AU2007203251A1 - Compositions and methods for targeting peptides in humans in vivo - Google Patents

Description

Australian Patents Act 1990 Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title ""Compositions and methods for targeting peptides in humans in vivo"" The following statement is a full description of this invention, including the best method of performing it known to me/us:- P/00/011 Q:\OPER\DND\005-June 200725607 div fil docs 30255182.doc 12/7/07 P:\OPERODND30255182 dlv spi pag 179doc.IO/O7/2007 COMPOSITIONS AND METHODS FOR TARGETING PEPTIDES IN HUMANS IN VIVO This is a divisional of Australian Patent Application No. 2001290662, the entire t contents of which are incorporated herein by reference.

(Ni BACKGROUND OF THE INVENTION This application claims the benefit of U.S. Provisional Patent Application No.

60/231,266 filed September 8, 2000, and U.S. Patent Application No. 09/765,101, filed January 17, 2001. This invention was made with government support under grants DAMD 17-98-1-8041 and 17-98-1-8581 from the U.S. Army and grants 1R01CA78512-01A1, 1R1CA90810-01 and 1R01CA82976-01 from the National Institutes of Health. The government has certain rights in this invention.

1. Field of the Invention The present invention concerns the fields of molecular medicine and targeted delivery of therapeutic agents. More specifically, the present invention relates to compositions and methods for identification and use of peptides that selectively target organs, tissues or cell types in the human body.

2. Description of Related Art Therapeutic treatment of many human disease states is limited by the systemic toxicity of the therapeutic agents used. Cancer therapeutic agents in particular exhibit a very low therapeutic index, with rapidly growing normal tissues such as skin and bone marrow affected at concentrations of agent that are not much higher than the concentrations used to kill tumor cells. Treatment of cancer and other organ, tissue or cell type confined disease states would be greatly facilitated by the development of compositions and methods for targeted delivery to a desired organ, tissue or cell type of a therapeutic agent.

Recently, an in vivo selection system was developed using phage display libraries to identify organ, tissue or cell type targeting peptides in a mouse model system. Phage display libraries expressing transgenic peptides on the surface of bacteriophage were initially developed to map epitope binding sites of WO 02/20723 PCT/US01/28044 iimmunoglobulins (Smith and Scott, 1986, 1993). Such libraries can be generated by Sinserting random oligonucleotides into cDNAs encoding a phage surface protein, I generating collections of phage particles displaying unique peptides in as many as 10 9 permutations. (Pasqualini and Ruoslahti, 1996, Arap et al, 1998a; Arap et al 1998b).

Vt Intravenous administration of phage display libraries to mice was followed by Cc the recovery of phage from individual organs (Pasqualini and Ruoslahti, 1996). Phage

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CI were recovered that were capable of selective homing to the vascular beds of different 0mouse organs, tissues or cell types, based on the specific targeting peptide sequences CI expressed on the outer surface of the phage (Pasqualini and Ruoslahti, 1996). A variety of organ and tumor-homing peptides have been identified by this method (Rajotte et al., 1998, 1999; Koivunen et al., 1999; Burg et al., 1999; Pasqualini, 1999). Each of those targeting peptides bound to different receptors that were selectively expressed on the vasculature of the mouse target tissue (Pasqualini, 1999; Pasqualini et al., 2000; Folkman, 1995; Folkman 1997). Tumor-homing peptides bound to receptors that were upregulated in the tumor angiogenic vasculature of mice (Brooks et al., 1994; Pasqualini et al., 2000). In addition to identifying individual targeting peptides selective for an organ, tissue or cell type (Pasqualini and Ruoslahti, 1996; Arap et al, 1998a; Koivunen et al., 1999), this system has been used to identify endothelial cell surface markers that are expressed in mice in vivo (Rajotte and Ruoslahti, 1999).

Attachment of therapeutic agents to targeting peptides resulted in the selective delivery of the agent to a desired organ, tissue or cell type in the mouse model system.

Targeted delivery of chemotherapeutic agents and proapoptotic peptides to receptors located in tumor angiogenic vasculature resulted in a marked increase in therapeutic efficacy and a decrease in systemic toxicity in tumor-bearing mouse models (Arap et al., 1998a, 1998b; Ellerby et al., 1999).

Previous studies with phage display libraries have relied on a mouse model system to identify targeting peptides and their receptors, under the assumption that human targeting peptides are homologuous. However, cell surface receptors may have WO 02/20723 PCT/US01/28044 CNI a very different distribution and function in humans than in mice. Further, the mouse Smodel system has been exploited to characterize targeting peptides for only a handful of C' specific organs. A need exists in the art for methods and compositions for identification of organ, tissue or cell type selective targeting sequences in humans that _can be of clinical use for targeted delivery of therapeutic agents and in vivo imaging.

SSUMMARY OF THE INVENTION The present invention solves a long-standing need in the art by providing compositions and methods for the identifying and using targeting peptides that are selective for human organs, tissues or cell types.

Certain embodiments of the present invention concern methods of identifying targeting peptides, comprising injecting a phage display library into the circulation of a human, obtaining a sample of one or more organs, tissues or cell types from said human and identifying one or more targeting peptides from phage present in said organ, tissue or cell type. In preferred embodiments, phage are recovered from an organ, tissue or cell type of a first human subject and further selected by reinjection into the circulation of a second human, followed by obtaining a sample of the same organ, tissue or cell type and identifying targeting peptides. Screening may be performed multiple times in order to obtain targeting phage of sufficient selectivity.

In another preferred embodiment, the human is brain dead or a terminal wean patient. More preferably, the individual is not a candidate organ donor. In other preferred embodiments, one or more primary phage libraries may be amplified in vitro, preferably to 1014 TU (transforming units) or higher, more preferably with a diversity of 2 x 108 or more different clones, prior to injection into a human subject. In an even more preferred embodiment, targeting peptide sequences are subjected to a custom designed statistical analysis, described in detail in the following Examples, to identify statistically significant targeting peptide sequences.

WO 02/20723 PCT/US01/28044 In yet another preferred embodiment, phage isolated from multiple organs, Stissues or cell types from a first human are mixed together and injected into the 1 circulation of a second human, followed by recovery and characterization of phage from the same organs, tissues or cell types of the second human.

tty In some embodiments, phage collected from an organ, tissue or cell type may be Cc, recovered by contacting a homogenate of the organ, tissue with an appropriate host cell,

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CI such as E. coli K91 kan, and allowing the phage to multiply within the host cell in Sbetween rounds of selection. In alternative embodiments, phage may be recovered by CI direct amplification, such as by PCR T M of the targeting peptide encoding sequences.

In certain embodiments, the targeting peptides of the present invention are of use for the selective delivery of therapeutic agents, including but not limited to gene therapy vectors and fusion proteins, to specific organs, tissues or cell types in human patients. The skilled artisan will realize that the scope of the claimed methods of use include any disease state that can be treated by targeted delivery of a therapeutic agent to a desired organ, tissue or cell type in a human patient. Although such disease states include those where the diseased cells are confined to a specific organ, tissue or cell type, such as non-metastatic cancer, other disease states may be treated by an organ, tissue or cell type-targeting approach.

One embodiment of the present invention concerns isolated peptides of 100 amino acids or less in size, comprising at least 3 contiguous amino acids of a targeting peptide sequence, selected from any of SEQ ID NO:5 through SEQ ID NO:325.

In a preferred embodiment, the isolated peptide is 50 amino acids or less, more preferably 30 amino acids or less, more preferably 20 amino acids or less, more preferably 10 amino acids or less, or even more preferably 5 amino acids or less in size.

In other preferred embodiments, the isolated peptide of claim 1 comprises at least 4, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous amino acids of a targeting peptide sequence, selected from any of SEQ ID through SEQ ID NO:325.

WO 02/20723 PCTUS01/28044 In certain embodiments, the isolated peptide is attached to a molecule. In preferred embodiments, the attachment is a covalent attachment In additional 7 embodiments, the molecule is a drug, a chemotherapeutic agent, a radioisotope, a proapoptosis 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, an imaging agent, a survival factor, an anti-apoptotic agent, a hormone antagonist, a virus, a bacterium, a yeast cell, a mammalian cell, a nucleic acid or an antigen. Those molecules are representative only. Molecules within the scope of the present invention include virtually any molecule that may be attached to a targeting peptide and administered to a human. In preferred embodiments, the pro-aptoptosis agent is gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK)2 (SEQ ID NO:1), (KLAKKLA) 2 (SEQ ID NO:2), AAKKAA) 2 (SEQ ID NO:3) or

(KLGKKLG)

3 (SEQ ID NO:4). In other preferred embodiments, the anti-angiogenic agent is angiostatin5, pigment epithelium-drived 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, CM101, Marimastat, pentosan polysulphate, angiopoietih 2 (Regeneron), 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 inhibitor (SU5416 or SU6668, Sugen, South San Francisco, CA), accutin, cidofovir, vincristine, bleomycin, AGM- 1470, platelet factor 4 or minocycline. In further preferred embodiments, the cytokine is interleukin 1 IL-2, IL-5, IL-10, IL-11, l.12, IL-18, interferon-y IF-a, IF-B, tumor necrosis factor-a (TNF-a), or GM-CSF (granulocyte macrophage colony stimulating factor). Such examples are representative only and are not intended to exclude other pro-apoptosis agents, anti-angiogenici agents or cytokines known in the art.

WO 02/20723 PCT/US01/28044 U In other embodiments, the isolated peptide is attached to a macromolecular Scomplex. In preferred embodiments, the attachment is a covalent attachment. In other preferred embodiments, the macromolecular complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle,, a nagnetic bead, a cell, a yeast cell or a microdevice. These are representative exam'ples only. Macromolecular complexes within the scope of the present invention include virtually any macromolecular complex that may be attached to a targeting peptide ahd administered to a human. In other preferred embodiments, the isolated peptide is attached to a eukaryotic expression 0vector, more preferably a gene therapy vector.

In another embodiment, the isolated peptide is attached to a solid support, preferably magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.

Additional embodiments of the present invention concern fusion proteins comprising at least 3 contiguous amino acids of sequence selected from any of SEQ ID NO:5 through SEQ ID NO:325.

Certain other embodiments concern compositions comprising the claimed isolated peptides or fusion proteins in a pharmaceutically acceptable carrier. Further embodiments concern kits comprising the claimed isolated peptides or fusion proteins in one or more containers.

Other embodiments concern methods of targeted delivery comprising selecting a targeting peptide for a desired organ, tissue or cell type, attaching said targeting peptide to a molecule, macromolecular complex or gene therapy vector, and providing said peptide attached to said molecule, complex or vetor to a human. Preferably, the targeting peptide is selected to include at least 3 contiguous amino acids from any of SEQ ID NO:5 through SEQ ID NO:325. In certain referred embodiments, the organ, tissue or cell type is bone marrow, skin, skeletal muscle, prostate or adipose tissue. In 6 WO 02/20723 PCT/US01/28044 other preferred embodiments, the molecule attached to the targeting peptide is a Schemotherapeutic agent, an antigen or an imaging agent The skilled artisan will realize c- that within the scope of the present invention any human organ, tissue or cell type can be targeted for delivery, using targeting peptides attached to any molecule, macromolecular complex or gene therapy vector.

c-i c Other embodiments of the present invention concern isolated nucleic acids of

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CN 300 nucleotides or less in size, encoding a targeting peptide. In preferred embodiments, c-- Sthe isolated nucleic acid is 250, 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20 or even C- 10 nucleotides or less in size. In other preferred embodiments, the isolated nucleic acid is incorporated into a eukaryotic or a prokaryotic expression vector. In even more preferred embodiments, the vector is a plasmid, a cosmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a virus or a bacteriophage. In other preferred embodiments, the isolated nucleic acid is operatively linked to a leader sequence that localizes the expressed peptide to the extracellular surface of a host cell.

Additional embodiments of the present invention concern methods of treating a disease state comprising selecting a targeting peptide that targets cells associated with the disease state, attaching one or more molecules effective to treat the disease state to the peptide, and administering the peptide to a human with the disease state.

Preferably, the targeting peptide includes at least three contiguous amino acids selected from any of SEQ ID NO:5 through SEQ ID NO:325. In preferred embodiments the disease state is diabetes mellitus, inflammatory disease, arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection, viral infection, cardiovascular disease, degenerative disease or macular degeneration.

Another embodiment of the present invention concerns compositions and methods of use of tumor targeting peptides against human cancers. Human tumor targeting peptides identified by the methods disclosed in the instant application may be attached to therapeutic agents, including but not limited to molecules or macromolecular assemblages and administered to a patient with cancer, providing for WO 02/20723 PCT/US01/28044 u 'increased efficacy and decreased systemic toxicity of the therapeutic agent Therapeutic ==agents within the scope of the present invention include but are not limited to c chemotherapeutic agents, radioisotopes, pro-apoptosis agents, cytotoxic agents, cytostatic agents and gene therapy vectors. Targeted delivery of such therapeutic agents to human tumors provides a significant improvement over the prior art for increasing C the delivery of the agent to the tumor, while decreasing the inadvertent delivery of the 0 agent to normal organs and tissues of the patient. In a preferred embodiment, the tumor targeting peptide is incorporated into the capsule of a phage gene therapy vector to 0target delivery of the phage to angiogenic endothelial cells in tumor blood vessels.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A. In vivo phage display screening for peptides that home to human tissues through the systemic circulation. Schematic flowchart of the study.

FIG. 1B. Phage recovery from various human tissues in vivo. Tissue samples were processed and phage recovered as described in Example 3. Shown are means standard errors of the mean of phage transducing units (TU) per gram of tissue obtained from each biopsy site.

FIG. 2A. Identification of extended homing motifs with the ClustalW program (European Molecular Biology laboratory; EMBL). Peptide sequences containing selected tripeptides (Table 3) enriched in each single tissue were aligned in clusters to obtain longer motifs shared between different peptides from each cluster. The software registers sequence identities and similarities among multiple peptide sequences and aligns the sequences by placing peptides with the most identity/similarity next to one another. The original and extended peptide motifs are shown highlighted in bold.

WO 02/20723 PCT/US01/28044 FIG. 2B. Identification of extended homing motifs with the ClustalW program S(European Molecular Biology laboratory; EMBL). Peptide sequences containing CI selected tripeptides (Table 3) enriched in multiple tissues were aligned in clusters to obtain longer motifs shared between different peptides from each cluster. The software registers sequence identities and similarities among multiple peptide sequences and CN aligns the sequences by placing peptides with the most identity/similarity next to one 0 another. The original and extended peptide motifs are shown highlighted in bold.

