ways in which gene therapy vectors may introduced into cells. In certain embodiments of the invention, the gene therapy vector comprises a virus. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome or be maintained episomally, and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubinstein, 1988.; Baichwal and Sugden, 1986; Temin, 1986). Preferred gene therapy vectors are generally viral vectors. DNA viruses used as gene therapy vectors include the papovaviruses (e.g., simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). [0115] One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include, but is not limited to, constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense or a sense polynucleotide that has been cloned therein.

[0116] Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991 ; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al, 1991; Rosenfeld et al, 1992), muscle injection (Ragot et al, 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic innoculation into the brain (Le Gal La Salle et al, 1993).

[0117] In preferred embodiments, certain advantages may be gained from coupling therapeutic molecules or substances to targeting moieties, e.g., APA targeting moieties, that target the vasculature of diseased tissues, e.g., tumors or neo-vascular beds. Specifically, moieties that home to tumor vasculature have been coupled to cytotoxic drugs or proapoptotic peptides to yield compounds were more effective and less toxic than the parental compounds in experimental models of mice bearing tumor xenografts (Arap et al, 1998; Ellerby et al, 1999). The insertion of the RGD-4C peptide into a surface protein of an adenovirus has produced an adenoviral vector that may be used for tumor targeted gene therapy (Arap et al, 1998).

[0118] A "fiber protein" according to the invention preferably comprises an adenoviral fiber protein. Any one of the serotypes of human or nonhuman adenovirus (e.g., a chimeric fiber protein) can be used as the source of the fiber protein or fiber gene. Optimally, however, the adenovirus is an Ad2 or Ad5 adenovirus, (see, U.S. Patent 6,649,407, which is incorporated herein by refernce in its entirety).

[0119] The fiber protein is "chimeric" in that it comprises amino acid residues that are not typically found in the protein as isolated from wild-type adenovirus (i.e., comprising the native protein, or wild-type protein). The fiber protein thus comprises a "nonnative amino acid sequence". "Nonnative amino acid sequence" means a sequence of any suitable length, preferably from about 3 to about 200 amino acids, optimally from about 3 to about 30 amino acids. Desirably, the nonnative amino acid sequence is introduced into the fiber protein at the level of gene expression (i.e., by introduction of a "nucleic acid sequence that encodes a nonnative amino acid sequence"). Such a nonnative amino acid sequence either is introduced in place of adenoviral sequences, or in addition to adenoviral sequences. Regardless of the nature of the introduction, its integration into an adenoviral fiber protein at the level of either DNA or protein, results in the generation of a peptide motif (i.e., a peptide binding motif) in the resultant chimeric fiber protein.

[0120] The peptide motif allows for cell targeting, for instance, by comprising a targeting moiety of the invention, and/or a ligand for a cell surface binding site. The peptide motif optionally can comprise other elements of use in cell targeting (e.g., a single-chain antibody sequence). The peptide binding motif may be generated by the insertion, and may comprise, for instance, native and nonnative sequences, or may be entirely made up of nonnative sequences. The peptide motif that results from the insertion of the nonnative amino acid sequence into the chimeric fiber protein can be either a high affinity peptide (i.e., one that binds its cognate binding site, e.g., APA, when provided at a relatively low concentration) or a low affinity peptide (i.e., one that binds its cognate binding site, e.g., APA, when provided at a relatively high concentration). Preferably, however, the resultant peptide motif is a high affinity motif, particularly one that has a high affinity for its cognate binding site due to its constraint within the adenovirus fiber protein.

[0121] Other gene transfer vectors may be constructed from retroviruses. (Coffin, 1990.) In order to construct a retroviral vector, a nucleic acid encoding protein of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes, but without the LTR and packaging components, is constructed (Mann et al, 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).

[0122] Other viral vectors may be employed as targeted gene therapy vectors. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), and herpes viruses may be employed.

[0123] In a further embodiment of the invention, gene therapy construct may be entrapped in a liposome. Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Wong et al, (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et al, (1987.) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.

[0124] Gene therapy vectors of the invention may comprise various transgenes, which are typically encoded DNA or RNA of an expression vector. Gene therapy may be used for the expression of a therapeutic gene, expression of APA to enhance neo-vascularization or for the inhibition of APA expression for the treatment of disease states associated with neovascularization. DNA may be in form of cDNA, in vitro polymerized DNA, plasmid DNA, parts of a plasmid DNA, genetic material derived from a virus, linear DNA, vectors (P 1 , PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, recombinant DNA, chromosomal DNA, an oligonucleotide, anti-sense DNA, or derivatives of these groups. RNA may be in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), in vitro polymerized RNA, recombinant RNA, chimeric sequences, anti-sense RNA, siRNA (small interfering RNA), ribozymes, or derivatives of these groups. An anti-sense polynucleotide is a polynucleotide that interferes with the function of DNA and/or RNA. Antisense polynucleotides include, but are not limited to: morpholinos, 2'-0-methyl polynucleotides, DNA, RNA and the like. SiRNA comprises a double stranded structure typically containing 15-50 base pairs and preferably 21-25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. Interference may result in suppression of expression. The polynucleotide can also be a sequence whose presence or expression in a cell alters the expression or function of cellular genes or RNA, e.g., APA. In addition, DNA and RNA may be single, double, triple, or quadruple stranded.

VI. Pharmaceutical Compositions [0125] Pharmaceutical compositions of the present invention comprise an effective amount of one or more compositions including a bacteriophage comprising a cell penetrating peptide and, optionally, an intracellular homing peptide, as described herein dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one composition of the present invention or an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0126] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0127] The therapeutic and diagnostic compositions of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intrapleurally, intratracheally, intratumorally, intramuscularly, intraperitoneally, subcutaneous ly, intravesicularlly, sublingually, by inhalation (e.g.. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0128] The actual dosage amount of a composition of the present invention administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0129] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 g/kg/body weight, about 5 g/kg/body weight, about 10 g/kg/body weight, about 50 g/kg/body weight, about 100 g/kg/body weight, about 200 g/kg/body weight, about 350 g/kg/body weight, about 500 g/kg/body weight, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, can be administered, based on the numbers described above.

[0130] In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0131] In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0132] Sterile injectable solutions are prepared by incorporating the APA targeting moiety or conjugate thereof in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

[0133] The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein. VII. Therapeutic Agents

[0134] In certain embodiments, therapeutic agents may be operatively coupled to an intracellular homing peptide for selective delivery to, a desired intracellular location or organelle. Agents or factors suitable for use may include any chemical compound that induces apoptosis, cell death, cell stasis and/or anti-angiogenesis. [0135] Regulators of Programmed Cell Death

[0136] Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al, 1972). The Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al, 1985; Cleary and Sklar, 1985; Cleary et al, 1986; Tsujimoto et al, 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists.

[0137] Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins that share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to Bcl-2 (e.g., BclXL, BclW, BclS, Mcl-1, Al, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

[0138] Angiogenic inhibitors

[0139] In certain embodiments the present invention may concern administration of targeting moieties operatively coupled to anti-angiogenic agents, such as angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, Gro-B, thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM 101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP- 470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.

[0140] Proliferation of tumors cells relies heavily on extensive tumor vascularization, which accompanies cancer progression. Thus, inhibition of new blood vessel formation with anti-angiogenic agents and targeted destruction of existing blood vessels have been introduced as an effective and relatively non-toxic approach to tumor treatment. (Arap et al, 1998; Arap et al, 1998; Ellerby et al, 1999). A variety of anti-angiogenic agents and/or blood vessel inhibitors are known, (e.g., Folkman, 1997; Eliceiri and Cheresh, 2001). [0141] Cytotoxic Agents

[0142] Chemotherapeutic (cytotoxic) agents may be used to treat various disease states, including cancer. Chemotherapeutic (cytotoxic) agents of potential use include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP 16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing. Most chemotherapeutic agents fall into the categories of alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof.

[0143] Chemotherapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art (see for example, the "Physicians Desk Reference", Goodman & Gilman's "The Pharmacological Basis of Therapeutics" and in "Remington's Pharmaceutical Sciences" 15th ed., pp 1035-1038 and 1570-1580, incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Of course, all dosages and agents described herein are exemplary rather than limiting, and other doses or agents may be used by a skilled artisan for a specific patient or application. Any dosage in-between these points, or range derivable therein is also expected to be of use in the invention. [0144] Alkylating agents

[0145] Alkylating agents are drugs that directly interact with genomic DNA to prevent cells from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific. An alkylating agent, may include, but is not limited to, a nitrogen mustard, an ethylenimene, a methylmelamine, an alkyl sulfonate, a nitrosourea or a triazines. They include but are not limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (Cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.

[0146] Antimetabolites [0147] Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. Antimetabolites can be differentiated into various categories, such as folic acid analogs, pyrimidine analogs and purine analogs and related inhibitory compounds. Antimetabolites include but are not limited to, 5- fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate. [0148] Natural Products

[0149] Natural products generally refer to compounds originally isolated from a natural source, and identified as having a pharmacological activity. Such compounds, analogs and derivatives thereof may be, isolated from a natural source, chemically synthesized or recombinantly produced by any technique known to those of skill in the art. Natural products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers.

[0150] Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors include, for example, docetaxel, etoposide (VP 16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.

[0151] Taxoids are a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia. Taxoids include but are not limited to compounds such as docetaxel and paclitaxel. Paclitaxel binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules.

[0152] Vinca alkaloids are a type of plant alkaloid identified to have pharmaceutical activity. They include such compounds as vinblastine (VLB) and vincristine. [0153] Antibiotics

[0154] Certain antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Examples of cytotoxic antibiotics include, but are not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin.

[0155] Miscellaneous Agents

[0156] Miscellaneous cytotoxic agents that do not fall into the previous categories include, but are not limited to, platinum coordination complexes, anthracenediones, substituted ureas, methyl hydrazine derivatives, amsacrine, L-asparaginase, and tretinoin. Platinum coordination complexes include such compounds as carboplatin and cisplatin (cz^'s-DDP). An exemplary anthracenedione is mitoxantrone. An exemplary substituted urea is hydroxyurea. An exemplary methyl hydrazine derivative is procarbazine (N-methylhydrazine, MIH). These examples are not limiting and it is contemplated that any known cytotoxic, cytostatic or cytocidal agent may be attached to targeting peptides and administered to a targeted organ, tissue or cell type within the scope of the invention.

EXAMPLES

[0157] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

FUNCTIONAL IPHAGE PARTICLES ENABLE RECEPTOR-INDEPENDENT CELL

ENTRY ACROSS MAMMALIAN MEMBRANES

[0158] The general structure of iPhage was generated on the backbone phage vector f88-4 (ref. 37; FIG. la) displaying pen (sequence RQIKIWFQNRRMKWKK; SEQ ID NO: l) on rpVIII (FIG. lb). As controls, the inventors used parental phage and site-directed mutant iPhage displaying a mutant loss-of-function form of pen (RQIKIAFQNRRMKAKK; SEQ ID NO:2), in which two critical tryptophan (W) residues (required for membrane translocation) were mutated to alanine (A) residues (FIG. lc). There was abundant iPhage production in K91 E. coli, a result indicating that iPhage constructs were correctly assembled and non-toxic to the host bacteria.

[0159] To demonstrate that iPhage can cross mammalian cell membranes through a receptor- independent mechanism, the inventors exposed human Kaposi Sarcoma (KS) 1767 cells to each of the three individual constructs. After 24 h, phage particles were detected in cells exposed to iPhage but not in cells incubated with either parental phage or mutant iPhage (FIG. Id). In addition, phage genomic DNA was detected only in cells infected with iPhage particles indicating that all viral components were internalized (FIG. le). Furthermore, pre-incubation of the soluble pen peptide on mammalian cells did not block iPhage particle internalization, a result indicative that the intracellular uptake is receptor-independent and non-saturable (FIG. 5). To test viability and eliminate the possibility that iPhage particles have either affinity or get trapped in the cell surface, the inventors isolated plasma membrane and cytosol fractions and recovered the viral pool by infection of host E. coli; a marked recovery of viable iPhage particles was observed in the cytosol after 24 h, in comparison to parental phage and mutant iPhage that served as negative controls (FIG. If). The inventors did not recover phage, iPhage, or mutant iPhage from the membrane fraction, a result indicative that phage particles were not non- specifically trapped within the cell surfaces. Moreover, to account for a possible uptake of iPhage particles by endocytosis, the inventors have performed a series of co-localization studies with markers of early (EEA1) and late (mannose 6-phosphate receptor) endosomes. Careful analysis of serial confocal images demonstrated that, upon cellular intake, iPhage particles do not totally reside in the endosomal compartment but rather, are evenly distributed in the cytosol. Notably, additional co-immunostaining studies with markers of intracellular organelles such as Golgi apparatus and mitochondrial confirmed these findings; iPhage particles showed a random intracellular distribution according to different subcellular markers detected by confocal microscopy (i.e., Golgi apparatus, mitochondria, early and late endosomes). (FIG. 6). Furthermore, cell internalization (FIG. lg-h) was not dependent on species (mouse or human respectively), transformation status (non-malignant or malignant cells), or tumor type [carcinoma (LLC), leukemia (K562), lymphoma (RMA), melanoma (B16F10), or sarcoma]. These results show that (i) pen-display mediates the internalization of phage particles into mammalian cells independently of a cell surface receptor and (ii) the internalized particles in the cytosol remain intact, viable, and functional within mammalian cells. Therefore, it appeared possible to target a specific organelle, or generate random peptide iPhage libraries to screen for internalizing homing peptides (iHoPe) in living cells (FIG. 2a) with this systematic approach. EXAMPLE 2

TARGETING MITOCHONDRIA WITH ORGANELLE-SPECIFIC LOCALIZATION

SIGNAL PEPTIDE

[0160] To test whether a defined localization signal peptide displayed by iPhage would target a distinct cellular compartment, the inventors produced an iPhage construct carrying an exemplary mitochondria localization signal (MLS), and used this construct to validate the predicted intracellular trafficking of targeted iPhage particles (Taylor et ah, 2001). First, the inventors confirmed that both iPhage and MLS-iPhage were both equally efficiently internalized compared to the negative control (parental phage) as observed in the cytosol fraction (FIG. 2b). Indeed, MLS-iPhage were detected and enriched by transducing unit (TU) recovery specifically within their corresponding organelle-targeted fraction compared to the nuclear fraction (FIG. 2b), and demonstrated by confocal microscopy (FIG. 2c). To minimize or eliminate the possibility of an off-target effect, the inventors repeated this proof-of-concept experiment with reduced Mitotracker concentration (5 -fold less) and/or shorter incubation time (2-fold less) and observed similar target results (FIG. 7). Moreover, co-localization index analysis revealed that MLS-iPhage co-localizes to mitochondria 10-fold more compared to non- targeted-iPhage particles and 1,000-fold more compared to parental phage (Fig 8). The inventors concluded that iPhage constructs displaying ligand-directed signal peptides penetrated mammalian cells, reached an intracellular equilibrium, and targeted specific compartments within mammalian cells. EXAMPLE 3

INTRACELLULAR SELECTION OF AN IPHAGE LIBRARY YIELDS A NEW

LIGAND-RECEPTOR

[0161] Having demonstrated targeting with a defined intracellular signal peptide, the inventors attempted to identify internalizing homing peptide (iHoPe) sequences that disrupt cellular viability as a convenient readout for biological activity. Thus, it was reasoned that targeting of organelles such as mitochondria and endoplasmic reticulum (ER) would likely be the most suitable way to select and identify candidate pathways involved in cell death³⁹. The inventors constructed a random peptide iPhage library [i.e., pen displayed on rpVIII and the combinatorial motif arrangement X₄YX₄ (X, any residue; Y, tyrosine) displayed on pill] and selected it on live KS 1767 cells (FIG. 3a). After 24 h, the inventors isolated the mitochondrial/ER-enriched fraction and recovered the pool of iPhage through infection of host E. coli. After three rounds of selection, there was an enrichment of differential iPhage clones in the subcellular fraction (Fig 3b). DNA sequencing and bioinformatic analysis revealed a large amount of single peptide iPhage clones in the mitochondria/ER-enriched fraction. Next, the inventors randomly selected ten peptide-iPhage clones and tested their biological activity (disruption of cell viability). The KS1767 cell line was starved overnight and was incubated with each peptide-iPhage clone; as negative control the inventors used insertless parental iPhage in complete MEM media. One should note that the concentration of each peptide-iPhage clone (i.e., 10⁹ TU per 100 μΐ) corresponds to only -60 nM of peptide; an extremely low molar concentration if compared to the amount of synthetic peptide utilized in subsequent cell death experiments (30 mM). After 24 h, there were morphological alterations and reduced cellular viability only with the YKWYYRGAA(SEQ ID NO:3)-iPhage clone (FIG. 3c-e).

[0162] Having shown that MLS-iPhage targets its respective organelle, and the combinatorial selection of YKWYYRGAA(SEQ ID NO:3)-iPhage in mitochondria/ER fraction, the inventors next evaluated the ability of iPhage particles to compete against non-targeted- iPhage particles. First, the inventors show that iPhage particles displaying an organelle-homing peptide (i.e., MLS-iPhage and YKWYYRGAA(SEQ ID NO:3)-iPhage) were selectively enriched in the mitochondria/ER fraction even in the presence of a 10,000-fold molar excess of non-targeted-iPhage particles (FIG. 9). Next, to rule out the possibility that particle selection might simply be an artifact (i.e., phage binding after the cell fractionation process), the inventors compared an immediate versus a 24 h post-incubation phage-cell fractionation. iPhage clones (i.e., MLS and YKWYYRGAA (SEQ ID NO: 3)) were enriched in the respective organelle fraction after overnight post- incubation compared to immediate cell fractionation (FIG. 10). Moreover, there were high amounts of phage in the cytosol when the cell lysate was immediately cell fractionated compared to 24 h post-incubation (FIG. 10), a result indicative of an intracellular steady-state equilibrium. Taken together, these results show that MLS-iPhage and YKWYYRGAA(SEQ ID NO:3)-iPhage particles are retained in the mitochondria/ER fraction irrespective of a competing non-targeted-iPhage, and that they target organelles prior to cell fractionation. Finally, internalizing versions of the YKWYYRGAA (SEQ ID NO:3) peptide and controls were generated through C-terminal chemical fusions to pen (Merrifield synthesis) and were subsequently evaluated for their intracellular entry and biological activity.

EXAMPLE 4

RPL29 IS A RECEPTOR FOR THE INTERNALIZING YKWYYRGAA (SEQ ID NO:3)

PEPTIDE [0163] The inventors used affinity chromatography to purify candidate receptors for the YKWYYRGAA (SEQ ID NO:3) peptide and subsequently probed the eluted and immobilized fractions for specific binding of iPhage displaying this ligand peptide (Figure 4a). SDS-PAGE identified a single protein with a relative molecular weight (Mr) of 22,000 only in target fraction number 43. Mass spectrometry revealed peptides corresponding unequivocally to the ribosomal protein L29 (RPL29; Figure 4b).