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

A "targeting peptide" is a peptide comprising a contiguous sequence of amino acids, that is characterized by selective localization to a human organ, tissue or cell type. 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. Administration to a human of a library of such phage that have been genetically engineered to express a multitude of such targeting peptides of different amino acid sequence is followed collection of one or more organs, tissues or cell types from the human and identification of phage found in that organ, tissue or cell type. A phage expressing a targeting peptide sequence is considered to be selectively locallized to a tissue or organ if it exhibits greater binding in that tissue or organ compared to a control tissue or organ. Preferably, selective localization of a targeting peptide will result in a two-fold or higher enrichment of the phage in the target organ, tissue or cell type, compared to a control organ, tissue or cell type. Alternatively, a phage expressing a targeting peptide sequence that exhibits selective localization preferably shows an increased enrichment in the target organ compared to a control organ when phage recovered from the target organ are reinjected WO 02/20723 PCT/US01/28044 into a second host for another round of screening. Further enrichment may be exhibited Jfollowing a third round of screening. Another alternative method to determine selective 1 localization is that phage expressing the putative target peptide preferably exhibit a two-fold, more preferably a three-fold or higher enrichment in the target organ compared to control phage that express a non-specific peptide or that have not been CI genetically engineered to express any putative target peptides. Another method to 0 determine selective localization is that locallization to the target organ of phage expressing the target peptide is at least partially blocked by the co-administration of a Ssynthetic peptide containing the target peptide sequence. 'Targeting peptide" and "homing peptide" are used synonymously herein.

A "phage display library" means a collection of phage that have been genetically engineered to express a set of putative targeting peptides on their outer surface. In preferred embodiments, DNA sequences encoding the putative targeting 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.

A "macromolecular complex" refers to a collection of molecules that may be random, ordered or partially ordered in their arrangement. The term encompasses biological organisms such as bacteriophage, viruses, bacteria, unicellular pathogenic organisms, multicellular pathogenic organisms and prokaryotic or eukaryotic cells. The term also encompasses non-living assemblages of molecules, such as liposomes, microcapsules, microparticles, magnetic beads and microdevices. The only requirement is that the complex contains more than one molecule. The molecules may be identical, or may differ from each other.

A "receptor" for a targeting peptide includes but is not limited to any molecule or complex of molecules that binds to a targeting peptide. Non-limiting examples of WO 02/20723 PCT/US01/28044 (N *receptors include peptides, proteins, glycoproteins, lipoproteins, epitopes, lipids, Jcarbohydrates, multi-molecular structures, a specific conformation of one or more C molecules and a morphoanatomic entity. 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.

Cc A "subject" refers generally to ;a mammal. In certain preferred embodiments, CN the subject is a mouse or rabbit In even more preferred embodiments, the subject is a 0 human.

Phage Display The methods described herein for identification of targeting peptides involve the in vivo administration of phage display libraries. Various methods of phage display and methods for producing diverse populations of peptides are well known in the art. For example, U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829, each of which is incorporated herein by reference, disclose methods for preparing a phage library. The phage display technique involves genetically manipulating bacteriophage so that small peptides can be expressed on their surface (Smith et al., 1985, 1993). The potential range of applications for this technique is quite broad, and the past decade has seen considerable progress in the construction of phage-displayed peptide libraries and in the development of screening methods in which the libraries are used to isolate peptide ligands. For example, the use of peptide libraries has made it possible to characterize interacting sites and receptor-ligand binding motifs within many proteins, such as antibodies involved in inflammatory reactions or integrins that mediate cellular adherence. This method has also been used to identify novel peptide ligands that serve as leads to the development of peptidomimetic drugs or imaging agents (Arap et al., 1998a). In addition to peptides, larger protein domains such as single-chain antibodies can also be displayed on the surface of phage particles (Arap et al., 1998a).

Amino acid sequences for a targeting given organ, tissue or cell type can be lI isolated by "biopanning" (Pasqualini and Ruoslahti, 1996; Pasqualini, 1999). In brief, a WO 02/20723 PCT/US01/28044 library of phage containing putative t'rgeting peptides is administered to an animal or human and samples of organs, tissues. or cell types containing phage are collected. In C, preferred embodiments utilizing filamentous phage, the phage may be propagated in vitro between rounds of biopanning in pilus-positive bacteria. The bacteria are not lysed by the phage but rather secrete multipl copies of phage that display a particular insert.

Phage that bind to a target molecule can be eluted from the target organ, tissue or cell Stype and then amplified by growing them in host bacteria. If desired, the amplified phage can be administered to a human host and samples of organs, tissues or cell types 0 again collected. Multiple rounds of biopanning may be performed until a population of selective binders is obtained. The amin. acid sequence of the peptides is determined by sequencing the DNA corresponding to the targeting peptide insert in the phage genome.

The identified targeting peptide can then, be produced as a synthetic peptide by standard protein chemistry techniques (Arap et al., 1998a, Smith et al., 1985). This approach allows circulating targeting peptides to!be detected in an unbiased functional assay, without any preconceived notions about' the nature of their target. Once a candidate target is identified as the receptor of a targeting peptide, it can be isolated, purified and cloned by using standard biochemical methods (Pasqualini, 1999; Rajotte and Ruoslahti, 1999).

Choice of phage display system Previous in vivo selection studies, performed in mice preferentially employed libraries of random peptides expressed as fusion proteins with the gene m capsule protein in the fUSE5 vector (Pasqualini and Ruoslahti, 1996). The number and diversity of individual clones present in a given library is a significant factor for the success of in vivo selection. It is preferred to use primary libraries, which are less likely -to have an over-representation of defective, phage clones (Koivunen et al., 1999). The preparation of a library may be amplified to between 108-109 transducing units Preferably, 1014 TU or more are prepared from a primary library for injection into human subjects. In certain embodiments, a bulk amplification strategy is applied between each round of selection.

12 WO 02/20723 PCT/US01/28044 Phage libraries displaying linear, cyclic, or double cyclic peptides may be used Swithin the scope of the present invention. However, phage libraries displaying 3 to C, random residues in a cyclic insert (CX 3 IC) are preferred, since single cyclic peptides tend to have a higher affinity for the target organ than linear peptides. Libraries displaying double-cyclic peptides (such as CX 3 C X 3

CX

3 C; Rojotte et al., 1998) have been successfully used. However, the production of the cognate synthetic peptides, although possible, can be complex due to the multiple conformers with different dissulfide bridge arrangements.

Ci Identification of homing peptides and receptors by in vivo phage display in mice.

In vivo selection of peptides from phage-display peptide libraries administered to mice has been used to identify targeting peptides selective for normal mouse brain, kidney, lung, skin, pancreas, retina, intestine, uterus, prostate, and adrenal gland (Pasqualini and Ruoslahti, 1996; Pasqualini, 1999; Rajotte et al., 1998). These results show that the vascular endothelium of normal organs is sufficiently heterogenous to allow differential targeting with peptide probes (Pasqualini and Ruoslahti, 1996; Rajotte et at, 1998). A means of identifying peptides that home to the angiogenic vasculature of tumors has been devised, as described below. A panel of peptide motifs that target the blood vessels of tumor xenografts in nude mice has been assembled (Arap et al., 1998a; reviewed in Pasqualini, 1999). These motifs include the sequences RGD-4C, NGR, and GSL. The RGD-4C peptide has previously been identified as selectively binding av integrins and has been shown to home to the vasculature of tumor xenografts in nude mice (Arap et al., 1998a, 1998b; Pasqualini et al., 1997).

The receptors for the tumor homing RGD4C targeting peptide has been identified as av integrins (Pasqualini et al., 1997). The av integrins play an important role in angiogenesis. The avP3 and avp5 integrins are absent or expressed at low levels in normal endothelial cells but are induced in angiogenic vasculature of tumors (Brooks et al., 1994; Hammes et al., 1996). Aminopeptidase N/CD13 has recently been WO 02/20723 PCT/US01/28044 Sidentified as an angiogenic receptor for the NGR motif (Burg et al., 1999).

SAminopeptidase N/CD13 is strongly expressed not only in the angiogenic blood vessels c of prostate cancer in TRAMP mice but also in the normal epithelial prostate tissue.

Tumor-homing phage co-localize with their receptors in the angiogenic Svasculature of tumors but not in non-angiogenic blood vessels in normal tissues (Arap et al., 1998b). Immunohistochemical evidence shows that vascular targeting phage bind to human tumor blood vessels in tissue sections (Pasqualini et al., 2000) but not to normal blood vessels. A negative control phage with no insert (fd phage) did not bind to normal or tumor tissue sections. The expression of the angiogenic receptors was evaluated in cell lines, in non-proliferating blood vessels and in activated blood vessels of tumors and other angiogenic tissues such as corpus luteum. Flow cytometry and immunohistochemistry showed that these receptors are expressed in a number of tumor cells and in activated HUVECs (data not shown). The angiogenic receptors were not detected in the vasculature of normal organs of mouseor human tissues.

The distribution of these receptors was analyzed by immunohistochemistry in tumor cells, tumor vasculature, and normal vasculature. Alpha v integrins, CD13, aminopeptidase A, NG2, and MMP-2/MMP-9 the known receptors in tumor blood vessels are specifically expressed in angiogenic endothelial cells and pericytes of both human and murine origin. Angiogenic neovasculature expresses markers that are either expressed at very low levels or not at all in non-proliferating endothelial cells (not shown).

The markers of angiogenic endothelium include receptors for vascular growth factors, such as specific subtypes of VEGF and basic FGF receptors, and av integrins, among many others (Mustohen and Alitalo, 1995). Thus far, identification and isolation of novel molecules characteristic of angiogenic vasculature has been slow, mainly because endothelial cells undergo dramatic phenotypic changes when grown in culture (Watson et al., 1995).

WO 02/20723 PCT/US01/28044 Many of these tumor vascular markers are proteases and some of the markers Salso serve as viral receptors. Alpha v integrins are receptors for adenoviruses C (Wickham et al., 1997c) and CD13 is a receptor for coronaviruses (Look et al., 1989).

MMP-2 and MMP-9 are receptors for echoviruses (Koivunen et al., 1999).

Aminopeptidase A also appears to be a viral receptor. Bacteriophage may use the same C cellular receptors as eukaryotic viruses. These findings suggest that receptors isolated Sby in vivo phage display will have cell internalization capability, a key feature for utilizing the identified peptide motifs as targeted gene therapy carriers.

C, Targeted delivery Peptides that home to tumor vasculature have been coupled to cytotoxic drugs or proapoptotic peptides to yield compounds that were more effective and less toxic than the parental compounds in experimental models of mice bearing tumor xenografts (Arap et al., 1998a; Elerby 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., 1998b).

BRASIL

In preferred embodiments, separation of phage bound to the cells of a target organ, tissue or cell type from unbound phage is achieved using the BRASIL technique (Provisional Patent Application No. 60/231,266 filed September 8, 2000; U.S. Patent Application entitled, "Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL)" by Arap, Pasqualini and Giordano, filed concurrently herewith, incorporated herein by reference in its entirety). In BRASIL (Biopanning and Rapid Analysis of Soluble Interactive Ligands), an organ, tissue or cell type is gently separated into cells or small clumps of cells that are suspended in an aqueous phase. The aqueous phase is layered over an organic phase of appropriate density and centrifuged. Cells attached to bound phage are pelleted at the bottom of the centrifuge tube, while unbound phage remain in the aqueous phase. This allows a more efficient separation of bound from unbound phage, while maintaining the binding interaction between phage WO 02/20723 PCT/US01/28044 and cell. BRASIL may be performed in an in vivo protocol, in which organs, tissues or Scell types are exposed to a phage display library by intravenous administation, or by an Sex vivo protocol, where the cells are exposed to the phage library in the aqueous phase before centrifugation.

itn In certain embodiments, a subtraction protocol is used with BRASIL or other Cc screening protocols to further reduce background phage binding. The purpose of

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C. subtraction is to remove phage from the library that bind to cells other than the cell of O interest, or that bind to inactivated cells. In alternative embodiments, the phage library C. may be screened against a control cell line, tissue or organ sample that is not the targeted cell, tissue or organ. After subtraction the library may be screened against the cell, tissue or organ of interest. In another alternative embodiment, an unstimulated, quiescent cell line, tissue or organ may be screened against the library and binding phage removed. The cell line, tissue or organ is then activated, for example by administration of a hormone, growth factor, cytokine or chemokine and the activated cell line screened against the subtracted phage library.

Other methods of subtraction protocols are known and may be used in the practice of the present invention, for example as disclosed in U.S Patent Nos.

5,840,841, 5,705,610, 5,670,312 and 5,492,807, incorporated herein by reference.

Preparation of large scale primary libraries In preferred embodiments, primary phage libraries are amplified before injection into a human subject. A phage library is prepared by ligating targeting peptide encoding sequences into a phage vector, such as fUSE5. The vector is transformed into pilus negative host E. coli such as strain MC1061. The bacteria are grown overnight and then aliquots are frozen to provide stock for library production. Use of pilus negative bacteria avoids the bias in libraries that arises from differential infection of pilus positive bacteria by different targeting peptide sequences.

WO 02/20723 PCT/US01/28044 To freeze, bacteria are pelleted from two thirds of a primary library culture liters) at 4000 x g for 10 min. Bacteria are resuspended and washed twice with 500 ml of 10% glycerol in water, then frozen in an ethanol/dry ice bath and stored at For amplification, 1.5 ml of frozen bacteria are inoculated into 5 liters of LB ^t medium with 20 jig/ml tetracycline and grown overnight Thirty minutes after Sinoculation, a serial dilution is plated on LB/tet plates to verify the viability of the

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C, culture. If the number of viable bacteria is less than 5-10 times the number of Sindividual clones in the library, (1-2 x 108) the culture is discarded.

c After growing the bacterial culture overnight, phage are precipitated. About /4 to 1/3 of the bacterial culture is kept growing overnight in 5 liters of fresh medium and the cycle is repeated up to 5 times. Phage are pooled from all cycles and used for injection into human subjects.

Humans The methods used for phage display biopanning in the mouse model system require substantial improvements for use with humans. A preferred example of the phage display protocol used for human patients, disclosing these improvements, is described in detail in Examples 1 and 2 below.

In general, humans suitable for use with phage display are either brain dead or terminal wean patients. The ainount of phage library (preferably primary library) required for administration musi be significantly increased, preferably to 1014 TU or higher, preferably administered intravenously in approximately 200 ml of Ringer lactate solution over about a 10 minute period.

The amount of phage reqlired for use in humans has required improvement of the mouse protocol, increasing tle amount of phage available for injection by five orders of magnitude. To produce such large phage libraries, the transformed bacterial pellets recovered from up to 500 t{ 1000 transformations are amplified up to 10 times in the bacterial host, recovering the' phage from each round of amplification and adding LB Tet medium to the bacterial pellet for collection of additional phage. The phage 17 WO 02/20723 PCT/US01/28044 U inserts remain stable under these conditions and phage may be pooled to form the large phage display library required for humans.

Samples of various organs and tissues are collected starting approximately minutes after injection of the phage library. Samples are processed as described below and phage collected from eacl organ, tissue or cell type of interest for DNA sequencing 1 to determine the amino acid sequences of targeting peptides.