[0164] To confirm the candidate receptor RPL29 as a target for the internalized YKWYYRGAA (SEQ ID NO:3) peptide, the inventors generated a recombinant GST fusion with RPL29 and performed binding assays with the YKWYYRGAA(SEQ ID NO:3)-displaying iPhage construct. Consistently, the YKWYYRGAA(SEQ ID NO:3)-displaying iPhage bound to RPL29 but not to control proteins that included a closely related recombinant GST-RPL30 fusion (FIG. 4c). Moreover, YKWYYRGAA (SEQ ID NO:3), but not controls, mediated concentration-dependent inhibition of binding (FIG. 4d).

[0165] Disruption of RPL29 promotes apoptosis induced by a caspase activation pathway in mammalian cells⁴⁰. Therefore, the inventors asked whether the YKWYYRGAA(SEQ ID NO:3)-pen peptide would disrupt RPL29 function and promote cell death. Cell viability assays revealed that YKWYYRGAA(SEQ ID NO:3)-pen, but not control (FIG. 4e) or non-conjugated admixtures of pen plus YKWYYRGAA (SEQ ID NO:3) peptides (FIG. 11), reduced cell survival; these results were not dependent on transformation status (non- malignant versus malignant cells), species of origin (mouse versus human), or pathological tumor cell type (carcinoma, leukemia, lymphoma, melanoma, or sarcoma), data suggestive of a general cellular mechanism of cell death (FIG. 12). The inventors first analyzed the cell surface expression of the established apoptotic marker phosphatidylserine. KS1767 cells were incubated with synthetic YKWYYRGAA(SEQ ID NO:3)-pen, pen alone, or YKWYYRGAA (SEQ ID NO:3), and were stained with annexin V-FITC an early cell death marker. After 6 h, FACS and fluorescence microscopy revealed that most cells treated with YKWYYRGAA-pen had undergone cell death (FIG. 4f, g). To show activation of the intracellular apoptotic pathways, the inventors evaluated the processing of caspase-7 and caspase-9 from inactive zymogen to active protease and observed caspase activation only in the presence of YKWYYRGAA(SEQ ID NO:3)-pen (FIG. 4h). Moreover, there were histone-associated DNA fragments in the cytosol of cells treated only with the YKWYYRGAA(SEQ ID NO:3)-pen peptide; these results suggest that chromatin fragmentation is activated by YKWYYRGAA(SEQ ID NO:3)-pen peptide via RPL29 (FIG. 4i). To begin to understand the molecular mechanism(s) of cell death in this new experimental system, the inventors subsequently evaluated biochemical markers related to autophagy and necrosis. In addition to apoptosis, we observed that the marker beclin-1 was upregulated only in cells exposed to YKWYYRGAA(SEQ ID NO:3)-pen peptide, a result suggestive of specific activation of an autophagy-dependent pathway (FIG. 4j). Finally, the inventors also investigated cellular necrosis by means of extracellular release of the high mobility group protein Bl (HMGB 1). The inventors detected HMGB1 in the supernatant of cells treated only with YKWYYRGAA(SEQ ID NO:3)-pen, a result indicative of necrosis activation pathway (FIG. 4k). Taken together, these results show that YKWYYRGAA(SEQ ID NO:3)-pen activates different molecular pathways including apoptosis, autophagy, and necrosis and indicate a complex mammalian cell death mechanism ultimately resulting in morphological alterations consistent with a late stage phenomena illustrated in KS 1767 cells by TEM (FIG. 41). After 6 h with the control peptides YKWYYRGAA (SEQ ID NO:3) or pen, the cells remained morphologically normal; in contrast, over 90% of the cells treated with YKWYYRGAA(SEQ ID NO:3)-pen showed the classical morphological signs of cell death (e.g., cytoplasmic vacuoles, swollen mitochondria, and chromatin condensation). Notably, during the cell death process, there was an increasingly translucent cytoplasm, swelling of organelles, and condensation of nuclear chromatin into small, irregular, circumscribed patches through different time points (i.e., 0.5, 1, 2, and 4 h; FIG. 13); these findings were only observed in cells treated with YKWYYRGAA(SEQ ID NO:3)-pen peptide. The inventors noticed normal and normal- vesicular mitochondria as early as 30 min. However at 1 to 2 h, most of the mitochondria showed vesicular-swollen and swollen forms upon exposure to the YKWYYRGAA(SEQ ID NO:3)-pen peptide (FIG. 14). These initial functional and morphologic analyses indicate that the internalizing RPL29-binding YKWYYRGAA (SEQ ID NO:3) peptide simultaneously activates several mammalian cell death pathways.

EXAMPLE 5

SIGNIFICANCE OF CERTAIN EMBODIMENTS OF THE INVENTION

[0166] The signal hypothesis and the existence of intracellular ZIP codes are both well-recognized fundamental concepts in cell biology (Blobel et al, 1970; Blobel et al, 1975; Pain et al, 1990). However, in contrast to ligand-directed targeting of cell membranes in vitro (Giordano et al, 2001; Cardo-Vila et al, 2003) or in vivo (Pasqualini et al, 1996; Arap et al, 1998; Arap et al, 2002; Kolonin et al, 2004; Mintz et al, 2009), combinatorial phage display- based technology has not yet been extended to the analysis of intracellular organelles and signaling or metabolic pathway targets in live cells. The inventors have approached this methodological gap through the display of established internalizing peptides (Howl et al, 2007; Joliot et al, 2008. The inventors chose pen, an internalizing peptide, which is derived from the homeodomain of the Drosophila melanogaster antennapedia protein (Derossi et al, 1998; Derossi et al, 1994. The inventors have shown that (i) expression of pen as a fusion recombinant protein on the major capsid rpVIII allows passage into cultured mammalian cells of viable, functional internalizing phage, named iPhage, without the participation of cell surface receptors; (ii) fusing a known signal peptide (Taylor et al, 2001) to the phage pie protein directs intact iPhage particles to the predicted intracellular location; (iii) simultaneous display of a random peptide on iPhage results in the selection and differential distribution of iPhage among the subcellular compartments, and (iv) peptides displayed in the iPhage particles can mimic their function with synthetic peptide counterparts. The inventors further demonstrate the power of this methodology by the identification of an iHoPe and its corresponding receptor mediating mammalian cell death.

[0167] RPL29 is a conserved protein involved in the assembly of the 60S ribosomal subunit, and studies in yeast have demonstrated that inactivation or mutation of the RPL29 gene retards protein synthesis (Delabre et al, 2002). RPL29 gene inactivation in mice showed an alteration in the cell cycle, and as a result, RPL29-null newborn pups were reduced to half in size Kirn-Safran et al, 2007). One may speculate that YKWYYRGAA(SEQ ID NO:3)- pen disrupts RPL29 function in ribosome assembly and protein synthesis, and thereby promotes cell death. Notably, the productive assembly of YKWYYRGAA(SEQ ID NO:3)-displaying iPhage particles indicates that this peptide is non-toxic to host bacteria and therefore does not appear to affect bacterial ribosomes, but rather targets eukaryotic ribosomes selectively. The YKWYYRGAA(SEQ ID NO:3)-pen peptide shows a remarkable activation of multiple mammalian cell death pathways at the biochemical level including apoptosis, autophagy and necrosis (Galluzzi et al, 2007; Kroemer et al, 2010; Kepp et al, 2011 ; Galluzzi et al, 2011). However one can dissect the inherent biologically complex death mechanism of the YKWYYRGAA(SEQ ID NO:3)-pen peptide via RPL29 in mammalian cells.

[0168] The relevance of subcellular targeting for drug delivery to improve drug efficiency, via the release of compounds within organelles, has recently been recognized (Rajendran et al, 2010). The iPhage technology originally reported here has not only the potential for subcellular delivery, but might also unveil iHoPe sequences relevant to intracellular trafficking in live cells. Finally, this internalizing strategy could also be combined with adeno- associated virus phage (AAVP)-based vectors for improved mammalian cell expression (Hajitou et al, 2006). In summary, this new internalizing combinatorial approach enables ligand-directed discovery of signal peptides and targeting of organelles or molecular pathways within live cells, with applications ranging at least from fundamental cell biology to drug development. EXAMPLE 6

EXEMPLARY METHODS

[0169] Reagents. The following antibodies were obtained from commercial sources: anti-bacteriophage antibody (Sigma), anti-caspase-7, anti-caspase-9, anti-atg-5, anti- atg-7, anti-beclin- 1 (Cell Signaling Technology), anti-HMG-B 1 (Millipore), anti-actin antibody (Novus Biologicals), anti-rabbit Cy3, anti-mouse FITC, anti-goat FITC (Zymed), anti-mouse HRP (Bio-Rad), Alexa Fluor 488 goat anti-rabbit (Invitrogen), and goat and mouse pre-immune sera (Jackson Immunoresearch). The following fluorescence probes were used: DAPI (Vector Laboratories), ProLong Gold antifade reagent, Orange MitoTracker (Invitrogen), and annexin V- FITC (BD Biosciences). Molecular biology reagents included restriction enzymes (New England Biolabs and Roche), T4 DNA ligase (Invitrogen), Taq DNA polymerase (Promega, Stratagene and Invitrogen); gel extraction, PCR purification, and plasmid isolation kits (Qiagen), isopropyl b-D-l-thiogalactopyranoside (IPTG; Fisher Scientific). The inventors used commercial recombinant ribosomal protein L30 (RPL30; Novus Biologicals). All the synthetic peptides were produced to our specifications (Polypeptide Laboratories).