C With humans, the opportunities for enrichment by multiple rounds of Sbiopanning are severely restricted, compared to the mouse model system. A substantial Simprovement in the biopanning technique involves polyorgan targeting.

Polyorgan targeting In the standard protocpl for phage display biopanning, phage from a single organ are collected, amplified And injected into a new host, where tissue from the same organ is collected for phage rescue and a new round of biopanning. This protocol is feasible in animal subjects. However, the limited availability and expense of processing samples from humans requires an improvement in the protocol.

As disclosed in a preferred embodiment, described in Example 3 below, it is possible to pool phage collected from multiple organs after a first round of biopanning and inject the pooled sample into a new subject, where each of the multiple organs may be collected again for phage resque. The polyorgan targeting protocol may be repeated for as many rounds of biopanning as desired. In this manner, it is possible to significantly reduce the number of subjects required for isolation of targeting peptides for multiple organs, while still achieving substantial enrichment of the organ-homing phage.

In preferred embodimentsj phage are recovered from human organs, tissues or cell types after injection of a ph ge display library into a human subject. In certain embodiments, phage may be recovered by exposing a sample of the organ, tissue or cell type to a pilus positive bacteriurt, such as E. coli K91. In alternative embodiments, WO 02/20723 WO 0220723PCT[USOI/28044 phage may be recovered by amplifying the phage inserts, ligating the inserts to phage DNA and producing new phage from the ligated DNA.

Proteins and Peptides 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.

In certain embodiments the size of the at least one protein or peptide may comprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 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, 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 110, about 120, aboout 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.

As used herein, an "amino acid residue" refers to any naturally occuring amino acid, any amino acid derivitive or any amino acid mimic known in the art In certain embodiments, the residues of the protein or peptide are sequential, without any non- WO 02/20723 WO 0220723PCTIUSOI/28044 amino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence may comprise one or more non-amino acid moieties. In particular embodiments, the sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.

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 those shown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3- Amninoadipic acid Hyl Hydroxylysine Bala f -alanine, 13-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 Afle allo-Isoleucine Aib 2-Aniinoisobutyric acid MeGly N-Methylglycine, sarcosine Baib 3-Arminoisobutyric 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'-Diaminopinielic acid Nie Norleucine Dpr 2,3-Diaminopropionic acid Orn Omithine EEtGlIy N-Ethyl glycine WO 02/20723 PCT/US01/28044 SProteins or peptides may be made by any technique known to those of skill in Jthe art, including the expression of proteins, polypeptides or peptides through standard ,I 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 0 ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/). The Scoding 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.

Peptide mimetics Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al., "Peptide Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, New York (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.

WO 02/20723 PCT/US01/28044 Fusion proteins Other embodiments of the present invention concern fusion proteins. These molecules generally have all or a substantial portion of a targeting peptide, linked at the N- or C-terminus, to all or a portion of a second polypeptide or proteion. For example, t fusions may employ leader sequences from other species to permit the recombinant c expression of a protein in a heterologous host. Another useful fusion includes the

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CN- addition of an immunologically active domain, such as an antibody epitope, to facilitate 0purification of the fusion protein. Inclusion of a cleavage site at or near the fusion CI 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 and 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.

Protein purification 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 WO 02/20723 PCT/US01/28044 C organ to polypeptide and non-polypeptide fractibns. The protein or polypeptide of Sinterest may be further purified using chromatographic and electrophoretic techniques C, 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, e, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. An 0 example of receptor protein purification by affinity chromatography is disclosed in U.S.

Patent No. 5,206,347, the entire text of which is incorporated herein by reference. A 0particularly efficient method of purifying peptides is fast protein liquid chromatography (FPLC) or even HPLC.

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 compositin, such as constituting about about 60%, about 70%, about 80%, about 90%, abott 95%, or more of the proteins in the composition.

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 SIS/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 WO 02/20723 PCT/US01/28044 particular assay technique chosen to follow the purification, and whether or not the expressed protein or peptide exhibits a detectable activity.

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 in sulphate, PEG, antibodies and the like, or by heat denaturation, followed by: CC centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, C1 hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; 0 and combinations of these and other techniques. As is generally known in the art, it is C. 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.

There is no general requirement that the protein or peptide always be provided in their most purified st te. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fever 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.

Affinity chromatoraphy is a chromatographic procedure that relies on the specific affinity between h substance to be isolated and a molecule to which it can specifically bind to. 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 altered pH, ionic strength, temperature, etc.). The matrix should be a substance WO 02/20723 PCT/US01/28044 U that itself does not adsorb molecules to any significant extent and that has a broad range Sof chemical, physical and thermal stability. The ligand should be coupled in such a way Sas 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.

Synthetic Peptides Because of their relatively small size, the targeting peptides of the invention can Sbe 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); Tam et al., (1983); Merrifield, (1986); and 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.

Antibodies In certain embodiments, it may be desirable to make antibodies against the identified targeting peptides or their receptors. The appropriate targeting peptide or receptor, 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 should be suitable for administration to humans, pharmaceutically acceptable. Preferred agents are the carriers are keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).

WO 02/20723 PCT/US01/28044 U The term "antibody" is used to refer to any antibody-like molecule that has an Santigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2 1 single domain antibodies (DABs), Fv, scPv (single chain Fv), and the like. Techniques for preparing and using various antibody-based constructs and fragments are well Sknown in the art. Means for preparing and characterizing antibodies are also well known in the art (See, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).

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SCytokines and chemokines C In certain embodiments, it may be desirable to couple specific bioactive agents to one or more targeting peptides for targeted delivery to an organ, tissue or cell type.

Such agents include, but are not limited to, cytokines, chemikines, pro-apoptosis factors and anti-angiogenic factors. The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. 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; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone 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-oa, and y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocytemacrophage-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 -10, IL-, IL11, IL-12; IL- 13, IL-14, IL-15, IL-16, L-17, IL-18, LIF, G-CSF, GM-CSF, M- CSF, EPO, kit-ligand WO 02/20723 PCT/US01/28044 or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT. As used herein, the term cytokine includes proteins from natural sources or from C recombinant cell culture and biologically active equivalents of the native sequence cytokines.

tt Chemokines generally act as chemoattractants to recruit immune effector cells aC to the site of chemokine expression. It may be advantageous to express a particular

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C1 chemokine gene in combination with, for example, a cytokine gene, to enhance the Srecruitment of other immune system components to the site of treatment. Chemokines CN include, but are not limited to, RANTES, MCAF, MIP1-alpha, MIP1-Beta, and The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines.

Imaging agents and radioisotopes In certain embodiments, the claimed peptides or proteins 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 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.

Non-limiting examples of paramagnetic ions of potential use as imaging agents include chromium (III), manganese iron (IIf), iron cobalt nickel (II), copper neodymium samarium ytterbium (III), gadolinium (Ill), vanadium terbium dysprosium holmium (Ifl) and erbium with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, WO 02/20723 PCT/US01/28044 include but are not limited to lanthanum gold lead and especially bismuth (Ill).

Radioisotopes of potential use as imaging or therapeutic agents include astatine 211 14carbon, "chromium, 3chlorine, 57 cobalt, 58 cobalt, copper 7 152Eu, i- gallium 6 3 hydrogen, iodine l 23 iodine 25 iodine 31 indium 11 59iron, 3phosphorus, cn rhenium 1 86 rhenium' 88 7 selenium, 35 sulphur, technicium 99 m and yttrium 9 0 1251 is often C.1 being preferred for use in certain embodiments, and technicium 99 m and indium"' are 0also often preferred due to their low energy and suitability for long range detection.

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, by incubating pertechnate, a reducing agent such as SNC1 2 a buffer solution such as sodium-potassium phthalate solution, and the peptide. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to peptides are diethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetracetic acid (EDTA). Also contemplated for use are fluorescent labels, including rhodamine, fluorescein isothiocyanate and renographin.

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 WO 02/20723 PCT/US01/28044

C

is described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; S4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.

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Cross-linkers Bifunctional cross-linking reagents have been extensively used for a variety of tI purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural C' studies. Homobifunctional reagents that carry two identical functional groups proved to 0 be highly efficient in inducing cross-linking between identical and different C1 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, amino, sulfhydryl, guanidino, indole, carboxyl specific groups. Of these, reagents dtirected 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.

Exemplary methods for cross-linking ligands to liposomes are described in U.S.

Patent 5,603,872 and U.S. Patent 5,401,511, each specifically incorporated herein by reference in its entirety). Various ligands lcan 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 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 purplses. Ligands such as epidermal growth factor (EGF) have been successfully linked with PE-liposomes. Ligands are bound 29 29 WO 02/20723 PCT/US01/28044 'covalently to discrete sites on the liposome surfaces. The number and surface density Sof these sites are dictated by the liposome formulation and the liposome type. The c1 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 C (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and a 0 water soluble carbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Through the complex chemistry of cross-linking, linkage of the 0amine residues of the recognizing substance and liposomes is established.

l In another example, heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described 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.

Nucleic Acids Nucleic acids according to the present invention may encode a targeting peptide, a receptor protein or a fusion protein. The nucleic acid may be derived from genomic DNA, complementary DNA (cDNA) or synthetic DNA. Where incorporation into an expression vector is desired, the nucleic acid may also comprise a natural intron or an intron derived from another gene. Such engineered molecules are sometime referred to as "mini-genes." 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 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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, WO 02/20723 PCTUS01/28044 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 96, 97, 98, 99, 100, about 110, about 120, aboout 130, about 140, about 150, about C1 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 nucleotide residues in length.

It is contemplated that targeting peptides, fusion proteins and receptors 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 (see Table 2 below). 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. Codon preferences for various species of host cell are well known in the art.

TABLE 2 Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutanic acid Glu E GAA GAG Phenylalanine Phe F UUC U UU Glycine Gly G GGA GGC GGO GGU WO 02/20723 PCT/US01/28044 Histidine Isoleucine Lysine Leucine Methionine Asparagine Proline Glutamine Arginine Serine Threonine Valine Tryptophan Tyrosine CAC CAU AUA AUC AUU le I

AAA

UUA

AAG

UUG CUA CUC CUG CUU Leu Met Asn Pro Gin Arg Ser Thr Val Trp Tyr

AUG

AAC AAU CCA CCC CCG CCU CAA CAG AGA AGG CGA CGC CGG CGU AGCAGU UCA UCC UCG UCU ACA ACC ACG ACU GUA GUC GUG GUU

UGG

UAC UAU In addition to nucleic acids encoding the desired targeting peptide, fusion protein or receptor amino acid sequence, the present invention encompasses complementary nucleic acids that hybridize under high stringency conditions with such coding nucleic acid sequences. High stringency conditions for nucleic acid hybridization are well known in the art. For example, conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCI at temperatures of about 50°C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the WO 02/20723 PCT/US01/28044 (N particular nucleic acid(s), the length and nucleotide content of the target sequence(s), Sthe charge composition of the nucleic acid(s), and to the presence or concentration of Sformamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.

tt3 Vectors for Cloning, Gene Transfer and Expression Cc In certain embodiments expression vectors are employed to express the targeting C-I peptide or fusion protein, which can then be purified and used. In other embodiments, l^the expression vectors are used in gene therapy. Expression requires that appropriate C-i signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are known.

Regulatory Elements The terms "expression construct" or "expression vector" are meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid coding sequence is capable of being transcribed. In preferred embodiments, the nucleic acid encoding a gene product is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent and under WO 02/20723 PCT/US01/28044 U the control of a promoter that is capable of being expressed in a human cell. Generally Sspeaking, such a promoter might include either a human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, t rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase promoter can be c used to obtain high-level expression of the coding sequence of interest. The use of

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Ci other viral or mammalian cellular or bacterial phage promoters which are well-known Sin the art to achieve expression of a coding sequence of interest is contemplated as well, C1 provided that the levels of expression are sufficient for a given purpose.

Where a cDNA insert is employed, typically one will typically include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed, such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression construct is a terminator. These elements can serve to enhance message levels and to minimize read through from the construct into other sequences.

Selectable Markers In certain embodiments of the invention, the cells containing nucleic acid constructs of the present invention may be identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.

Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers.

Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid WO 02/20723 PCT/US01/28044 encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.

Delivery of Expression Vectors There are a number of ways in which expression vectors may introduced into it cells. In certain embodiments of the invention, the expression construct comprises a Cc virus or engineered construct derived from a viral genome. The ability of certain

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CN viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell 0genome, and express viral genes' stably and efficiently have made them attractive N candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). Preferred gene therapy vectors are generally viral vectors.

Although some viruses that ban accept foreign genetic material are limited in the number of nucleotides they can accommodate and in the range of cells they infect, these viruses have been demonstrated to successfully effect gene expression. However, adenoviruses do not integrate their genetic material into the host genome and therefore do not require host replication for gene expression making them ideally suited for rapid, efficient, heterologous gene expression. Techniques for preeparing replication infective viruses are well known in the art.

I

In using viral delivery systenrs, one will desire to purify the virion sufficiently to render it essentially free of undesirable contaminants, such as defective interfering viral particles or endotoxins and other pyrogens such that it will not cause any untoward reactions in the cell, animal or individual receiving the vector construct. A preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.

DNA viruses used as gene vectors include the papovaviruses simian virus bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).

WO 02/20723 PCTUSO1/28044 0 One of the preferred methods for in vivo delivery involves the use of an Sadenovirus expression vector. Although adenovirus vectors are known to have a low CI 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 support packaging of the construct and to express an antisense or a sense polynucleotide that has been cloned therein.

The expression vector comprises a genetically engineered form of adenovirus.

NC Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to retroviral infection, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early and late regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late WO 02/20723 PCT/US01/28044 C promoter (MLP). The MLP, (located at 16.8 is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a c tripartite leader (TPL) sequence which makes them preferred mRNAs for translation.

In currently used systems, recombinant adenovirus is generated from in homologous recombination between shuttle vector and provirus vector. Due to the Cc possible recombination between two proviral vectors, wild-type adenovirus may be

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c, generated from this process. Therefore, it is critical to isolate a single clone of virus Sfrom an individual plaque and examine its genomic structure.

Generation and propagation of adenovirus vectors which are replication deficient depend on a unique helper cell line, designated 293, which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the E3, or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vectorborne cytotoxicity. Also, the replication deficiency of the El-deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., WO 02/20723 PCT/US01/28044 (NI Vero cells or other monkey embryonic mesenchymal or epithelial cells. As discussed, Sthe preferred helper cell line is 293.

Racher et al., (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) Cc containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is

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CI estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, SStone, UK) (5 g/1) are employed as follows. A cell innoculum, resuspended in 5 ml of C1 medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking is initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking is commenced for another 72 hr.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

A typical vector applicable to practicing the present invention is replication defective and will not have an adenovirus El region. Thus, it are most convenient to introduce the polynucleotide encoding the gene at the position from which the Elcoding sequences have been removed. However, the position of insertion of the WO 02/20723 PCT/US01/28044 construct within the adenovirus sequences is not critical. The polynucleotide encoding Sthe gene of interest may also be inserted in lieu of the deleted E3 region in E3 1 replacement vectors as described by Karlsson et al., (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.

lt Adenovirus is easy to grow and manipulate and exhibits broad host range in C vitro and in vivo. This group of viruses can be obtained in high titers, 109-101 CI plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus Sdoes not require integration into the host cell genome. The foreign genes delivered by CN adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

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). Animal studies have suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993). 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).