[0170] Cell Culture. Human Kaposi sarcoma (KS 1767) cells were maintained in Modified Eagle's Medium (MEM) containing 10% FBS, MEM-vitamins, non-essential amino acids, penicillin G (100 units), streptomycin S0₄ (100 mg/ml), and 2.7 mM L-glutamine (Invitrogen) at 37 °C in a 5% CO2 humidified incubator.

[0171] Phage Internalization Assay. For mammalian cell internalization, KS1767 cells were grown in 8-well tissue-chamber slides and incubated with 10⁹ TU of iPhage, mutant iPhage, or insertless phage in MEM containing 1% BSA at 37 °C. After a 24 h incubation, the population of cell membrane-bound phage was removed by acidic washing (20 mM glycine, pH 2.3). Cells were washed with PBS and were fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 15 min. After washes with PBS, fixed cells were rendered permeable with PBS containing 0.1% Triton X-100, blocked with PBS containing 1% BSA, and incubated with anti-bacteriophage (1 :200 dilution) in PBS containing 1% BSA for 2h at room temperature. Next, Cy3 -conjugated anti-rabbit (1 :200 dilution) was added, and the incubated at room temperature for 1 h. Finally, cells were washed with PBS, fixed with PBS containing 4% PFA, mounted in the presence of DAPI, and visualized under a fluorescence or confocal microscope. [0172] Generation of the iPhage Peptide Display Library. To generate iPhage constructs, we transformed the M13-derived vector f88-4 (AF218363) into MC1061 E. coli. Single colonies selected on LB agar plates containing tetracycline (40 mg/ml) and streptomycin (50 mg/ml) were cultured overnight. Each plasmid DNA was first isolated by standard plasmid purification kit (Qiagen) and was subsequently re-purified through a CsCl gradient. Next, annealed oligonucleotides (encoding the penetratin peptide, sense 5'- cacaagctttgccaacgtccctcgacagat

[0173] aaagatttggttccaaaacggcgcatgaagtggaagaagcctgcagcaca-3 '(SEQ ID NO:4); antisense 5'-tgtgc tgcaggcttcttc [0174] cacttcatgcgccggttttggaaccaaatctttatctgtcgagggacgttggcaaagcttgtg; SEQ ID

NO:5) and f88-4 plasmid were digested with HindHl and Pstl restriction endonucleases. DNA fragments were gel-purified (Qiagen) and ligated at various vector: insert molar ratios. Restriction digested- and sequence-verified individual clones were electroporated into MCI 061 E. coli; iPhage particles were cultured in LB media containing 1 mM IPTG, tetracycline (40 mg/ml), and streptomycin (50 mg/ml), and were purified by the PEG-NaCl method (53). To produce an iPhage library displaying the peptide insert in the minor pill coat protein (general arrangement X₄YX₄; SEQ ID NO: 6), the inventors fused the iPhage construct (described above) and fUSE5 genomes to create a chimeric vector. Both plasmids were digested with BamHl and Xbal, and the products were resolved on a 0.8% agarose gel. A DNA fragment of 3,925 bp from fUSE5 (containing the rpIII) and another 5,402 bp fragment of iPhage (containing the rpVIII) were gel-purified and ligated overnight at 16 °C. Ligated products were precipitated in ethanol and electroporated into MC1061 E. coli; these bacteria were plated on LB-agar containing tetracycline and streptomycin. Next, we performed a CsCl plasmid purification of the iPhage/fUSE5 chimeric vector, which was digested with Sfil restriction endonuclease. In parallel, we performed a PCR amplification of the degenerate oligonucleotide (5'- cactcggccgacggggctnriknnlmriknriktatnnlmriknnknnkggggccgctggggccgaa-3 '; SEQ ID NO: 7) insert sequence containing Bgll restriction sites at both DNA ends, as previously described (54). Resulting PCR products were purified on Qiagen columns, digested with Bgll, and gel-purified. 5yZI-linearized iPhage/fUSE5 and double-stranded oligonucleotide inserts were prepared for large-scale ligations. After overnight incubation, ligated products were precipitated in ethanol and reconstituted in sterile water. We performed over 250 electroporations in MCI 061 E. coli and transferred them into Super Optimal Broth with Catabolite repression (SOC) medium at 37 °C for 1 h. Finally, the bacterial culture was amplified in LB medium (4 liters) containing 1 mM IPTG, tetracycline (40 mg/ml), and streptomycin (50 mg/ml); phage particles were recovered to yield an X₄YX₄-iPhage display library. [0175] iPhage Library Intracellular Selection. KS1767 cells were incubated with 5 x 10¹¹ TU of the X₄YX₄ iPhage library overnight at 37 °C. The next day, cells were washed extensively with pre-warmed PBS and were subsequently detached with trypsin. Cells were washed with ice-cold PBS, incubated with hypotonic buffer [10 mM NaCl, 1.5 mM MgCi₂, 10 mM Tris-HCl (pH 7.5)] for 15 min, and placed in a standard Dounce homogenizer to disrupt cell membranes. Next, we added stabilization buffer [525 mM mannitol, 175 nM sucrose, 2.5 mM EDTA (pH 7.5), and 12.5 mM Tris-HCl (pH 7.5)]. The resulting organelle suspension was centrifuged at 1,300 g for 5 min at 4 °C, the supernate was transferred to a new tube, and centrifuged at 17,000 g for 15 min; this pellet contained the mitochondrial/ER enriched fraction. The subcellular fraction-bound phage population was recovered through infection of log-phase k91kan E. coli for 1 h at room temperature. Serial dilutions of the infected bacteria were plated on Luria-Bertani (LB) plates containing tetracycline (40 mg/ml) and kanamycin (100 mg/ml) to determine the recovery phage titer. In addition, infected bacteria were grown overnight in LB containing tetracycline and kanamycin, and phage was recovered by PEG/NaCl precipitation (Kepp et ah, 201 1; Galluzzi et ah, 2011). After three rounds of selection, 96 bacterial colonies from each round were randomly selected for DNA sequencing.

[0176] Confocal Imaging. The cells were imaged with an inverted Olympus FV1000 (Olympus Inc, Center Valley, PA) laser scanning confocal microscope using a 60x 03 PLAPO oil objective. The imaging and 3D reconstruction was performed by taking a series of horizontal scans through the cells, arranged as a vertical stack/z-stack by means of image analysis software (FV1000 version 1.6, Olympus Inc). Optical sections were acquired at 0.44 mm intervals, with an imaging stack consisting of 22 optical sections. The image size was an 800 ^' 800 pixel matrix/image (132 ^' 132 mm) with a color depth of 12 bits/pixel. In this study, we used the UV LD405nm laser (Olympus America Inc, Center Valley PA) as well as the Argon ion 488nm and green HeNe 543nm lasers (Melles Griot, Albuquerque, NM) and suitable filter sets. [0177] Binding Assay and Ligand-Receptor Purification. By coupling the selected synthetic peptide to a Sepharose column (Carboxy-link kit, Pierce) and exposing the columns to KS1767 cell-derived lysates, we purified a candidate receptor. After extensive washing, the receptor was eluted with the corresponding competitive peptide at a concentration of 5 mM. Eluted fractions were analyzed by absorbance [optical density (OD) at 280 nm], dialyzed, and concentrated at 4 °C. Equal amounts of protein (5 mg/well in 50 ml PBS) were immobilized on a 96-well plate overnight at 4 °C. Wells were washed twice with PBS, blocked with PBS containing 2% BSA for 2 h at room temperature, and incubated with targeted iPhage or insertless iPhage (10⁹ TU each) in 50 ml PBS containing 0.1% BSA. After 2 h at room temperature, wells were washed ten times with PBS, and each phage population was recovered by host bacterial infection.

[0178] Cell Death assays and Western blot. KS1767 cells grown in 48-well plates were maintained for 24 h in non-supplemented MEM. The medium was subsequently supplemented with 10% FBS plus each respective test peptide. Cellular viability was assessed by measurement of cellular metabolism by the MTT assay (Roche), and WST-1 (Roche) at 37 °C according to standard protocols. Staining for annexin V (Clontech) was performed to monitor early stages of apoptosis. KS 1767 cells grown in 8-well slide chambers were incubated with the indicated peptides for 6 h. Cells were next incubated with annexin V-FITC, washed, and analyzed under a fluorescence microscope. For flow cytometry analysis, cells were detach and wash twice with cold PBS and then resuspended in binding buffer (Clontech) at a concentration of 10⁶ cells/ml. We used transfer 100 ml of the solution to a 5 ml culture tube and add annexin- V-FITC and incubate for 15 min room temperature. The cells were washed twice with binding buffer and analyzed by flow cytometry. The inventors used Western blotting for analysis of caspase activation, autophagy and necrosis pathways. Total cell extracts, prepared from KS1767 cells treated with or without peptides, were solubilized by lysis in radio immuno precipitation assay (RIP A) buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% triton X-100). The protein content of cell lysates was determined by the Bradford method (BioRad). Lysates were electrophoretically resolved on 4%-20% gradient sodium dodecyl sulfate polyacrylamide gels (BioRad), and proteins were transferred to nitrocellulose membranes. The membranes were blocked and incubated with primary antibodies diluted in blocking buffer [5% milk in 0.1% Tween-20, 100 mM NaCl, lOmM Tris-HCl, pH (7.4)]. The primary antibodies were diluted 1 : 2,000 (Cell Signaling Technology). Membranes were washed and incubated with secondary antibodies (horseradish peroxidase-linked conjugate of anti-rabbit IgG, Cell Signaling Technology). Reactive bands were visualized by the use of an enhanced chemiluminescence reagent (ECL; Amersham).

[0179] Statistical Analysis. Student's t-tests were used for statistical analysis as indicated.

ADDITIONAL METHODS

[0180] Reagents. The following antibodies were obtained from commercial sources: anti-EAAl, anti- M6RP, anti-ERAB, and anti-Golgi antibodies (Abeam).

[0181] Membrane and cytosol fractionation. KS1767 cells were incubated with 5 x 10⁹ TU of the Phage, iPhage and mutant-iPhage overnight at 37 °C. The next day, cells were washed extensively with pre-warmed PBS and were subsequently detached with trypsin. Cells were washed with ice-cold PBS, incubated with hypotonic buffer [10 mM NaCl, 1.5 mM MgCi₂, 10 mM Tris-HCl (pH 7.5)] for 15 min, and placed in a standard Dounce homogenizer to disrupt cell membranes. Next, the inventors added stabilization buffer [525 mM mannitol, 175 nM sucrose, 2.5 mM EDTA (pH 7.5), and 12.5 mM Tris-HCl (pH 7.5)]. The resulting organelle suspension was centrifuged at 1,300 g for 5 min at 4 °C, the supernate was transferred to a new tube, and centrifuged at 17,000 g for 15 min; this pellet contained the mitochondrial/ER enriched fraction.

[0182] Next, the supernate was centrifugated 100,000 g for 30 min at 4°C. The pellet contained the enriched plasma membrane and the supernate the cytosol fraction. The subcellular fractionbound phage population was recovered through infection of log-phase k91kan E. coli for 1 h at room temperature. Serial dilutions of the infected bacteria were plated on Luria-Bertani (LB) plates containing tetracycline (40 mg/ml) and kanamycin (100 mg/ml) to determine the recovery phage titer.

[0183] Intracellular iPhage competition assay. KS1767 cells were cultured in 6- well plate and incubated with increasing concentrations of iPhage (10⁵, 10⁷, 10⁹ TU) were mixed with decreasing concentrations of YKWYYRGAA (SEQ ID NO:3) or MLS-iPhage (10⁹, 10⁷, 10⁵ TU) respectively [i.e., 10⁵ iPhage + MLS-iPhage 10 9 (1 : 10,000), 10⁷ iPhage + 10⁷ MLS- iPhage (1 : 1), 10⁹ iPhage + MLS-iPhage 10⁵ (10,000: 1)]. After 24 h incubation, cells were washed, trypsinized and wash with ice-cold PBS. Then, the cell pellet was incubated with hypotonic buffer [10 niM NaCl, 1.5 niM MgCl₂, 10 niM Tris-HCl (pH 7.5)] for 15 min, and placed in a standard dounce homogenizer to disrupt cell membranes. Next, the inventors added stabilization buffer [525 mM mannitol, 175 nM sucrose, 2.5 mM EDTA (pH 7.5), and 12.5 mM Tris-HCl (pH 7.5)]. The resulting organelle suspension was centrifuged at 1,300 g for 5 min at 4 °C, the supernate was transferred to a new tube, and centrifuged at 17,000 g for 15 min; this pellet contained the mitochondrial/ER enriched fraction. The subcellular fraction-bound phage population was recovered through infection of log-phase k91kan E. coli for 1 h at room temperature. Serial dilutions of the infected bacteria were plated on LB plates containing tetracycline (40 mg/ml) and kanamycin (100 mg/ml) to obtain single bacteria clones. Next, the inventors randomly pick 96 colonies per competition assay, each clone was phage-PCR and DNA sequenced. [0184] DNA fragmentation assay. KS1767 cells were cultured in 96-well plate and exposed to different peptides (30 μΜ) or media as a negative control. The microplate was incubated for 4 h at 37°C and 5%C0₂. Next, the inventors used the cell death detection ELISA plus and follow the manufacturer's recommendations (Roche). The assay was run in triplicate.

[0185] Protein Expression and Purification. The recombinant GST-fusion expressing ribosomal protein L29 (RPL29) was generated by PCR cloning, with forward and reverse primers respectively (5'-CACAGAATTCATGGCGAAGTCCAAGAACCACACC-3' (SEQ ID NO:8));

[0186] 5 '-CACAGCGGCCGCCTACTCTGAAGCCTTTGTAGGGGCCTGG-3 ' (SEQ ID NO:9)). Five nanograms of plasmid (human RPL29 clone, Invitrogen) were used as a template in a 50 [11 PCR reaction with high fidelity Taq polymerase (Stratagene). The cycling parameters consisted of one cycle of 94 °C for 3 min, 35 cycles of 94 °C for 0.5 min, 60 °C for 1 min, and 68 °C for 1 min, followed by a single 10 min cycle at 72 °C for extension. The PCR product was purified on Qiagen columns, and both plasmid (pGEX4T-l; Amersham) and PCR product were digested with EcoRI and Not I, gel-purified, and ligated overnight at 16 °C. Ligated products were precipitated in ethanol and electroporated into DH5a E. coli; these bacteria were plated on Luria-Bertani (LB) agar containing carbenicillin (50 mg/ml). GST- RPL29 and GST expressing plasmids were transformed into BL21 E. coli for recombinant protein expression. In brief, BL21 E. coli were cultured in rich liquid media (2XYT) supplemented with 50 mg/ml of carbenicillin until an OD590nm -0.6 was reached, at which point GST expression was induced with 1 mM IPTG. After overnight incubation at 30 °C, bacteria were collected by centrifugation (5,000 g for 10 min) and lysed with Bugbuster reagent (EMD Biosciences, CA). Purified recombinant proteins were analyzed by staining with Coomassie blue, and by Western blotting with anti-GST antibody (Amersham).

[0187] Southern blot. KS1767 cells were grown in 6-well tissue-plate and incubated with 10⁹ transducing units (TU) of iPhage, mutant iPhage, or parental phage in MEM containing 1% FBS at 37 °C. After a 24 h incubation, the cells were washed, and trypsinized. The genomic DNA was extracted by using a commercial kit (QIAGEN DNeasy blood & tissue kit). The DNA was analyzed in a 0.8% agarose gel and treated with acidic (0.25 M HC1), denaturing (0.5N NaOH; 1M NaCl) and neutralizing (0.5M Tris pH 7.4; 1.5M NaCl) solutions. Next, genomic DNA was transferred into a nylon membrane by overnight capillarity using 10X saline-sodium citrate (SSC) buffer. The membrane was UV crosslinked and washed with 2X SSC buffer for 5 min. Then, the inventors generated a phage genomic probe by using the PCR primers forward 5 '-TTTATACGGGCACTGTTACTCAAG-3 ' (SEQ ID NO: 10) and reverse 5'- TTTCATCGGCATTTTCGGTCATAG-3 ' (SEQ ID NO: 11). After PCR amplification, the 322 bp fragment was gel purified (QIAquick, QIAGEN) and radiolabeled using a commercial kit (Megaprime DNA labeling kit, Amersham). Finally, the nylon membrane containing the genomic DNA was hybridized with ³²P-probe, washed under stringent conditions and exposed to X-ray film overnight at -80°C for 24 h.

[0188] Transmission Electron Microscopy. Cells were fixed with a solution containing 3% glutaraldehyde plus 2% PFA in 0.1 M cacodylate buffer (pH 7.3) for 1 h. After fixation, the samples were washed, treated with 0.1% Millipore-filter cacodylate-buffered tannic acid, and post-fixed with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated and embedded in LX-112 medium, and polymerized in a 70°C oven for 2 days. Ultra-thin sections were cut on an ultracut microtome (Leica), stained with uranyl acetate and lead citrate in a Leica EM stainer and examined on a JEM 1010 transmission electron microscope (JEOL). Digital images were captured at an accelerating voltage of 80 kV (Advanced Microscopy Techniques Corp).

EXAMPLE 7

PEPTIDE-LIGAND TARGETING OF INTRACELLULAR ANNEXIN A2 DISRUPTS

ADHESION AND MIGRATION OF TUMOR CELLS

[0189] Adhesion and migration are mechanisms used by tumor cells to promote metastasis in distal organ sites. These mechanisms are regulated by cytoskeletal-interacting proteins, which are relevant therapeutic targets for cancer. Therefore, targeting intracellular pathways by delivery of drugs to specific cell compartments is useful as a potent and selective therapeutic approach. However, the discovery of drugs targeting the cytoskeleton components has been a currently challenging task. As described herein, the inventors provide a new family of peptide-targeted internalizing-phage (iPhage) libraries that enter mammalian cells through a receptor-independent, and enables peptide-ligand selection for organelle receptor targeting. The present example concerns the receptor and functional validation of the LGRFYAASG (SEQ ID NO: 12)-internalizing homing peptide (iHoPe) that undergoes receptor-independent internalization and disrupts adhesion and migration via Annexin A2. Furthermore, cancer cells preloaded with LGRFYAASG(SEQ ID NO: 12)-penetratin (LGRFYAASG-pen) peptide and administrated intravenously decreased the formation of experimental lung metastasis. In specific embodiments of the invention, LGRFYAASG(SEQ ID NO: 12)-pen has the ability to disrupt adhesion and migration via cytoskeleton-Annexin A2 function and is a useful peptidomimetic to disrupt metastasic capacity of cancer cells.