Other gene transfer vectors may be constructed from retroviruses. The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reversetranscription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration 'results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env. that code WO 02/20723 PCT/US01/28044 for capsid proteins, polymerase enzyme, and envelope components, respectively. A Ssequence found upstream from the gag gene contains a signal for packaging of the c genome into virions. Two long terminal repeat (LTR) sequences are present at the and 3' ends of the viral genome. These contain strong promoter and enhancer Ssequences, and also are required for integration in the host cell genome (Coffin, 1990).

n In order to construct a retroviral vector, a nucleic acid encoding protein of N 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 Scell 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).

There are certain limitations to the use of retrovirus vectors. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al., 1981). Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This may result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are now WO 02/20723 PCT/US01/28044 u available that should greatly decrease the likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

Other viral vectors may be employed as expression constructs. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; lt Coupar et al., 1988), adeAo-associated virus (AAV) (Ridgeway, 1988; Baichwal and CSugden, 1986; Hermonat and Muzycska, 1984), and herpes viruses may be employed.

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CI They offer several attractie features for various mammalian cells (Friedmann, 1989; 0Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., CN 1990).

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are, contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et at, 1986; Potter et al., 1984), direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

In a further embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phosphlipids are suspended in an excess of aqueous solution.

The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes.

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, WO 02/20723 PCT/US01/28044 HeLa, and hepatoma cells. Nicolau et al., (1987) accomplished successful liposome- Smediated gene transfer in rats after intravenous injection.

A number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine tt) phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively. Also, anti- Cc metabolite resistance can be used as the basis of selection for dhfr: that confers Cl resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that 0confers resistance to the aminoglycoside G418; and hygro, that confers resistance to C. hygromycin.

Pharmaceutical compositions Where clinical applications are contemplated, it may be necessary to prepare pharmaceutical compositions expression vectors, virus stocks, proteins, antibodies and drugs in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of impurities that could be harmful to humans or animals.

One generally will desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also are employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the protein or peptide, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.. Such compositions also are referred to as innocula. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the proteins or peptides of the present WO 02/20723 PCT/US01/28044

C

N invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the t present invention are via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, Sadministration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial or intravenous injection. Such compositions normally would be administered as pharmaceutically acceptable compositions, described supra.

The pharmaceutical forms suitable for 'injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringabilit exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria add fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride.. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients 43 WO 02/20723 PCT/US01/28044 enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile Svehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any Sadditional desired ingredient from a previously sterile-filtered solution thereof.

STherapeutic agents In certain embodiments, chemotherapeutic agents may be attached to a targeting peptide or fusion protein for selective delivery to a tumor. Agents or factors suitable for use may include any chemical compound that induces DNA damage when applied to a cell. Chemotherapeutic agents include, but are not limited to, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP16), 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 following categories: alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof.

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", 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 WO 02/20723 PCT/US01/28044 being treated. The person responsible for administration will, in any event, determine Sthe appropriate dose for the individual subject. Examples of specific chemotherapeutic Sagents and dose regimes are also described herein. Of course, all of these 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- C between these points, or range derivable therein is also expected to be of use in the 0 invention.

0 Alkylating agents Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell 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.

Antimetabolites 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 cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.

Natural Products Natural products generally refer to compounds originally isolated from a natural source, and identified has 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 WO 02/20723 PCT/US01/28044 u *products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes _and biological response modifiers.

Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate lt during a specific phase during the cell cycle. Mitotic inhibitors include, for example, C docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and

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Cl vinorelbine.

O 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.

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

Antitumor Antibiotics Antitumor 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 antitumor antibiotics include, but are not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin.

Hormones Corticosteroid hormones are considered chemotherapy drugs when they are implemented to kill or slow the growth of cancer cells. Corticosteroid hormones can increase the effectiveness of other chemotherapy agents, and consequently, they are frequently used in combination treatments. Prednisone and dexamethasone are examples of corticosteroid hormones.

WO 02/20723 PCT/US01/28044 U Progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, Sand megestrol acetate have been used in cancers of the endometrium and breast.

C'I Estrogens such as diethylstilbestrol and ethinyl estradiol have been used in cancers such as breast and prostate. Antiestrogens such as tamoxifen have been used in cancers such as breast. Androgens such as testosterone propionate and fluoxymesterone have also C been used in treating breast cancer. Antiandrogens such as flutamide have been used in the treatment of prostate cancer. Gonadotropin-releasing hormone analogs such as leuprolide have been used in treating prostate cancer.

C1 Miscellaneous Agents Some chemotherapy agents do not fall into the previous categories based on their activities. They include, but are not limited to, platinum coordination complexes, anthracenedione, substituted urea, methyl hydrazine derivative, adrenalcortical suppressant, amsacrine, L-asparaginase, and tretinoin. It is contemplated that they may be used within the compositions and methods of the present invention.

Platinum coordination complexes include such compounds as carboplatin and cisplatin (cis-DDP).

An anthracenedione such as mitoxantrone has been used for treating acute granulocytic leukemia and breast cancer. A substituted urea such as hydroxyurea has been used in treating chronic granulocytic leukemia, polycythemia vera, essental thrombocytosis and malignant melanoma. A methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH) has been used in the treatment of Hodgkin's disease. An adrenocortical suppressant such as mitotane has been used to treat adrenal cortex cancer, while aminoglutethimide has been used to treat Hodgkin's disease.

Regulators of Programmed Cell Death 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 WO 02/20723 PCT/US01/28044 have been demonstrated to be important regulators and effectors of apoptosis in other Ssystems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays 1a 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 C conserved Bcl-2 protein now is recognized to be a member of a family of related 0 proteins, which can be categorized as death agonists or death antagonists.

SSubsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death C1 triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to Bcl-2 BclxL, Bclw, Bcls, Mcl-1, Al, Bfl-1) or counteract Bcl-2 function and promote cell death Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

Non-limiting examples of pro-apoptosis agents contemplated within the scope of the present invention include gramicidin, magainin, mellitir, defensin, cecropin,

(KLAKLAK)

2 (SEQ ID NO:1), (KLAKKLA) 2 (SEQ ID NO:2), (KAAKKAA) 2

(SEQ

ID NO:3) or (KLGKKLG) 3 (SEQ ID NO:4).

Angiogenic inhibitors In certain embodiments the present invention may concern administration of targeting peptides attached 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, CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferonalpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, Docetaxel (Taxotere), polyamines, a proteasome inhibitor, a kinase inhibitor, a signaling inhibitor WO 02/20723 PCT/US01/28044 SU5416 or SU6668 from Sugen, South San Francisco, CA), accutin, angiostatin, Scidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.

Dosages The skilled artisan is directed to "Remington's Pharmaceutical Sciences" Cl Edition, chapter 33, and in particular to pages 624-652. Some variation in dosage will O 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 0individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA Office of Biologics standards.

EXAMPLES

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. Biopanning with Phage Display Libraries Using Human Patients Certain of the methods and compositions of the present invention concern identification of targeting peptides for human organs, tissues or cell types by in vivo biopanning. Generally, protocols used in animal subjects, such as mice, are not suited for humans. Further, ethical considerations play a large role in human protocols. The following novel methods are preferred for use with humans, although the skilled artisan WO 02/20723 PCT/US01/28044 will realize that variations on the methods and compositions disclosed herein may be Z used within the scope of the present invention.

Human preparation Patients were selected for the protocol according to inclusion and exclusion ttn criteria. Inclusion criteria include: patient legally declared brain dead or terminal Cc wean patient; approval of attending and/or treating physicians; and approved

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C' written informed consent form signed by the patient's legally responsible family Smember. Exclusion criteria were: the absence of a responsible family member; (2) C' HIV positive patient; patient with active tuberculosis infection; acute or chronic hepatitis B or C infections; or patient was a potential organ transplant donor. In preferred embodiments, the patient was not on antibiotics for at least the previous 6 hrs, preferably the last 24 hrs, in order to avoid detrimental effects on the bacterial hosts used to propagate the phage used for the peptide display library.

After informed consent and before the patient was prepared for the procedure, relatives of the patient were asked to leave the room the patient was in. The patient had a well running IV line (preferably central) with nothing but saline running through the channel of application of the phage library. Personnel required for the procedure were notified intervention radiologist, internist, surgeon, nurse, possibly neurologist or neuroradiologist). Materials needed for biopsies were collected: bone marrow aspiration needle, lumbar puncture kit, skin biopsy kit, materials for taking biopsies of any organ, tissue or cell type used for targeted peptide identification, such as liver, fat and tumor, materials for transabdominal prostate biopsy, 50 ml syringe with 40 ml saline for blood sample, 10 ml tube containing heparin and 10 ml serum collection tube to draw blood sample for lab tests. Before phage library injection, blood samples were drawn for routine screening of liver function, bicarbonate, electrolytes and blood count, unless test results from the day of the injection were available.

In the laboratory, 120 large dishes with LB-tet/kan agar as well as 200 regular LB tet/kan plates (100 mm) were prepared (tetracycline concentration 40 itg/ml, kanamycin concentration 50 E. coli K91 kan were grown in 10 independent WO 02/20723 PCT/US01/28044 tubes, each containing 10 ml TB medium plus supplements. Growth of bacteria was started approx. 15-60 min prior to beginning the biopsies. About 1014 TU of the C (preferably primary) phage library were diluted in 200 ml ringer lactate at room temperature and aspirated under clean but not necessarily sterile conditions into four ml syringes. LB-tet/kan dishes or plates were warmed in a 370C incubator. One liter of C- LB medium containing 0.2 /g/ml tet and 100 p/g/ml kan was warmed in the waterbath 0 at 37 0 C. One liter LB medium containing 40 Ag/ml tet and 100 /g/ml kan was warmed to 370C and 8 more liters were prepared at room temperature. Thirty glass grinders A Sand B size as well as suitable glass tubes were autoclaved. Three 50 ml Falcon tubes were prepared for each of the organs for which biopsies were to be taken. Tubes were filled with 10 ml DMEM-PI DMEM containing PMSF (1 mM), aprotinin and leupeptin (1l/g/ml) and put on ice approximately 15 minutes before tissue collection. For each of the 4 teams taking over in the lab after the tissue samples were collected, one autoclaved set of surgicals at least one forceps and one pair of scissors and a scalpel) were prepared in order to trim, divide or mince organ samples.

Phage library injection All drugs running through the intended port of application of the phage library were discontinued during library injection. If possible without compromising the patient's hemodynamic stability, all IV drugs running through different ports were discontinued during library injection as well. A running saline infusion ensured that the IV line for the library injection was open and was left running during the injection.

The 200 ml library solution was manually injected over a period of 10 minutes while monitoring and protocoling the patient's vital functions such as breathing (if not mechanically ventilated), heart rate and blood pressure. The injection was stopped any time the running saline infusion stopped dripping, indicating obstruction of the line.

Fifteen minutes after beginning the injection, tissue sample collection (biopsies) was initiated. Preferred biopsy sites included bone marrow aspirate, liver, prostate, skin, skeletal muscle, tumor (if applicable), adipose tissue, blood (as positive control), blood (for red/white blood cells) and cerebral-spinal fluid (CSF).

WO 02/20723 PCT/US01/28044 The samples were taken under very clean if not sterile conditions to reduce Scontamination with bacteria. To the extent possible, the different samples were taken C simultaneously. For small samples, triplicate biopsies were preferred. The time elapsed between beginning of injection and the collection of a particular tissue sample was recorded. Tissue samples were placed in the prepared 50 ml tubes containing DMEM- C PI and stored on ice. For bone marrow, a regular diagnostic sample (undiluted into a 0 syringe with heparin) was taken in addition to the samples diluted in 40 ml saline to confirm aspiration of bone marrow as opposed to blood. If needed, all IV drugs, 0including antibiotics, were continued after removal of tissue samples.

All organ samples that were not taken in triplicate were divided under clean conditions to obtain three different pieces of tissue. The three samples of each organ were handled as follows. One piece was stored at -80 0 C as a backup. One piece was forwarded to the histology/pathology department to cut cryosections (or to make smears for bone marrow) and perform HE staining (Pappenheim staining for bone marrow) as well as phage staining to confirm that the samples contained the organ of interest. In some cases the histology sample was divided in two one for regular HE staining and one for LCM (laser capture microscopy) or LPC (laser pressure catapulting). The last of the three original pieces was processed for bacterial infection to recover phage.

After freezing of backup tissue and saving material for pathology, samples for phage rescue were weighed. Samples were kept on ice at all times. Sample was transferred to 1ml DMEM-PI in a glass tube and homogenized with a grinder. Some organs such as bone marrow, blood, or CSF do not require homogenization, whereas other organs like muscle need to be minced before they can be efficiently homogenized.

Lysis of erythrocytes for blood samples was preferred. Homogenized samples were transferred to autoclaved 2 ml Eppendorf tubes.

Tissue samples were washed 3 times with ice cold DMEM-PI containing 1% BSA by mixing the tissue with DMEM-PI and vortexing for 30 seconds. After centrifugation at 4,000 rpm for 3 min, supernatant was carefully discarded, leaving the tissue pellet undisturbed. A small amount of medium was left on the surface of the WO 02/20723 PCT/US01/28044 u pellet. Samples were vortexed again for 30 seconds before adding more medium to Sfacilitate resuspension of the tissue. After adding 1.5 ml of DMEM-PI plus BSA the CI samples were centrifuged again. When processing multiple samples, the tissues were kept on ice at all times.

After 3 washes, the pellet was briefly vortexed and the dissolved pellet was

C

warmed briefly to 37 0 C before adding bacteria. The washed tissue samples were incubated with 1.5 ml of competent K91-kan bacteria (OD 0 0 o 0.2 in 1:10 dil.) for one hour at room temperature, then transferred to Falcon tubes containing 10 ml of LB Smedium with 0.2 g/ml tetracycline. After 20 min at RT, multiple aliquots were plated on LB tet/kan plates or dishes containing 40 g/ml of tetracycline and 100 Ag/ml kanamycin. The following quantities (per organ sample) were plated: 2 dishes with 3 ml; 2 dishes with 1 ml; 3 dishes with 300 3 dishes with 100 Gl; 3 dishes with 30 pl.

The beads that were used for plating were passed on to two subsequent 10 cm LB tet/kan plates to recover every potentially phage infected bacterial clone that might be trapped on the bead surface. Dishes were incubated overnight at 37 0

C.