[0190] Cell adhesion and motility require a dynamic remodeling of the actin cytoskeleton in response to extracellular stimuli (Parson et al, 2010). The regulation of the cytoskeleton involves the coordinated activity of numerous of proteins, among which members of the annexin family of Ca²⁺ and phospholipid binding proteins play an important role. Studies have shown that annexin A2 (ANXA2) is implicated in the organization of membrane domains and cytoskeleton contacts that mediate adhesion and migration (Gerke et al, 2005). Molecularly, ANXA2 binds to cytoskeletal proteins F-actin and non-erythroid spectrin in a Ca²⁺-dependent manner (Gerke and Weber, 1984), and mediates cell scattering, branching morphogenesis via cofilin activation (de Graauw et al, 2008). Moreover, clinical studies have shown that ANXA2 is overexpressed in several types of tumors, including gastric carcinoma, colorectal cancer, pancreatic cancer, breast cancer, high-grade gliomas, kidney cancer and vascular tumors, in which can be used as a prognostic marker (Mussunoor and Murray, 1008; Lokman et al, 2011). Because in vivo studies have described the functional role of anxA2 in homing and invasion activity of cancer cells (Zhao et al, 2010; Sharma et al, 2010; Shiozawa et al, 2008; Jung et al, 2007), in particular aspects of the invention ANXA2 is a target for therapeutic intervention.

[0191] Phage display has been a versatile technology to fingerprint cell surface receptors in vitro and in vivo conditions. For instance, the inventors have determined that conditionally immortalized lung endothelial cells surprisingly recapitulate in vitro at least some of their tissue-specific molecular diversity, a discovery that enabled them to return to the living mouse and target the pulmonary microcirculation by injection of a lung endothelial cell-selective synthetic peptide ligand fused to a proapoptotic agent, resulting in generation of an emphysema disease model (Giordano et al, 2008). Recently, the inventors have reported a vascular ligand- receptor mapping by direct combinatorial phage display selection in cancer patients. The inventors have validated several ligand-receptors among multiple tissues (integrin a4/annexin A4 and cathepsin B/apolipoprotein E3) and restricted in normal tissue (prohibitin/annexin A2) or cancer (RAGE/leukocyte proteinase-3). These results mean that these intracellular molecules are exposed in the cell surface under specific pathological settings (Staquicini et al, 2011). Importantly, the peptide sequences displayed in the phage mimic intracellular proteins indicate that these molecules are relevant targets to disrupt intracellular interactions. However, targeting some of these intracellular molecules (i.e., AnxA2, cathepsin B) involved in the cytoskeleton regulation by using phage display technology has not been addressed yet.

[0192] As described herein, there is a new type of filamentous phage-based reagents that integrate the penetratin peptide as a fusion protein with the recombinant major coat protein (rpVIII) that enable receptor-independent phage particle entry into mammalian cells. The inventors have termed this new family of reagents "internalizing phage" (iPhage). Moreover, random peptide iPhage libraries allowed the intracellular selection of an internalizing homing peptide (iHoPe) isolated from the mitochondria/endoplasmic reticulum fraction that activates several cell death pathways (Rangel et al, in press). Therefore, the iPhage technology can target intracellular components involved in the cytoskeleton regulation that are abundantly located in the cytosol fraction of cancer cells. [0193] The present Example reports the discovery of the LGRFYAASG (SEQ ID NO: 12)-iHoPe selectively enriched in the cytosol fraction of the Kaposi sarcoma (KS) cell line. The LGRFYAASG (SEQ ID NO: 12) targeting motif chemically fused to penetratin disrupts adhesion and migration via ANXA2 in different tumor and primary cells. LGRFYAASG(SEQ ID NO: 12)-pen is useful to disrupt adhesion and migration via cytoskeleton-ANXA2 function and is useful to disrupt metastasis.

[0194] Exemplary Material and Methods

[0195] Reagents

[0196] The following fluorescence probes were used: 4',6-diamidino-2- phenylindole (DAPI; Vector Laboratories), Alexa Fluor® 488 Phalloidin conjugate (Invitrogen). The invenotrs used several commercially available recombinant proteins: ANXA1, 2, 4, and 5 (Amprox); fibronectin and vitronectin (R&D Systems). The recombinant GST protein was produced from the vector pGEX4T-l (Amersham) and was purified according to their instructions. All the synthetic peptides were produced to specifications (Polypeptide Laboratories).

[0197] Cell culture

[0198] Human Kaposi sarcoma KS1767 cells (a gift from Dr. S. Leventon-Kriss) were maintained in Modified Eagle's Medium (MEM) containing 10% fetal bovine serum (FBS), MEM-vitamins, non-essential amino acids, and penicillin G plus streptomycin S0₄ plus L-glutamine (Gibco) at 37°C in a 5% CO₂ humidified incubator. The B 16F 10 melanoma cell line was obtained from the American Type Culture Collection and culture according to their recommendations .

[0199] iPhage library intracellular selection

[0200] KS1767 cells were infected with 5 x 10¹¹ TU of the X₄YX₄ iPhage library overnight at 37°C. The next day, cells were washed extensively with pre- warmed PBS and were subsequently detached with added trypsin. Cells were washed with ice-cold PBS, incubated with hypotonic buffer [10 mM NaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl (pH 7.5)] for 15 minutes, and placed in a standard Dounce homogenizer to disrupt cell membranes. Next, the inventors added a 2.5x stabilization buffer [525 mM mannitol, 175 nM sucrose, 2.5 mM EDTA (pH 7.5), and 12.5 mM Tris-HCl (pH 7.5)]. The resulting organelle suspension was centrifuged at 1,300 g for 5 minutes at 4°C to enrich for the nuclear fraction. The supernatant was transferred to a new tube and was centrifuged at 17,000 g for 15 minutes; this pellet contained the mitochondrial/ER fraction. Finally, the remaining supernatant was centrifuged at 100,000 g for 40 minutes to obtain the cytosol fraction. The subcellular fraction-bound phage population was recovered through infection of log-phase k91kan E. coli for 1 hour at RT. Serial dilutions of the infected bacteria were plated on LB plates containing tetracycline (40 mg/ml) and kanamycin (100 mg/ml) to determine the recovery phage titer. In addition, infected bacteria were grown overnight in LB containing tetracycline and kanamycin, and phage was recovered by the PEG/NaCl precipitation method. After three rounds of selection, 96 bacterial colonies from each fraction were randomly selected for DNA sequencing.

[0201] Binding assay and ligand-receptor purification

[0202] The synthetic peptides were coupled to a Sepharose column (Carboxy-link kit, Pierce) and the columns were exposed to KS1767 cell-derived lysates. After extensive washing, each receptor was eluted with the corresponding competitive peptide at a concentration of 5 mM. Eluted fractions were analyzed by absorbance [optical density (OD) at 280 nm], dialyzed, and concentrated at 4°C. Equal amounts of protein (5 mg/well in 50 ml PBS) were immobilized on a 96-well plate overnight at 4°C. Wells were washed twice with PBS, blocked with PBS containing 2% BSA for 2 hours at RT, and incubated with targeted iPhage and insertless iPhage (10⁹ TU each) in 50 ml PBS containing 0.1% BSA. After 2 hours at RT, wells were washed ten times with PBS, and each phage population was recovered by host bacterial infection.

[0203] Cell viability assay [0204] KS1767 cells grown in 48-well plates were maintained for 24 hours in non- supplemented MEM. The medium was subsequently supplemented with 10% FBS plus each respective test peptide (1, 3, and 30 mM). Cellular viability was assessed by using the MTT assay (Roche). [0205] Cell adhesion assay

[0206] For cell adhesion assays, the inventors coated 48-well flat-bottom plates with 100 ml of fibronectin (20 mg/ml) or vitronectin (10 mg/ml) in PBS overnight at 4°C. The wells were washed twice with PBS and blocked with PBS containing 1% BSA for 2 hours at RT. Cells were removed and loaded with different internalizing peptides or control peptides for 30 minutes at 37°C. A total of 50,000 cells in 200 ml per well were incubated at 37°C for 1 hour. Non-adherent cells were removed by extensive washing with PBS. Cells were counted immediately under the microscope (at least 10 random fields). Finally, the inventors added 20 μΐ of WST-1 reagent and, after 2 hours, we measured the absorbance (OD at 450 nm). [0207] Wound-closure assay

[0208] Cells were grown in 6-well plates until 100% confluence, at which point they formed a continuous monolayer. The inventors created a scratch ("wound") with a pipetman tip. The cells were washed and the medium was replaced with or without peptides (i.e., pen, LGRFYAASG(SEQ ID NO: 12)-pen, LGRFYAASG (SEQ ID NO: 12)). Serial pictures of the wound were taken in corresponding regions of the plate. After a 16-hour incubation, we examined and scored the degree of closure by phase contrast microscopy.

[0209] Confocal imaging.

[0210] KS1767 cells were grown in 16-well tissue-chamber slides and were maintained for 24 h in non-supplemented MEM. The medium was subsequently supplemented with 10% FBS plus each respective test peptide (i.e., pen, LGRFYAASG(SEQ ID NO: 12)-pen, LGRFYAASG (SEQ ID NO: 12) peptides) or media as control. After 6 h of incubation, cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 15 min. After washes with PBS, fixed cells were permeabilized with PBS containing 0.1% Triton X-100 (Sigma) for 5 min, and blocked with PBS containing 1% BSA (10 min). Incubate with incubated with Alexa Fluor® 488 Phalloidin conjugate for 20 min at roo temperature. The cells were washed with PBS and mounted with DAPI and seal. The cells were imaged with an inverted Leica 600B (Leica Mircosystems, Buffalo Grove, IL) laser scanning confocal microscope using a 60x oil objective. [0211] Tumor models.

[0212] B16F 10 melanoma cells were incubated with different peptides [(i.e., pen, LGRFYAASG(SEQ ID NO: 12)-pen, LGRFYAASG (SEQ ID NO: 12) (30 mM)] for 30 min. Then, the cells were washed with plain MEM media. C57BL/6 male mice 8-12 week were injected intravenously with 10⁵ B16F10 melanoma cells.