The remaining 2-3 ml of infected culture (including the homogenized tissue) was transferred to 10 ml of LB medium containing 40/g/ml tetracycline and 100/g/ml kanamycin (LB tet/kan) and shaken at 37 0 C for 2 hr. This approximately 12 ml culture was transferred to 1 liter LB tet/kan and grown overnight in a 37 0 C shaker.

The next day, phage were rescued from the bulk amplified bacterial culture according to standard protocols and saved for a potential second round of in vivo selection. From the plates/dishes in the incubator, 1500 well separated colonies were picked for each organ plated and transfered to 96 well plates containing 20 pl PBS/well for sequencing. This assumed a readout of about 2 out of 3 picked colonies to obtain 1000 sequences.

After picking 1500 colonies, the remainder of colonies on the dishes/plates were grown in 1000 ml LB tet/kan overnight in the 37 0 C shaker. Then phage were harvested as before for a second round of selection. Alternatively, the plates were stored in the refrigerator and 1000-2000 individual colonies grown at a time. Alternatively, the WO 02/20723 PCT/US01/28044 remainder of colonies were transferred to PBS and stored frozen to infect and amplify as needed.

SNumerous non-limiting examples of human organ, tissue or cell type selective targeting peptides have been identified by in vivo biopanning using the present methods, as disclosed below. A non-limiting example of human biopanning using the methods of the present invention, along with targeting peptide sequences identified by Sthe present methods, is disclosed in Example 3.

SExample 2. Mapping the Human Vasculature by In vivo Phage Display The in vivo selection method was used to screen a phage library in a human subject. A pattern recognition analysis program was used to survey 47,160 tripeptide motifs within peptides that localized to the human bone marrow, fat, skeletal muscle, prostate, or skin. The results of this large-scale screening indicated that the distribution of circulating peptide motifs to different organs is non-random. High-throughput analysis of peptide motifs enriched in individual tissues revealed similarities to sequences present in candidate ligands for differentially expressed vascular receptors.

These data represent a major step towards the construction of a ligand-receptor map of human vasculature and may have broad implications for the development of targeted therapies. Many therapeutic targets may be expressed in very restricted-but highly specific and accessible--locations in the vascular endothelium. Potential targets for intervention may be overlooked in high-throughput DNA sequencing or in gene arrays because these approaches do not usually take into account cellular location and anatomical, and functional context. The human in vivo phage display screening methods disclosed herein are uniquely suited to identification of naturally occurring ligand-receptor pairs that may provide the basis for highly selective therapies against various disease states.

WO 02/20723 PCT/US01/28044 Materials and Methods A 48 year-old male Caucasian patient who had been diagnosed with Waldenstrom macroglobulinemia (a B cell malignancy) was previously treated by splenectomy, systemic chemotherapy (fludarabine, mitoxantrone, and dexamethasone), and immunotherapy (anti-CD20 monoclonal antibody). In the few months prior to his admission, the disease became refractory to treatment and clinical progression occurred with retroperitoneal lymphadenopathy, pancytopenia, and marked bone marrow Sinfiltration by tumor cells. The patient was admitted with massive intracranial bleeding Ssecondary to thrombocytopenia. Despite prompt craniotomy and surgical evacuation of a cerebral hematoma, the patient remained comatose with progressive and irreversible loss of brainstem function until the patient met the formal criteria for brain-based determination of death, as evaluated by an independent clinical neurologist. Because of his advanced cancer, the patient was rejected as transplant organ donor. After surrogate written informed consent was obtained from the legal next of kin, in vivo phage display was performed.

In Vivo Phage Display A large-scale preparation of a CX 7 C cysteine; X, any amino acid residue) phage display random peptide library was optimized to create the highest possible insert diversity (Pasqualini et al., 2000). The diversity of the library was about 2x10 8 and its final titer was about 1012 transducing units (TU)/ml. Short-term intravenous infusion of the phage library (a total dose of 1014 phage TU suspended in 100 ml of saline) into the patient was followed by multiple representative tissue biopsies. Prostate and liver samples were obtained by needle biopsy under ultrasonographic guidance. Skin, fat tissue, and skeletal muscle samples were obtained by surgical excision. Bone marrow needle aspirates and core biopsies were also obtained. Histopathological diagnosis was determined by examination of frozen sections processed from tissues obtained at the bedside.

Triplicate samples were processed for host bacterial infection, phage recovery, and histopathological analysis. In brief, tissues were weighed, ground with a glass WO 02/20723 PCT/US01/28044 Dounce homogenizer, suspended in 1 ml of Dulbecco Modified Eagle's medium Z (DMEM) containing proteinase inhibitors (DMEM-prin; 1 mM PMSF, 20 /ig/ml C" aprotinin, and 1 /g/ml leupeptin), vortexed, and washed three times with DMEM-prin.

The human tissue homogenates were incubated with 1 ml of host bacteria (log phase E.

coli K91kan; OD600 Aliquots of the bacterial culture were plated onto Luria- C" Bertani agar plates containing 40 pg/ml tetracycline and 100 g/ml of kanamycin.

Plates were incubated overnight at 37 0 C. Bacterial colonies were processed for sequencing of phage inserts recovered from each tissue and from unselected phage 0 library. Human samples were handled with universal blood and body fluid precautions.

Statistical Analysis A high-throughput character pattern recognition program (MJD. Anderson Cancer Center, Biostatistics, Houston, TX) was developed to automate the analysis of the peptide motifs derived from phage screenings. By using SAS (version 8, SAS Institute) and Perl (version the program conducts an exhaustive amino acid residue sequence count and tracks the relative frequencies of N distinct tripeptide motifs representing all possible n 3 overlapping tripeptide motifs in both directions n 3 This was applied for phage recovered from each target tissue and for the unselected

CX

7 C random phage display peptide library.

With defined as the probability of observing a particular tripeptide motif under total randomness, and q=l-p, the probability of observing K sequences characterized as a particular tripeptide motif out of n 3 total tripeptide motif sequences is binomial (n 3 That probability may be approximated by the formula: pK 4[(k+l)/sqrt(n 3 pq)] c[k/sqrt(n 3 where (c is the cumulative Gaussian probability. The value PK may be treated as a P-value in testing for total randomness of observing exactly K sequences of a particular tripeptide motif. However, this test requires exact knowledge of the true value of p, which it is difficult to obtain in practice.

In order to identify the motifs that were enriched in the screening, the count for each tripeptide motif within each tissue was compared with the count for that tripeptide WO 02/20723 PCT/US01/28044 motif within the unselected library. Starting from a CX 7 C peptide insert, counts were recorded for all overlapping interior tripeptide motifs, subject only to reflection and single-voting restrictions. No peptide was allowed to contribute more than once for a single tripeptide motif (or a reflected tripeptide motif). Each peptide contributed five tripeptide motifs. Tripeptide motif counts were conditioned on the total number for all C1 motifs being held fixed within a tissue. The significance of association of a given 0 allocation of counts was assessed by Fisher's exact test (one-tailed). Results were considered statistically significant at P 0.05. In summary, to test for randomness of Sdistribution, the relative frequencies of a particular tripeptide motif from each target was compared to the frequencies of the motifs from the unselected library. This approach is a large-scale contingency table association test.

Results The general procedure followed in human phage display biopanning is illustrated in FIG. 1A. For biopanning with human subjects, use of a large-scale phage display library (diversity about 2 x 10 8 is advantageous compared to the smaller scale libraries used in mouse studies. The protocol followed was as described above.

The feasibility of producing phage display random peptide libraries in very large-scale and of selecting phage clones that home to different human organs in vivo through the systemic circulation is shown in FIG. IB. Phage localizing to fat, skin, bone marrow, skeletal muscle, prostate and liver were recovered from a human subject (see FIG. This is the first demonstration of the feasibility of in vivo phage display in humans.

To determine the distribution of the peptide inserts homing to specific sites after intravenous administration, the relative frequencies of every tripeptide motif from each target tissue were compared to the frequencies from the unselected library. The 4,716 phage inserts recovered from representative samples of five tissues (bone marrow, fat, skeletal muscle, prostate, and skin) and from the unselected library were analyzed.

Tripeptide motifs were chosen for the phage insert analysis because three amino acid residues appear to provide the minimal framework for structural formation and protein- WO 02/20723 PCT/US01/28044 protein interaction (Vendruscolo et al., 2001). Examples of biochemical recognition units and binding of tripeptide ligand motifs to receptors include RGD (Ruoslahti, C_ 1996), LDV (Ruoslahti, 1996), and LLG (Koivunen et al., 2001) to integrins, NGR (Pasqualini et al., 2000) to aminopeptidase N/CD13, and GFE (Rajotte and Ruoslahti, 1999) to membrane dipeptidase.

C

Each phage insert analyzed contained seven amino acid residues and contributed five potential tripeptide motifs. Taking reflection into account, a total of 47,160 tripeptide motifs were surveyed. Comparisons of the motif frequencies in a given organ Srelative to those frequencies in the unselected library showed the non-random nature of the peptide distribution (Table This is particularly noteworthy given that only a single round of in vivo screening was performed. Of the tripeptide motifs enriched in tissues, some were enriched in a single site WO 02/20723 WO 0220723PCT[US01/28044 Table 3. Peptide motifs isolated by in vivo phage display in humans Target organ/motif Motif frequency P-value Unselected Library _none N/A N/A Bone Marrow CIGFS* 1.1 0.0244 LWS* 1.0 0.0453 ciARL 1.0 0.0453 FGG 1.0 0.0453 GVL 1.1 0.0453 ciSGT 2.3 0.0 137 GGG* 2.3 0.0350 Fat EGG* 1.3 0.0400 LLV* 1.0 0.0269 LS*0.9 0.0402 EGR 1.1 0.0180 FGV 0.9 0.0402 Muscle LVS* 2.1 0.0036 GER 0.9 0.0344 Prostate AGG* 2.5 0.0340 EGR 1.0 0.0185 GER 0.9 0.03 82 GVL 2.3 0.0079 Skin GRR* 2.9 0.0047 GGH* 0.9 0.0341 GTV* 0.8 0.0497 ARL 0.8 0.0497 FGG 1.3 0.0076 FGV 1.0 0.0234 SGT 1.0 0.0234 WO 02/20723 PCT/US01/28044 U whereas others were enriched in multiple sites. The data are consistent with some of Z the peptides homing in a tissue-specific manner while other peptides targeted several C.l tissues.

Table 3 lists motifs occurring in peptides isolated from target organs, but not from the unselected phage library (Fisher's exact test, one-tailed; P 0.05). The number of peptide sequences analyzed per organ was: unselected library, 446; bone O marrow, 521; fat, 901; muscle, 850; prostate, 1018; skin, 980. Aserisks indicate motifs that were enriched only in a single tissue. The abbreviation N/A means not applicable.

Cl Tripeptide motifs appeared to be targeted to specific human organs, tissues or cell types including bone marrow (GGG, SEQ ID NO:5; GFS, SEQ ID NO:6; LWS, SEQ ID NO:130), adipose tissue (EGG, SEQ ID NO:7; LSP, SEQ ID NO:8, LLV, SEQ ID NO:9), skeletal muscle (LVS, SEQ ID NO:10), prostate (AGG, SEQ ID NO:11) and skin (GRR SEQ ID NO:12; GGH, SEQ ID NO:13; GTV, SEQ ID NO:14) (FIG. 2A, Table Tripeptide motifs that appeared to be targeted to multiple organs, tissues or cell types included GVL (SEQ ID NO:15), EGR (SEQ ID NO:16), FGV (SEQ ID NO:17), FGG (SEQ ID NO:18), GER (SEQ ID NO:19) SGT (SEQ ID NO:20) and ARL (SEQ ID NO:21) (FIG. 2B, Table Each of these motifs showed a statistically significant localization to one or more target organs, tissues or cell types, compared to the unselected phage display library.

The ClustalW program (European Molecular Biology Laboratory; EMBL) was used to analyze the original cyclic phage peptide inserts of seven amino acid residues containing the tripeptide motifs. The analysis revealed four to six residue motifs that were shared among multiple peptides isolated from a given organ (FIG. 2A, Table 4).

Each of the motifs was searched for in on-line databases (including BLAST, SWISSPROT, PROSITE, PRODOM, and BLOCKS) through the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp. html#proteindatabases). Some appeared within previously known human proteins and others were not found in the databases. As these motifs are likely to represent sequences present in circulating WO 02/20723 PCT/US01/28044 u ligands (either secreted proteins or surface receptors in circulating cells) that home to C~vascular receptors, a panel of candidate human proteins potentially mimicked by c selected peptide motifs was compiled (Table 4).

WO 02/20723 WO 0220723PCTIUSOI/28044 Table 4 Examples of human proteins potentially mimilcked by peptide motifs Extended Human protein Protein description Accession motif number Bone Marrow

PGGG

PGGG

Bone morphogenetic protein 3B Fibulin 3 GHI{SFG Microsialin Fat EGGT LTBP-2 TGGE Sortilin GPSLH Protocadherin gamma C3 growth, factor, TGF-beta family member fibrillin- and EGF-like macrophage antigen, glycoprotein fibrillin- and EGF-like, TGFbeta Interactor adipocyte differentiationinduced receptor cell adhesion intercellular adhesion molecule endothelial growth factor receptor NP_004953 Q12805 NP_001242 CAA86030 CAA66904 AAD43784 P05362 CAA48290 Muscle

GGSVL

LYSGY

ICAM-1 Flt4 Prostate

RRAGGS

RRAGG

Sin

GRRG

HGG+G

+PHGG

PHGG

+PHGG

Interleukin 11 Smad6 cytokine Smad family member TOF-beta 1 Neuropilin-l Pentaxin Macrophage inhibitory cytokine- 1 Desmoglein 2 growth factor, TGF-beta family member endothelial growth factor receptor infection/trauma-induced glycoprotein growth, factor, TGF-beta family member epithelial cell junction protein epidermal cell lunction protein NP_000632 AAB94137 XP_!008912 AAFP44344 CAA45 158 AAB88673 S38673 AAC838 17 VTG+SG Desmoglein 1 WO 02/20723 WO 0220723PCT[US01/28044 Mulliple Orans

EGRG

GRGE

NFGVV

GERIS

SIREG

+GVLW

WvLVG+

GGFR

GGFF

+SGGF

PSGTS

+TGSP

MMP-9 ESM-1

CDO

BPA1 Wnt- 16 Sialoadhesin 1L,5 receptor gelatinase endothelial cell-specific molecule surface glycoprotein, Ig- and fibronectin-like basement membrane protein glycoprotein Ig-ike lectin soluble interleukin 5 receptor endothelial focal junctionlocalized protein TNF family member EGF-like protein intercellular adhesion glycoprotein vascular repair heparan sulfate proteoglycan AAH06093 XP_003781 NP_058648 NP_001714 Q9UBV4 AAK00757 CAA44081 CA91 196 AAC5 1762 T00209 Q14773 XP _001825 Plectin 1

TRANCE

MIEGF8 ICAM-4 Perlecan Extended targeting motifs homologous to known proteins were identified from bone marrow (GHHSFG, SEQ ID) NO:22, PGGG, SEQ ID NO:23), fat (EGOT, SEQ IID NO:24, TGGE, SEQ ID NO:25, GPSLH-L SEQ ID NO:26), skeletal muscle (GGSVL, SEQ ID) NO:27; LVSGY, SEQ ID NO:28), prostate (RRAGGS, SEQ ID) NO:29; RRAGG, SEQ ED) NO:30), skin (GRRG, SEQ IID NO:31; HGGXG, SEQ ID NO:32; PHG(G, SEQ ID NO:33; VTGXSG, SEQ DD NO:34) and from multiple organs (GRGE, SEQ ID NQ:35; NFGVV, SEQ II) NO:36; GERIS, SEQ ID) NO:37; SIREG, SEQ IID WO 02/20723 PCT/US01/28044 NO:38; GVLW, SEQ ID NO:39; WLVG, SEQ ID NO:40; GGFR, SEQ ID NO:41; GGFF, SEQ ID NO:42, SGGF, SEQ ID NO:43; PSGTS, SEQ ID NO:44; TGSP, SEQ ID NO:45, EGRG, SEQ ID NO:131).

Table 4 shows sequences corresponding to regions of 100% sequence identity Sbetween the peptide selected and the candidate protein. Conserved amino acid Ssubstitutions are indicated as by a Tripeptide motifs from Table 3 are shown in 0 bold.

O The homologous proteins thus identified may represent natural ligands for the C1 human receptors that bound targeting phage. For example, a peptide contained within bone morphogenetic protein 3B (BMP 3B) was recovered from phage localized to bone marrow. BMP 3 B is a growth factor that is known to regulate bone development (Daluiski et al., 2001). Thus, this protein is a candidate ligand mimicked by a peptide homing to bone marrow tissue. Also, interleukin 11 has been shown to interact with receptors within endothelium and prostate epithelium (Mahboubi et al., 2000). IL-11 may be mimicked by a targeting peptide recovered from the prostate (Table In addition to secreted ligands, motifs were also found in several extracellular or transmembrane proteins that may operate selectively in the target tissue, such as sortilin in fat (Lin et al., 1997). Certain motifs appear to be enriched in multiple organs. One such peptide is a candidate mimeotope of perlecan (Table which is a protein known to maintain vascular homeostasis.

These results were confirmed by in situ staining, using polyclonal antibodies against IL-11 receptor alpha. IL-11 is a cytokine that is apparently mimicked by the peptide motif RRAGGS (SEQ ID NO:29), a human prostate targeting peptide. This suggests that the IL-11 alpha receptor (IL-llRa) should be overexpressed in prostate blood vessels. Studies with cultured cells have shown that IL-11 interacts with receptors in endothelium and prostate epithelium (Mahboubi et al., 2000; Campbell et al., 2001). However, expression of IL-lRa in prostate blood vessels has not previously been examined.

WO 02/20723 PCT/US01/28044 Immunostaining of prostate thin sections with antibodies against IL-11Ra F. showed that IL-llRRa is present in the luminal prostate epithelium and in prostate blood _vessels (not shown). This result validates the human biopanning results and shows that the presence of cell surface receptors identified by targeting peptide binding can be confirmed by antibodies against the receptor protein.

C In vivo phage display in humans may reveal heterogeneity at the level of

O

C individual patients. For example, it is unclear whether the malignant B cell infiltration Sin the tested patient influenced the outcome of the screening of the bone marrow, given C1 that the patient had Waldenstrim macroglobulinemia. However, if vascular targeting ligands are to be developed for clinical use, they must resist degradation and be robust enough to target blood vessels in diseased tissue in vivo. Thus, a considerable advantage of the method described here is that the selected targeting peptides bind to native receptors, as they are expressed in vivo. Even if a ligand-receptor interaction is mediated through a conformational (rather than a linear) epitope, it is still possible to select binders in the screening. As it is difficult to ensure that transmembrane proteins expressed by recombinant systems (such as in protein arrays) maintain the correct structure and folding after purification in vitro, peptides selected in vivo is likely to be more suitable to clinical applications, such as identification of novel inhibitors or activators of native receptor proteins.

Precedent exists to suggest that phage can be safely administered to patients, as bacteriophage were widely used in humans during the pre-antibiotic era. Ultimately, it may be possible to determine molecular profiles of human blood vessels by infusing phage libraries systemically before resections of lung, prostate, breast, or colorectal carcinomas or even regionally before resection of limb sarcomas. The methods disclosed herein will allow the construction of a molecular map outlining vascular diversity in each human organ, tissue, or disease.

Human targeting peptide sequences identified by the methods described above and directed to fat, skeletal muscle, skin bone marrow, prostate or to multiple organs are listed in Table WO 02/20723 WO 0220723PCT[USOI/28044 Table 5. Human Targeting Peptide Sequences Sequence SEQ E) Tissue AEEGGTS SEQ ID NO:46 Fat EGGSFNW SEQ ID NO:47 Fat IEGGQVG SEQ ID NO:48 Fat EGGS VfS SEQ II) NO:49 Fat EGGIIFWH SEQ ID NO:50 Fat EGGLSGC SEQ ID NO:51 Fat CAEGGAS SEQ ID NO:52 Fat AEGGVRG SEQ ID NO:53 Fat AEGGRVY SEQ ID NO:54 Fat VVEGGVK SEQ ID NQ:55 Fat VLVGEGG SEQ ID NO:56 Fat TKKLEGG SEQ ID NO:57 Fat GGLSPNW SEQ ID NO:58 Fat TGBLSPG SEQ ID NO:59 Fat VLSPGLG SEQ ID NO:60 Fat LSPGVKG SEQ ID NO:61 Fat LSPWKKR SEQ ID NO:62 Fat AWLSPAR SEQ ID NO:63 Fat AWRRLSP SEQ ID NO:64 Fat WO 02/20723 WO 0220723PCT/USO1/28044 LSPDDAL SEQ ID NO:65 Fat LVSGGMA SEQ ID NO:66 Skeletal muscle LVSGCNT SEQ ID NO:67 Skeletal muscle DLVSGYG SEQ ID NO:68 Skeletal muscle LVSTSAT SEQ ID NO:69 Skeletal muscle TALVSQT SEQ ID NO:70 Skeletal muscle WLVSGIG SEQ ID NO:71 Skeletal muscle LVSSVFP SEQ ID NQ:72 Skeletal muscle PSLVSSV SEQ ID NO:73 Skeletal muscle GVSLVST SEQ ID) NON7 Skeletal muscle QLVSGEP SEQ ID NO:75 Skeletal muscle NLVSRiRL SEQ 11) NQ:76 Skeletal muscle LVSW~vRGS SEQ ID NO:77 Skeletal muscle DHFLVSP SEQ ID NO:78 Skeletal muscle GRGLVSL SEQ ID NQ:79 Skeletal muscle FPVALVS SEQ ID NO:80 Skeletal muscle RWSSLVS SEQ ID NO: 81 Skeletal muscle WSKSLVS SEQ ID NO:82 Skeletal muscle PGRSLVS SEQ ID NO: 83 Skeletal muscle GRRGSAG SEQ ID NO: 84 Skin RPGRRGS SEQ ID NO: 85 Skin SGRRGPR SEQ ID NO:86 Skin GLGRRNG SEQ ID NO:87 Skin WO 02/20723 WO 0220723PCTIUS01/28044 GGRRSQT SEQ ID NO: 88 Skin LWDGRRH SEQ ID NO: 89 Skin GRRSVLT SEQ HI) NO:90 Skin FGRRNLF SEQ ED NO:91 Skin GAGRRYW SEQ ID) NO:92 Skin GRRLWAT SEQ D NO:93 Skin GVGRRFG SEQ ED NO:94 Skin LEMVGRR SEQ H) NO:95 Skin LSSIGRR SEQ ID NO:96 Skin GRRWIDV SEQ ID NQ:97 Skin GRREEGL SEQ ID NO:98 Skin GRRVLOR SEQ ID NQ:99 Skin RGLMGRR SEQ ID NO: 100 Skin RIFLLGRR SEQEDNO:1Q1 Skin PGVGRRL SEQ ID NO: 102 Skin GVIDGRR SEQ ID NO: 103 Skin ADGRRLG SEQ ID NO: 104 Skin AGRRAQI SEQ ID NO: 105 Skin YGRRARE SEQ DNO: 106 Skin PGRRLRM SEQ ID NO: 107 Skin GGRRVTL SEQ ID NO:108 Skin EQGGRRL SEQ ID NO: 109 Skin SGRRLHP SEQ ID NO: 110 Skin WO 02/20723 PCTfUSOI/28044 FDHSGRR SEQIDNO:lll Skin GRRDVAI SEQ ID NO:112 Skin GGBPRLA SEQ ID NO:113 Skin GGHWRVN SEQ ID NO:114 Skin GGHILEV SEQIDD NO:115 Skin GGHRAQS SEQ ID NO:116 Skin GDGGHRP SEQ IDNO:117 Skin SCVGGHS SEQ ID NO:118 Skin GSGVGGH SEQ ID NO:119 Skin VRGWGGH SEQ ID NO:120 Skin WRGWGGH SEQ ID NO:121 Skin WGSKGTV SEQ I NO:122 Skin TGSLGTV SEQ ID NO:123 Skin WGTVSDA SEQ ID NO:124 Skin ATGTVGP SEQ ID NO:125 Skin VVGTVAW SEQ ID NO:126 Skin WVVGTVT SEQ ID NO:127 Skin RVVHGTV SEQ ID NO:128 Skin GTVRFFS SEQ ID NO:129 Skin SGGGPGV SEQ ID NO:132 Bone Marrow RLGGQLA SEQ ID NO:133 Bone Marrow WWGGGVS SEQ ID NO:134 Bone Marrow GSARGGG SEQ ID NO:135 Bone Marrow WO 02/20723 WO 0220723PCT[U SO1/28044 ARGGGIR SEQ ID NO: 136 Bone Marrow RAAGGGG SEQ ID NO: 137 Bone Marrow GSSAGGG SEQ ID NO: 138 Bone Marrow LOEAGGG SEQ ID NO: 139 Bone Marrow GGLEGGG SEQ ID NO: 140 Bone Marrow GNGGGES SEQ ID NO: 141 Bone Marrow STGOCS SEQ ID NO: 142 Bone Marrow LGGGEEW SEQ ID NO: 143 Bone Marrow HGFSHHG SEQ ID NO: 144 Bone Marrow RRGFSLG SEQ ID NO: 145 Bone Marrow GGFSPWL SEQ ID NO: 146 Bone Marrow GRLVGFS SEQ ID NO: 147 Bone Marrow TTrGVGFS SEQ ID NO: 148 Bone Marrow GRRAGGS SEQ ID NO: 149 Prostate TRRAGGG SEQ ID) NO: 150 Prostate SRAGGLG SEQ ID NO: 151 Prostate SYAGGLG SEQ ID NO: 152 Prostate DVAGGLG SEQ ID NO: 153 Prostate GAGGLGA SEQ ID NO: 154 Prostate GAGGWGV SEQ ID NO: 155 Prostate AGGTFKP SEQ ID NO: 156 Prostate LGEVAGG SEQ ID NO: 157 Prostate GSNDAGG SEQ ID NO: 158 Prostate WO 02/20723 PCT/US01/28044 YRGIAGG SEQ ID NO:159 Prostate AGGVAGG SEQ ID NO:160 Prostate GGLAGGF SEQ ID NO:161 Prostate LLAGGVL SEQ ID NO:162 Prostate LVVSAGG SEQ ID NO:163 Prostate RTQAGGV SEQ ID NO:164 Prostate AGGFGEQ SEQ ID NO: 165 Prostate AGGLIDV SEQ ID NO:166 Prostate AGGSTWT SEQ ID NO:167 Prostate AGGDWWW SEQ ID NO:168 Prostate AGGGLLM SEQ ID NO:169 Prostate VAAGGGL SEQ ID NO:170 Prostate LYGAGGS SEQ ID NO:171 Prostate CALAGGC SEQ ID NO:172 Prostate IGAGGVH SEQ ID NO:173 Prostate PKHGVLW SEQ ID NO:174 Multiple Organ SGVLWYH SEQ ID NO:175 Multiple Organ GVLWAFG SEQ ID NO:176 Multiple Organ QARGVLW SEQ ID NO:177 Multiple Organ GVLVSRM SEQ ID NO:178 Multiple Organ GTVGVLV SEQ ID NO:179 Multiple Organ VGVLLPA SEQ ID NO:180 Multiple Organ GGVLLLS SEQ ID NO:181 Multiple Organ WO 02/20723 WO 0220723PCT/US01/28044 SGVLIHD SEQ IID NO: 182 Multiple Organ PYFGVLA SEQ IID NO: 183 Multiple Organ FFVSGVL SEQ ED NO: 184 Multiple Organ LLAGGVL SEQ ID NO: 185 Multiple Organ GEMGGVL SEQ ID NO: 186 Multiple Organ GRAYGVL SEQ IID NO: 187 Multiple Organ SGVLDGR SEQ lID NO: 188 Multiple Organ WSGGVLH SEQ ID NO: 189 Multiple Organ WSGGVLH SEQ ID NO: 190 Multiple Organ SRQGVILR SEQ IID NO: 191 Multiple Organ GVLTSYQ SEQ ID NO: 192 Multiple Organ RGVLTSQ SEQ ID NO: 193 Multiple Organ RVPGVIS SEQ ID NO: 194 Multiple Organ LGVLSYR SEQ ID) NO: 195 Multiple Organ KRGVLGW SEQ ID NO: 196 Multiple Organ GVLGLGF SEQ ID NO: 197 Multiple Organ FLGVLGR SEQ ID NO: 198 Multiple Organ EGVLETS SEQ ID NO: 199 Multiple Organ WWGGVLG SEQ ID NO:200 Multiple Organ VWSRGVL SEQ ID NO:201 Multiple Organ GVLRGVS SEQ ID NO:202 Multiple Organ SFGVLRG SEQ ID NO:203 Multiple Organ KGSVGVL SEQ ID NO:204 Multiple Organ WO 02/20723 WO 0220723PCTfUS01/28044 GGHFGVL SEQ ID NO:205 Multiple Organ WMDVGVL SEQ ID NO:206 Multiple Organ AFRVGVL SEQ ID NO:207 Multiple Organ GVGVLRK SEQ ID NO:208 Multiple Organ MEGRGAG SEQ ID NO:209 Multiple Organ SEGRGFM SEQ ID NO:210 Multiple Organ VEGRNSK SEQ ID NO:21 1 Multiple Organ VEGRYTP SEQ ID NO:212 Multiple Organ FNEGRQM SEQ IID NO:213 Multiple Organ FEGRSRS SEQ ID NO:214 Multiple Organ DHVVEGR SEQ IID NO:215 Multiple Organ WDGTEGR SEQ ID NO:216 Multiple Organ U)W~vREGR SEQ IID NO:217 Multiple Organ RGCEGRV SEQ ED NO:218 Multiple Organ MTPEGRV SEQ ED NO:219 Multiple Organ RLFBGRV SEQ ED NO:220 Multiple Organ RBGRRMC SEQ ID NO:221 Multiple Organ TQFEGRR SEQ ED NO:222 Multiple Organ SMEGRMF SEQ ID NO:223 Multiple Organ PGSAEGR SEQ ID NO:224 Multiple Organ GEGRILA SEQ ID NO:225 Multiple Organ EGRFSAW SEQ ID NO:226 Multiple Organ EGRSDIW SEQ ID NO:227 Multiple Organ WO 02/20723 WO 0220723PCT[US01/28044 EGRARWL SEQ ID NO:228 Multiple Organ EGRERWRM SEQ ID NO:229 Multiple Organ CQCGFGV SEQ ID NO:230 Multiple Organ RGGFGVR SEQ ID NO:23 1 Multiple Organ AVGFGVI SEQ ID NO:232 Multiple Organ AVGFGVI SEQ ID NO:233 Multiple Organ IVGFGVA SEQ ID NO:234 Multiple Organ GNFGVVW SEQ ID NO:235 Multiple Organ DEPFGVA SEQ ID NO:236 Multiple Organ VWFGVGS SEQ ID) NO:237 Multiple Organ WFGVSLS SEQ ED NO:238 Multiple Organ FGVGQWA SEQ ED NO:239 Multiple Organ SMRFGVS SEQ ID NO:240 Multiple Organ RFGVWTG SEQ ID NO:241 Multiple Organ RFGVGRV SEQ ID NO:242 Multiple Organ SGLFGVY SEQ ED NO:243 Multiple Organ MKGVFGV SEQ ED NO:244 Multiple Organ AFGVVSD SEQ ID NO:245 Multiple Organ LYAFGVV SEQ ID NO:246 Multiple Organ KVFGVVE SEQ ID NO:247 Multiple Organ FGVRTDL SEQ ID NO:248 Multiple Organ TIFGVRR SEQ ID NO:249 Multiple Organ VWPRFGG SEQ ID NO:250 Multiple Organ WO 02/20723 WO 0220723PCTIUS01/28044 SRFGGRV SEQ ID NO:251 Multiple Organ MKFGGRL SEQ ID NO:252 Multiple Organ RFGGAILR SEQ ID NO:253 Multiple Organ ERFGGDE SEQ ID NO:254 Multiple Organ FGGSVAP SEQ ID NO:255 Multiple Organ WFGGSVQ SEQ ID NO:256 Multiple Organ FGGSWSL SEQ ID NO:257 Multiple Organ LLFGGSA SEQ ID NO:258 Multiple Organ.