[0213] After 21 days, the lungs were removed, rinsed with phosphate buffer saline (PBS), and weighed. Black metastatic foci were counted under a binocular dissecting microscope. The Institutional Animal Care and Use Committee of the University of Texas M. D. Anderson Cancer Center approved all animal experiments. [0214] Statistics

[0215] Test for statistical significance between groups were carried out using Student t-tests, as indicated, with the level of significance defined as a value of P > 0.05.

[0216] Exemplary Results

[0217] Intracellular screening of an iPhage library yields an iHoPe in the cytosol compartment

[0218] To uncover internalizing homing peptides (iHoPe's) targeting molecules associated to the cytoskeleton, the inventors performed an iPhage library screening on live KS 1767 cells, and recover the cytosol fraction, in which most of the cytoskeleton proteins are enriched. After 24 hours, the inventors recovered the cytosolic fraction and recovered the pool of iPhage through infection of host E. coli. After three rounds of synchronous selection (FIG. 21 A), they observed a marked number of an iPhage clones in the cellular compartment (FIG. 2 IB). By the third round of selection, bioinformatic analysis revealed two peptides found with high frequency in the cytosol fraction, as well as unique, lower-frequency peptide motifs selective to the cell compartment (Table 1A). After two additional rounds of synchronous selection, an iPhage displaying the LGRFYAASG (SEQ ID NO: 12) peptide was recovered from cytosolic fraction (Table IB). By solid-phase (Merrifield) synthesis, internalizing versions of the peptide were generated through C-terminal fusions to pen and were subsequently evaluated for their intracellular entry, and biological activity. [0219] Table 1A: Cytosol peptide sequences isolated after Round III

[0221] Annexin A2 is a receptor for the internalizing LGRFYAASG (SEQ ID NO: 12) peptide

[0222] The inventors used peptide-affinity chromatography to purify the receptor for LGRFYAASG (SEQ ID NO: 12) peptide from KS1767 cell lysates, and subsequently probed the eluted and immobilized fractions for specific binding of iPhage displaying this ligand peptide (FIG. 15 A). SDS-PAGE analysis identified a set of proteins with relative molecular masses (Mr) of 33,000, 36,000 and 38,000 only in the target fraction 46 (FIG. 22). Mass spectrometry revealed several receptor candidates: F-actin capping protein alpha- 1 subunit (CAPZA1), Lim SH3 protein 1 (LASP1), and annexin A2 (ANXA2) (FIG. 15B). Because they eluted CAPZA1- LASP1-ANXA2 proteins by peptide-affinity chromatography, in specific aspects of the invention these molecules interact and form a protein complex as predicted also by using bioinformatic software (FIG. 15C). This data provides experimental evidence that these protein- protein interactions might be occurring at the cellular level.

[0223] The inventors tested the binding of iPhage displaying LGRFYAASG (SEQ ID NO: 12) to these proteins, as well as to other related annexins (FIG. 16A, B), and found LGRFYAASG (SEQ ID NO: 12) to be specific for ANXA2 only; binding was negligible to LASP1, CAPZA1, and other control annexins (ANXA1, ANXA4, or ANXA5). Accordingly, they evaluated the specificity of this peptide-protein interaction by standard biochemical and genetic approaches. The concentration-dependent inhibition of binding of LGRFYAASG(SEQ ID NO: 12)-iPhage to ANXA2 by the synthetic peptide LGRFYAASG (SEQ ID NO: 12) was indicative of specificity (FIG. 16C); moreover, alanine-scanning of LGRFYAASG (SEQ ID NO: 12) revealed that the arginine (R3) and phenylalanine (F4) residues were critical for the specificity of binding to ANXA2 (FIG. 16D). Given these results, we concluded that the ANXA2 is the receptor of the LGRFYAASG(SEQ ID NO: 12)-pen peptide. [0224] The internalizing LGRFYAASG (SEQ ID NO: 12) peptide disrupts cell adhesion and migration

[0225] ANXA2 is an abundant protein on the cytoplasmic side of the plasma membrane (Liu and Vishwanatha, 2007). It interacts with cofilin, actin, alpha-actinin, ezrin, and moesin, among other molecules, to regulate cytoskeletal architecture and dynamics (Bellagamba et al, 1997; harder et al, 1997; Oliferenko et al, 1999; Eberhard et al, 2001; Liu et al, 2003; Jacob et al, 2004; Hayes et al, 2004; Lauvrak et al, 2005; Hayes et al, 2006; Lorusso et al, 2006). Mammalian cells treated with LGRFYAASG(SEQ ID NO: 12)-pen showed a reduced cell F-actin protrusions, whereas internalizing control peptides had no detectable effects on the cells (FIG. 17 A). In certain embodiments of the invention, intracellular delivery of LGRFYAASG (SEQ ID NO: 12) modifies cytoskeletal organization via ANXA2 and subsequent cell detachment and disruption of cell protrusions. In concordance with this interpretation, cell adhesion assays revealed that LGRFYAASG(SEQ ID NO: 12)-pen at log-dose increasing concentrations progressively inhibited cell attachment to fibronectin or vitronectin, whereas internalizing control peptides again had no detectable effects on cell adhesion (FIG. 17B). The effects observed on cell adhesion were not dependent on transformation status (non-malignant or tumor cells), species (mouse or human), or tumor type (melanoma, sarcoma, or carcinoma) (FIG. 23).

[0226] To evaluate peptide-mediated effects on the migration of attached cells, the inventors used an in vitro assay in which cell migration is tested across an abraded area of the dish. KS 1767 cells treated with LGRFYAASG(SEQ ID NO: 12)-pen did not cover the abraded areas; in fact, they became detached from the dish, whereas cells treated with control internalizing peptides showed a normal migratory dose-response (FIG. 18A-B). At increasing molar concentrations of internalizing peptide controls or LGRFYAASG(SEQ ID NO: 12)-pen, cells remained viable for more than 96 hours, making non-specific toxicity an unlikely possibility (FIG. 18C). These data indicate that the internalizing LGRFYAASG (SEQ ID NO: 12) peptide binds to ANXA2 and alters cytoskeletal function, as evidenced by impaired cell attachment, spreading, and migration.

[0227] ANXA2 targeting disrupts metastasis in an experimental lung models

[0228] Previous studies have shown that inactivation of ANXA2 prevents homing of stem cells and disrupts the metastatic activity of cancer cells (Zhao et al, 2010; Sharma et al, 2010; Shiozawa et al, 2008; Jung et al, 2007). Because the LGRFYAASG-pen peptide alters adhesion and migration of cancer cells, in certain aspects of the invention B16F 10 melanoma cells preloaded with the LGRFYAASG(SEQ ID NO: 12)-pen peptide will reduce the formation of lung metastasis when the cancer cells are administrated intravenously. To study this aspect, B 16F10 melanoma cells were preloaded with different peptides for 30 minutes and inoculated intravenously into C57BL/6 mice (FIG. 19). As predicted, only melanoma cells treated with the LGRFYAASG(SEQ ID NO: 12)-pen peptide prevent the formation of cancer colonies in the lung, such are reflected in tumor weights and number of lung metastasic foci (FIG. 20 A-C). Hematoxilin and eosin staining served to validate the identity of malignant colonies in the lungs of mice. Importantly, histological analysis confirmed that media and control peptides (i.e., pen, LGRFYAASG (SEQ ID NO: 12)) developed large colonies compared to fewer but also much smaller metastatic foci in melanoma cells treated with the internalizing LGRFYAASG (SEQ ID NO: 12)peptide (FIG. 20D). Collectively, these findings reveal that the LGRFYAASG(SEQ ID NO: 12)-pen peptide targets ANXA2 by altering the mechanism of cell adhesion and preventing cancer cells to home and growth in the lung endothelium.

[0229] Significance of Certain Embodiments of the Invention

[0230] Targeted phage constructs that penetrate eukaryotic cells through a receptor-independent mechanism provide a novel discovery platform for selection, evaluation, and validation of intracellular molecular interactions in live cells. In embodiments of this work, the inventors discover the LGRFYAASG (SEQ ID NO: 12) iHoPe that binds to ANXA2 and disrupts cytoskeletal functions as cell adhesion and migration.

[0231] In most cells, ANXA2 is present as a cytosolic monomer and as a heterotetrameric complex comprising two molecules of ANXA2 and two of S 100A10 (Johnson et al, 1988). The heterotetramer locates constitutively to the membrane-cytoskeleton where, it might be involved in the organization of receptor cytoplasmatic domains with the subplasma membrane cytoskeleton. Moreover, ANXA2 regulates actin filament turnover by monomer sequestration and barbed-end capping activities (Hayes et al, 2006). Thus, ANXA2 is a modulator of actin dynamics in vivo. Our results add a relevant contribution to the body of evidence for the role of ANXA2 in cell adhesion homing and migration as described as well by others (Zhao et al, 2010; Sharma et al, 2010; Shiozawa et al, 2008; Jung et al, 2007). In addition, the inventors have validated a peptide-ligand able to interact and disrupt ANXA2 function in tumor or normal cells.

[0232] Over the past two decades, phage selection in vitro and in vivo has consistently contributed to cell-surface biology, in the form of previously unrecognized functions for known proteins, novel multi-protein complexes, or targetable expression patterns in pathologic settings, by our own groups (Pasqualini and Ruoshlahti, 1996; Arap et al, 1998; Pasqualini et al, 2000; Arap et al, 2002; Mintz et al, 2003; Kolonin et al, 2004; Zurita et al, 2004; Kolonin et al, 2006; Mintz et al, 2009; Staquicini et al, 2009; Moeller et al, 201 1; Barnhart et al, 2011) and by others (Laakkonen et al, 2002; Higgins et al, 2004; Ballard et al, 2006; Zhang et al, 2006; Hardy et al, 2008). Here the inventors show that intracellular processes and pathways in living ells can be similarly interrogated with an iPhage-based technology. The inventors found that an initially identified peptides have produced striking morphological and functional phenotypes after delivery to the intracellular microenvironment. The inventors reasoned that LGRFYAASG (SEQ ID NO: 12) targets at least one protein involved in modulation of the cytoskeleton, due to the observed disruption of cellular protrusions. Affinity chromatography identified three proteins (CAPZA1, LASP1, and ANXA2) that form a supramolecular protein complex involved in cytoskeletal organization and further identified ANXA2, but not other closely related annexins, as a specific target.