MIZLFGGT SEQ ID NO:259 Multiple Organ FGGFFMY SEQ ID NO:260 Multiple Organ FGGFFMY SEQ ID NO:261 Multiple Organ EFGGQMN SEQ ID NQ:262 Multiple Organ TFGGL1L SEQ ID NO:263 Multiple Organ GNSFGGW SEQ ID NO:264 Multiple Organ RTFGGAG SEQ ID NO:265 Multiple Organ VFGGKS SEQ ID NO:266 Multiple Organ RGFGGLS SEQ ID NO:267 Multiple Organ LWPSFGO SEQ ID NO:268 Multiple Organ GEMIGP SEQ ID NO:269 Multiple Organ GERLSSR SEQ ID NQ:270 Multiple Organ TEGERAG SEQ ID NO:271 Multiple Organ WWLGERV SEQ ID NO:272 Multiple Organ WAWAGER SEQ ID NO:273 Multiple Organ WO 02/20723 WO 0220723PCTIUS01/28044 GVISGER SEQ ID NO:274 Multiple Organ (3PGGERG SEQ ID NO:275 Multiple Organ LGGGERD SEQ ID NO:276 Multiple Organ DIAGERV SEQ ID NO:277 Multiple Organ SRSKGER SEQ ID NO:278 Multiple Organ KRKGERV SEQ ID NO:279 Multiple Organ SRPGERQ SEQ ID NO:280 Multiple Organ CMIRRGER SEQ ID NO:281 Multiple Organ TIRGERN SEQ ID NO:282 Multiple Organ FGERNRI SEQ ID NO:283 Multiple Organ RGERW~DL SEQ ID NO:284 Multiple Organ GERTALL SEQ ID NO:285 Multiple Organ PSGTSSW SEQ ID NO:286 Multiple Organ SMSGTGM SEQ ID NO:287 Multiple Organ LFDVSGT SEQ ID NO:288 Multiple Organ VTGLSGT SEQ I) NO:289 Multiple Organ NMVIGT SEQ ID NO:290 Multiple Organ GVSGTLG SEQ ID NO:291 Multiple Organ RSGTGK SEQ ID NO:292 Multiple Organ GRSGTSG SEQ ID NO:293 Multiple Organ IYSGTLW SEQ ID NO:294 Multiple Organ CSGTLFC SEQ ID NO:295 Multiple Organ RSGTLQT SEQ ID NO:296 Multiple Organ WO 02/20723 PCT/US01/28044 LGSGTWS SEQ ID NO:297 Multiple Organ ESGTATG SEQ ID NO:298 Multiple Organ FTERSGT SEQ ID NO:299 Multiple Organ RYLRSGT SEQ ID NO:300 Multiple Organ PLGSSGT SEQ ID NO:301 Multiple Organ The tripeptide motifs listed in Table 3 above were identified using a one-tail Fisher's test. The identification of statistically significant human targeting peptide motifs was dependent in part on the type of statistical analysis applied. Using a two-tail Fisher's test, a somewhat different set of tripeptide motifs was identified, shown in Table 6.

WO 02/20723 WO 0220723PCTJUS01/28044 Table 6. Human tripeptide motifs identified using a two-tail Fisher's test Tissues Motifs Target organ vs. Target organ other organs vs. unselected library Adipose tissue EVS (SEQ ID NO:302) 0.0059 0.0297 TAG (SEQ ID NO:303) 0.0022 0.0018 VTV (SEQ ID NO:304) 0.0012 0.0073 GEV (SEQ ID NQ:305) 0.0397 0.0147 LPG (SEQ ID NO:306) 0.0242 0.0297 LSP (SEQ ID NO:8) 0.0040 0.0147 POL (SEQ ID NO:307) 0.0402 0.0297 RGT (SEQ ID NO:308) 0.033 1 0.0367 VLL (SEQ ID NO:309) 0.0204 0.0297 Skeletal muscle PAV (SEQ ID NO:3 10) 0.03 17 0.0255 AAV (SEQ ID NO:31 1) 0.0100 0.0255 LVS (SEQ ID NO: 10) 0.0042 0.0031 RGP (SEQ ID NO:3 12) 0.0488 0.0255 VRM (SEQ ID NO:3 13) 0.0015 0.0255 Prostate LLL (SEQ ID NO:3 14) 0.0056 0.0169 RAV (SEQ ID NO:3 15) 0.0040 0.0084 RGE (SEQ ID NO:3 16) 0.0433 0.0324 Skin ARL (SEQ ID NO:21) 0.0444 0.0080 KGG (SEQ ID NO:3 17) 0.0225 0.0317 GAR (SEQ ID NO:3 18) 0.0016 0.0004 WO 02/20723 PCTUSO 1/28044 GGR (SEQ ID NO:319) 0.0001 0.0262 GRR (SEQ ID NO:12) 0.0284 0.0322 WGG (SEQ ID NO:320) 0.0172 0.0078 Bone marrow GGG (SEQ ID NO:5) 0.0469 0.0070 GGM (SEQ ID NO:321) 0.0404 0.0067 GVD (SEQ ID NO:322) 0.0182 0.0183 LGF (SEQ ID NO:323) 0.0062 0.0183 SWV (SEQ ID NO:324) 0.0388 0.0183 SWR (SEQ ID NO:325) 0.0388 0.0183 Several tripeptide motifs were identified as significant by either statistical analysis (indicated in bold in Table While the two statistical analyses identified somewhat different sets of tripeptide motifs, it is considered that tripeptide sequences identified by either statistical analysis will be of use for targeting applications within the scope of the present invention.

The skilled artisan will realize that the multiple targeting peptide sequences identified in the present Example will be of use for numerous applications within the scope of the present invention, including but not limited to targeted delivery of therapeutic agents or gene therapy, in vivo imaging of normal or diseased organs, tissues or cell types, identification of receptors and receptor ligands in organs, tissues or cell types, and therapeutic treatment of a number of human diseases.

Example 3. Polyorgan Targeting In standard protocols, developed with animal model systems, targeting phage are collected from a single organ, tissue or cell type, amplified and reinjected into a second animal, then recollected from the same organ, tissue or cell type. With humans this process is inefficient. In certain alternative embodiments, the efficiency of human organ, tissue or cell type targeting may be improved by polyorgan targeting, in which WO 02/20723 PCT/US01/28044 phage may be collected from multiple organs, tissues or cell types of a first human, Spooled and injected into a second human.

It is possible to perform multiple rounds of selection in humans if the phage recovered from each biopsy is prepared for reinjection as a pool. This protocol has been I successfully demonstrated in the mouse model, using the following exemplary Sprocedure.

O

C A phage library (CX7C) was intravenously injected into anesthetized mice. The Sinput was approximately 1010 transducing units. After 15 minutes and no perfusion (to C1 emulate the human protocol), a variety of organs were removed, including brain, kidney, pancreas, uterus, skeletal muscle, and intestine. After grinding with a Dounce glass homogenizer, tissue samples were washed. After washing, the tissues are incubated with starved competent E. coli K91 and serial dilutions of bacteria were plated on LB tet/kan plates or dishes containing 40 /g/ml of tetracycline and 100 fg/ml kanamycin. Different dilutions for each organ sample were plated, as disclosed above.

The dishes were then incubated overnight at 37 0 C. After the desired enrichment of the phage pool to a certain organ was obtained, about 50 phage clones from the last two rounds of selection were sequenced. Encoded peptide sequences were compared to identify enriched peptides or peptide motifs. The selectivity of selected clones was validated by individually comparing their homing to other organs and also to the homing of an insertless phage clone.

The remaining 2-3 ml of infected culture (including the homogenized tissue) were transferred to 10 ml of LB medium containing 40 ig/ml tetracycline and 100 /tg/ml kanamycin (LB tet/kan) and shaken at 37 0 C for 2 hr. This approximately 12 ml culture was transferred to 1 liter of LB tet/kan and grown overnight in the 37 0 C shaker.

The next day, phage were rescued from the bulk amplified bacterial culture according to standard protocols. These samples were pooled and reinjected for the polyorgan targeting.

In the second and third rounds of selection, 50-100 microliters out of 1 ml of the total bulk phage recovered from each organ was pooled. The resulting mixture was WO 02/20723 PCT/US01/28044 'reinjected in another mouse and the same organs were recovered after 10 minutes (no Sperfusion). Plating of the phage from each organ revealed enrichment for each site 1 except uterus, showing that selection was possible with the polyorgan protocol.

Progressive enrichment of targeting phage in other organs as a function of the number of rounds of polyorgan targeting (not shown).

Peptide motifs of phage targeting skeletal muscle and kidney by polyorgan targeting are listed in Table 7 below.

STable 7. Consensus Peptide Motifs Identified by Polyorgan Targeting in Mice Skeletal Muscle VGV SEQ ID NO:326 VGVG SEQ ID NO:327 IGS SEQ ID NO:328 GGA SEQ ID NO:329 VGA SEQ ID NO:330 WHG SEQ ID NO:331 SGEA SEQ ID NO:332 SNEA SEQ ID NO:333 SLRD SEQ ID NO:334 SVRD SEQ ID NO:335 SARD SEQ ID NO:336 HVV SEQ ID NO:337 RTG SEQ ID N0422 RLG SEQ ID NO:423 RWG SEQ ID NO:338 LRWG SEQ ID NO:339 WO 02/20723 WO 0220723PCTJUS01/28044 C1RNS SEQ ID NO:344) RSG SEQ ID NO:341 GRSG SEQ ID NO:342 _RSGL SEQ ID NO:343 YGR SEQ ID NO:344 ASL SEQ ID NO:345 SGA SEQ ID NO:346 SGR SEQ ID NO:347 DSG SEQ ID NQ:348 SSRV SEQ H) NQ:349 SSRI SEQ ID NO:350 FGSR SEQ ID NO:351 FCSR SEQ ID NO:352 FQS SEQ H)DNO:353 PPV SEQ ID NQ:354 LFQ SEQ ID NO:355 VVA SEQ ID NO:356 AVY SEQ H) NO:357 VAS SEQ ID NQ:358 EVSFSR SEQ ID NO:359 EPSLFLR SEQ ID NO:360 POL SEQ ID NO:361 PGI SEQ ID NO:362 GLA SEQ ID NO:363 WO 02/20723 WO 0220723PCTIUSOI/28044

VGLAV

VGPAV

TRG

GLS

DWR

RGA

RGG

ARGG

NGR

ALAKG

ALARG

'1TQ

GYR

G'YL

DRT

lys

WLS

II=E

IFQTES

LARP

IVRP

HRP

LRPI

KNWAIBhR SEQ ID NO:364 SEQ ID NO:365 SEQ ID NO:366 SEQ ID NO:367 SEQ ID NO:368 SEQ ID NQ:369 SEQ ID NQ:370 SEQ ID NO:371 SEQ ID NO:372 SEQ ID NO:373 SEQ ID NO:374 SEQ ID NO:375 SEQ ID NO:376 SEQ ID NO:377 SEQ ID NO:378 SEQ ID NO:379 SEQ ID NO:380 SEQ ID NO:381 SEQ ID NQ:382 SEQ ID NO:383 SEQ ID NO:384 SEQ ID NO:385 SEQ ID NO:386 SEQ H) NQ:387 WO 02/20723 WO 0220723PCTIUS01/28044 rlASLR SEQ ID NO:388 AGLR SEQ ID NQ:389 AAV SEQID NO:390 QLDRI{ SEQ ID NO:391 N QWDRII SEQ H) NO:392 VEV SEQ ID NO:393 FRYL SEQ ID NQ:394 FRSL SEQ ID NQ:395 ARI SEQ ID NO:396 ARL SEQ ID NO:397 ARLG SEQ ID NO:398 ARIG SEQ ID NO: 399 RSA SEQ ID NO:400 ADWF SEQ ID NO:401 ADAF SEQ ID NO:402 GWS SEQ ID NO:403 TWM SEQ ID NO:404 Kidney LPS SEQ ID GLP SEQ ID NO:406 LLG SEQ ID NO:407 IDS SEQ ID NO:408 GLR SEQ ID NO:409 TYs SEQ ID WO 02/20723 PCT/US01/28044 GGGR SEQ ID NO:411 SGVR SEQ ID NO:412 VWG SEQID NO:413 VGG SEQ ID NO:414 SRVR SEQ ID NO:415 YVR SEQ ID NO:416 0 VFG SEQ ID NO:417 PQL SEQ ID NO:418 RAA SEQ ID NO:419 RWA SEQ ID NO:420 LGS SEQ ID NO: 421 This example showed the feasibility of polyorgan targeting in a single subject to which a mixture of organ targeting phage was administered as a pool. This method is particularly relevant for vascular targeting in humans. Due to the shortage of potential subjects and the expense of processing each one, it is impractical to inject a human with a single phage population to target a single organ at a time.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it are apparent to those of skill in the art that variations maybe applied to the COMPOSITIONS, METHODS and APPARATUS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it are apparent that certain agents that are both chemically and physiologically related may be P:\OPER\DND3025 I 2 dlv peci pgcl 179doc o -86substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Ni Ccr Throughout this specification and the claims which follow, unless the context requires C<K otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps Ci but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

PAOPER\DND00255I82 d c sp., p~g 179 dmc -86A-

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by t reference.