[0233] Microtubules are dynamic filamentous cytoskeletal proteins composed of tubulin and are important therapeutic target in tumor cells. Agents that bind to microtubules have been part of the pharmacopoeia of anticancer therapy for decades (Jordan et al, 1993; Tian et al, 1996; Wahl et al, 1996; McCarroll et al, 2006; Jordan and Kamath, 2007). All of the currently approved compounds bind directly to tubulin, therefore the need to identify novel drugs to target alternative molecules that regulate cytoskelton dynamics is needed. Here we report a peptide compound that affects cytoskeleton structures affecting cell adhesion and migration, by targeting ANXA2. Future peptidomimetics studies will unveil its clinical applications for different pathological settings.

[0234] The exemplary studies with iPhage have identified the LGRFYAASG (SEQ ID NO: 12) peptide that when delivered intracellularly modulates cytoskeletal reorganization; it is now clear that many such functionally relevant peptides can be discovered with this new platform. In certain embodiments of the invention, one can combine receptor-targeting peptides that provide tissue selectivity with intracellular bioactive peptides discovered by iPhage; after specific delivery, such constructs can modulate cell function in a tissue- and organ-specific fashion. The technological resource described here can target intracellular ZIP codes, interrogate signal transduction pathways, and participate in developing the foundation of an organelle-targeted cell biology and pharmacology in mammalian and possibly other eukaryotic cells.

EXAMPLE 8

EXEMPLARY CELL-PENETRATING PEPTIDES [0235] In some embodiments of the invention, the compositions and methods employ one or more cell-penetrating peptides. The cell-penetrating peptides may be of any suitable length and content, although in specific embodiments only one or more of the cell- penetrating peptides from Table 3 are utilized.

[0236] Table 3. Sequences of the Cell-penetrating peptides (Deshayes et al, 2005)

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[0238] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims

1. An isolated peptide of 50 amino acids or less that selectively binds an intracellular organelle, wherein the isolated peptide comprises a peptide sequence of SEQ ID NO: 3 or SEQ ID NO: 12.

2. The isolated peptide of claim 1, wherein the isolated peptide is operatively coupled to a cell penetrating peptide.

3. The isolated peptide of claim 1, wherein the isolated peptide is operatively coupled to a specific tissue/cell targeting moiety.

4. The isolated peptide of claim 3, wherein the specific tissue/cell targeting moiety comprises a peptide.

5. The isolated peptide of claim 3, wherein the specific tissue/cell targeting moiety comprises an antibody.

6. The isolated peptide of claim 1 , further comprising a therapeutic agent, an imaging agent, a diagnostic agent, or a combination thereof.

7. The isolated peptide of claim 6, wherein said therapeutic agent, imaging agent, or diagnostic agent is a drug, small molecule, a chemotherapeutic agent, a radioisotope, a pro- apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a cytotoxic agent, a cytocidal agent, a cytostatic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, a hormone antagonist, a nucleic acid or an antigen.

8. The isolated peptide of claim 7, wherein the anti-angiogenic agent is selected from the group consisting of thrombospondin, angiostatin5, pigment epithelium-derived factor, angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP- 10, Gro-B, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CMlOl, Marimastat, pentosan polysulphate, angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP- 470, endostatin, paclitaxel, Docetaxel, polyamines, a proteasome inhibitor, a kinase inhibitor, a signaling peptide, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline.

9. The isolated peptide of claim 7, wherein said pro-apoptosis agent is selected from the group consisting of etoposide, ceramide sphingomyelin, Bax, Bid, Bik, Bad, caspase-3, caspase- 8, caspase-9, fas, fas ligand, fadd, fap-1, tradd, faf, rip, reaper, apoptin, interleukin-2 converting enzyme or annexin V.

10. The isolated peptide of claim 7, wherein said cytokine is selected from the group consisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon- (IF- ), IF-a, IF-β, tumor necrosis factor-a (TNF-a), or GM-CSF (granulocyte macrophage colony stimulating factor).

11. The isolated peptide of claim 6, wherein the diagnostic agent is selected from the group consisting of an enzyme, a fluorescent label, a near infrared label, a luminescent label, a bioluminescent label, a magnetic label, and biotin.

12. The isolated peptide of claim 6, wherein the imaging agent is selected from the group consisting of an enzyme, a fluorescent label, a near infrared label, a luminescent label, a bioluminescent label, a magnetic label, and biotin.

13. The isolated peptide of claim 7, wherein said radioisotope is selected from the group consisting of 64Cu, l l lln 213Bi, 103Pd, 133Xe, 1311, 68Ge, 57Co, 65Zn, 85Sr, 32P, 35S, 90Y, 153Sm, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 1 13Sn, 1 17Sn, 186Re, 166Ho and 188Re.

14. The isolated peptide of claim 7, wherein said nucleic acid agent comprises siR A, shRNA, miRNA, or antisense RNA.

15. The isolated peptide of claim 1, wherein said peptide is attached to or is part of a molecular complex.

16. The isolated peptide of claim 15, wherein said complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a yeast cell, a mammalian cell or a cell.

17. The isolated peptide of claim 15, wherein said complex is a virus or a bacteriophage.

18. The isolated peptide of claim 17, wherein said virus is selected from the group consisting of adenovirus, retrovirus and adeno-associated virus.

19. The isolated peptide of claim 17, wherein said virus is further defined as containing a gene therapy vector.

20. The isolated peptide of claim 1, wherein said peptide is attached to a eukaryotic expression vector.

21. The isolated peptide of claim 20, wherein said vector is a gene therapy vector.

22. The isolated peptide of claim 1, wherein the peptide is 40 amino acids or less in length.

23. The isolated peptide of claim 1, wherein the peptide is 30 amino acids or less in length.

24. The isolated peptide of claim 1, wherein the peptide is 20 amino acids or less in length.

25. The isolated peptide of claim 1, wherein the peptide is 10 amino acids or less in length.

26. A pharmaceutical composition comprising a peptide according to any one of claims 1-25.

27. An in vitro method of targeting an entity to an organelle in a cell by providing to the cell a composition comprising the entity and a peptide comprising SEQ ID NO:3, wherein the organelle is the mitochondria or endoplasmic reticulum.

28. The method of claim 27, wherein the composition further comprises a cell penetrating peptide.

29. The method of claim 27, wherein the composition is further defined as comprising a specific tissue/cell targeting moiety.

30. The method of any one of claims 27 to 29, wherein the entity comprises a therapeutic agent, imaging agent, or diagnostic agent.

31. The method of claim 30, wherein the therapeutic agent is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a cytotoxic agent, a cytocidal agent, a cytostatic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, a hormone antagonist, a nucleic acid or an antigen.

32. The method of claim 27, wherein the composition is further defined as being a bacteriophage.

33. An in vitro method of targeting an entity to an organelle in a cell by providing to the cell a composition comprising the entity and a peptide comprising at least one of SEQ ID NOS: 12- 31, wherein the organelle is the cytoskeleton.

34. The method of claim 33, wherein the composition further comprises a cell penetrating peptide.

35. The method of claim 33, wherein the composition is further defined as comprising a specific tissue/cell targeting moiety.

36. The method of claim 33, wherein the composition is further defined as being a bacteriophage.

37. The method of claim 33, wherein the entity comprises a therapeutic agent, imaging agent, or diagnostic agent.

38. The method of claim 37, wherein the therapeutic agent is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a cytotoxic agent, a cytocidal agent, a cytostatic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, a hormone antagonist, a nucleic acid or an antigen.

39. A composition for use in treating or imaging a subject comprising an isolated peptide according to any one of claim 1-15.

40. An isolated bacteriophage, comprising a recombinant protein comprising a major coat protein and a cell-penetrating peptide.

41. The phage of claim 40, wherein the major coat protein comprises pVIII.

42. The phage of claim 40, wherein the cell-penetrating peptide comprises penetratin.

43. The phage of claim 40, wherein the phage further comprises an intracellular homing peptide.

44. The phage of claim 43, wherein the intracellular homing peptide selectively binds an organelle.

45. The phage of claim 43, wherein the intracellular homing peptide has cell death activity, adhesion activity, and/or migration activity.

46. The phage of claim 40, further defined as comprising a recombinant protein comprising a minor coat protein and an intracellular homing peptide.

47. The phage of claim 43, wherein the intracellular homing peptide comprises SEQ ID NO:3 or SEQ ID NO: 12.

48. The phage of claim 43, wherein the intracellular homing peptide is 50 amino acids or less and comprises SEQ ID NO:3 or SEQ ID NO: 12.

49. The phage of claim 46, wherein the minor coat protein comprises pill.

50. The phage of any one of claims 40-49, further comprising a therapeutic and/or diagnostic agent.

51. A method of producing phage having cell penetrating activity and intracellular localization activity, said method comprising the steps of:

obtaining a population of bacteriophages in accordance with claim 40, said bacteriophages comprising randomized peptides inserted within a sequence of the minor pill coat protein to produce test phages; and

assaying the test phage for intracellular localization activity.

52. The method of claim 51, wherein the randomized peptides are linear or cyclic.