CN Anand-Apte B, Pepper MS, Voest E, Montesano R, Olsen B, Murphy G, Apte SS and SZetter B. Inhibition of angiogenesis by tissue inhibitor of metallopeinase-3. Invest.

CN Opthamol. Vis. Sci. 38: 817-823, 1997 Arap W, Pasqualini R, and Ruoslahti E. Chemotherapy targeted to tumor vasculature. Curr.

Opin. Oncol., 1998b.

Arap, Pasqualini and Ruoslahti, E. Cancer treatment by targeted drug delivery to tumor vasculature. Science 279: 377-380, 1998a.

Arap, Pasqualini, R. Ruoslahti, E. Chemotherapy targeted to tumor vasculature.

Curr Opin Oncol 10, 560-565 (1998).

Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum Press, pp. 117-148, 1986.

Baldwin, R. W. et al. Monoclonal antibody-defined antigens on tumor cells.

Biomembranes 11, 285-312 (1983).

Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, pp. 1-284, 1979.

WO 02/20723 PCT/US01/28044 Bartlett, Kleinschmidt, Boucher, R.C. Samulski, R.J. Targeted adeno- Sassociated virus vector transduction of nonpermissive cells mediated by a bispecific C1 Fab'gamma)2 antibody. Nat Biotechnol 17, 181-186, 1999.

BERGELSON, CUNNINGHAM, DROGUETT, KURT-JONES, E.A., KRITHIVAS, HONG, HORWITZ, CROWELL, and C FINBERG, R.W. (1997). Isolation of a common receptor for coxsackie B viruses 0 and adenoviruses 2 and 5. Science 275; 1320-1322.

SBielenberg, D. M. F. McCarty, C. D. Bucana, S. H. Yuspa, D. Morgan, J. M. Arbeit, C, L. M. Ellis, K. R. Cleary, and I. J. Fidler. 1999. Expression of interferon-beta is associated with growth arrest of murine and human epidermal cells. J Invest Dermatol 112:802-9.

Boehm T, Folkman J, Browder T, and O'Reilly MS. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390:404-407, 1997 Boon, T. Old, L. J. Cancer Tumor antigens. Curr Opin Immunol 9, 681-683 (1997).

Bossemeyer, Engh, Kinzel, Ponstingl, H. and Huber, R.

Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn 2+ adenylyn imidiophosphate and inhibitor peptide PKI(5-24). EMBO J. 12:849-859, 1993.

Brodt et. al, The role of marrow endothelium in the localization of metastastic cancer cells to bone. In Bone Metastasis- mechanisms and pathophysiology, ppl7-23, 1996. (Orr and Singh, eds.) Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin avB3 for angiogenesis. Science 264:569-571, 1994a.

WO 02/20723 PCT/US01/28044 U Brooks PC, Stromblad S, Klemle R, Visscher D, Sarkar FH, and Cheresh DA. Anti- Jintegrin av83 blocks human breast cancer growth and angiogenesis in human skin. J. Clin. Invest. 96:1815- 1822, 1995.

Brooks, P.C. et al. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell 85, 683-693, 1996.

Brooks, Montgomery Rosenfeld, Reisfeld, Hu, Klier, and Cheresh D.A. Integrin avp3 antagonists promote tumor regression by inducing 0apoptosis of angiogenic blood vessels. Cell 79, 1157-1164, 1994b Brousset, S. Chittal, D. Schlaifer, J. Icart, C. Payen, F. Rigal-Huguet, J. J. Voigt, and G. Delsol. 1991. Detection of Epstein-Barr virus messenger RNA in Reed-Sternberg cells of Hodgkin's disease by in situ hybridization with biotinylated probes on specially processed modified acetone methyl benzoate xylene (ModAMeX) sections. Blood 77:1781-6.

Burg M, Pasqualini R, Arap W, Stallcup W, and Ruoslahti E. Identification of NG2 proteoglycan-binding peptides that home to tumor neovasculature. Cancer Res 58:2869-2874, 1999.

Burg, Pasqualini, Arap, Ruoslahti, E. Stallcup, W.B. NG2 proteoglycan-binding peptides target tumor neovasculature. Cancer Res 59, 2869- 2874, 1999.

Campbell et al., Am. J. Pathol., 158:25-32, 2001.

Cao Y. O'Reilly MS. Marshall B. Flynn E. Ji RW and Folkman J. Expression of angiostatin cDNA in a murine fibrosarcoma suppresses primary tumor growth and produces long-term dormancy of metastases. J. Clin. Invest. 101:1055-1063, 1998.

Carter, H. Piantadosi, S. Isaacs, J. T. Clinical evidence for and implications of the multistep development of prostate cancer. J Urol 143, 742-746 (1990).

WO 02/20723 PCT/US01/28044 C' Chang, K. and L. M. Weiss. 1996. The association of the Epstein-Barr virus with Z malignant lymphoma. Biomed Pharmacother 50:459-67.

Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987.

Chen et al., J. Cell. Biochem., 78:404-416,2000.

SChinni et al., Clin. Cancer Res. 3:1557-64, 1997.

0 Clark, E.A. and Brugge, J.S. Integrins and signal transduction pathways: the road taken.

Science 268:233-238,1995.

Coffin, In: Virology, Fields et aL, eds., Raven Press, New York, pp. 1437-1500, 1990.

Cortese, I. et al. Identification of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using phage libraries. Proc Natl Acad Sci U S A 93, 11063- 11067 (1996).

Couch et al., Am. Rev. Resp. Dis., 88:394-403, 1963.

Coupar et al., Gene, 68:1-10, 1988.

Cox, D. R. Regression models and life tables. Journal of the Royal Statistical Society 74, 187-220 (1972).

Curiel, D.T. Strategies to adapt adenoviral vectors for targeted delivery. Ann N Y Acad Sci 886, 158-171, 1999.

Defilippi, Bozzo, Volpe, Romano, Venturino, Silengo, L. and Tarone, G. Integrin-mediated signal transduction in human endothelial cells: analysis of tyrosine phosphorylation events. Cell Adh. Commun. 87:75-86, 1994.

Delannet, Martin, Bossy, Cheresh, Reichardt, L.F. and Duband, J.L.

Specific roles of the av1l, avB3, and avB5 integrins in avian neural crest cell adhesion and migration on vitronectin. Development. 120:2687-702, 1994.

Delpino et al., Mol. Membr. Biol. 15:21-26, 1998.

WO 02/20723 PCT/US01/28044 'Dente, Vetriani,C., Zucconi, Pelicci, Lanfrancone, Pelicci, P.G. and SCesareni, G. Modified phage peptide libraries as a tool to study specificity of C phosphorylation and recognition of tyrosine containing peptides. J. Mol. Biol.

269:694-703,1997.

ln Derossi, Chassaing, G. and Prochiantz, A. Trojan peptides: the penetratin system for c intracellular delivery. Trends Cell Biol. 8:84-87, 1998.

O

Derossi, Joliot, Chassaing, G. and Prochiantz, A. The third helix of SAntennapedia homeodomain translocates through biological membranes. J. Biol.

Chem. 269:10444-10450, 1994 DMITRIEV, KRASNYKH, MILLER, WANG, KASHENTSEV,A.E., MIKHEEVA, BELOUSOVA, and CURIEL, D.T. (1998). An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackie virus and adenovirus receptor-independent cell entry mechanism. J. Virol. 72; 9706-9713.

DOUGLAS, ROGERS, ROSENFELD, ME., MICHAEL, FENG, M., and CURIEL, D.T. (1996). Targeted gene delivery by tropism-modified adenoviral vectors. Nature Biotechnol. 14; 1574-1578.

Dunn, I.S. Mammalian cell binding and transfection mediated by surface-modified bacteriophage lambda. Biochimie 78, 856-861, 1996.

Dybwad, Forre, Kjeldsen-Kragh, Natvig, J. B. Sioud, M. Identification of new B cell epitopes in the sera of rheumatoid arthritis patients using a random nanopeptide phage library. Eur J Immunol 23, 3189-3193 (1993).

Eisen, T. et al. Continuous low dose Thalidomide: a phase II study in advanced melanoma, renal cell, ovarian and breast cancer. Br J Cancer 82, 812-817, 2000.

Ellerby HM, Arap W, Ellerby L, Kain R, Andrusiak R, Rio G, Krajeswki S, Lombardo C, Rao R, Ruoslahti E, Bredesen D, and Pasqualini R. Anti-cancer Activity of Targeted proapoptotic peptides. Nature Med 9:1032-1038, 1999 WO 02/20723 PCT/US01/28044 C' Enblad, K. Sandvej, E. Lennette, C. Sundstrom, G. Klein, B. Glimelius, and G.

SPallesen. 1997. Lack of correlation between EBV serology and presence of EBV in C1 the Hodgkin and Reed-Stemberg cells of patients with Hodgkin's disease. Int J Cancer 72:394-7.

n Engelstlidter M et al. Targeting human T cells by retroviral vectors displaying antibody cdomains selected from a phage display library. Hum Gene Ther. 2000; 11: 293-303.

O

C' Engerman, R.L. and Kern, T.S. (1986) Hyperglycemia as a cause of diabetic retinopathy.

0 Metabolism 35(S1), 20-23.

C. Ferrara, N. and Davis-Smyth, T. (1997) The biology of vascular endothelial growth factor.

Endocr. Rev., 18,4-25.

Filardo, E.J. and Cheresh, D.A. A 8 turn in the cytoplasmic tail of the integrin av subunit influences conformation and ligand binding of avB3. J. Biol. Chem.

269:4641-4647, 1994a.

Filardo, E.J. and Cheresh, D.A. A structural basis for bidirectional integrin signalling.

Princess Takamatsu Symp. 24:106-117, 1994b.

Filardo, Brooks, Deming, Damsky, C. and Cheresh, D.A. Requirement of the NPXY motif in the integrin 13 subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo. J. Cell Biol. 130:441-450, 1995.

Folkman J. Addressing tumor blood vessels. Nature Biotechnol. 15: 510, 1997.

Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med 1:27-31, 1995 Folkman, J. Antiangiogenic gene therapy. Proc Natl Acad Sci U S A 95, 9064-9066, 1998.

Friedlander M, Brooks PC, Sharffer RW, Kincaid CM, Vamer JA, and Cheresh DA.

Definition of two angiogenic pathways by distinct av integrins. Science, 270: 1500- 1502, 1995.

WO 02/20723 PCT[US01/28044 Friedlander M, Theesfeld CL, Sugita M, Fruttiger M, Thomas MA, Chang S, Cheresh DA. Involvement of integrins axvJ3 and cxvP5 in ocular neovascular diseases. Proc.

Nati. Acad. Sci. USA 93:9764-9769, 1996.

Friedmann, Science, 244:1275-1281, 1989.

tt~ Frisch SM. And Ruoslahti E. Integrins and anoikis. Cur. Opin. in Cell Biol. 9:701-706, 1997.

Furuya et al., Cancer Res. 54:6167-75, 1994.

Ghosh-Choudhury et al., EMBO 6:1733-1739, 1987.

Gingrich JR, Barrios RT, Morton RA, Boyce BF, DeMayo FJ, Finegold MJ, Angelopoulou R, Rosen 3M and Greenberg NM. Metastatic prostate cancer in a transgenic mouse. Cancer Res. 56:4096-4102, 1996.

Girod A et al. Genetic capsid modifications allow efficient re-targeting of adenoassociated virus type 2. Nat Med 1999; 5: 1052-1056.

Gold R. Differenitation between Cellular Apoptosis ad Necrosis by the Combined Use of In Situ Tailing Translation Techniques. Lab. Invest. 71: 219, 1994 Goldman CK et al. Targeted gene delivery to Karposi's sarcoma cells via the fibroblast growth factor receptor. Cancer Res 1997; 57: 1447-145 1.

GOLDMAN, C.K, ROGERS, DOUGLAS, SOSNOWSKI YING, W., SIEGAL, BAIRD, CAMPAIN, and CURIEL, D.T. (1997). Targeted gene delivery to Karposi's sarcoma cells via the fibroblast growth factor receptor.

Cancer Res. 57; 1447-145 1.

Gomez-Foix et al., J. Biol. Chem., 267:25 129-25134, 1992.

Gopal, Mol. Cell Biol., 5:1188-1190, 1985.

Grace, M. L. Xie, M. L. Musco, S. Ciii, M. Gumnani, R. DiGiacomo, A. Chang, S.

Indelicato, J. Syed, R. Johnson, and L. L. Nielsen. 1999. The use of laser scanning WO 02/20723 PCT/US01/28044 U *cytometry to assess depth of penetration of adenovirus p53 gene therapy in human Z xenograft biopsies. Am J Pathol 155:1869-78.

Graham and Prevec, In: Methods in Molecular Biology: Gene Transfer and Expression Protocol, E.J. Murray, ed., Humana Press, Clifton, NJ, 7:109-128, 1991.

n Graham and van der Eb, Virology, 52:456-467, 1973.

0 Graham et al., J. Gen. Virol., 36:59-72, 1977.

Gram, Schmitz, Zuber, J.F. and Baumann, G. Identification of phosphopeptide ligands for Src-homology 2 (SH2) domain of Grb2 by phage display. Eur. J.

Biochem. 246:633-637, 1997.

Greenberg NM, DeMayo F, Finegold MJ, Medina D, Tilley WD, Aspinall JO, Cunha GR, Donjacour AA, Matusik RJ and Rosen JM. Prostate cancer in a transgenic mouse. Proc. Natl. Acad. Sci. USA 92:3439-3443, 1995.

Griscelli F. Li H. Bennaceur-Griscelli A. Soria J. Opolon P. Soria C. Perricaudet M.

Yeh P and Lu H. Angiostatin gene transfer- inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest.

Proc. Natl. Acad. Sci. USA 95:6367-72, 1998 Grunhaus and Horwitz, Seminar in Virology, 3:237-252, 1992.

Gunge et al., Mol. Gen. Genet. 263:846-854, 2000.

Hall, Williams, EJ., Moore, SE., Walsh, FS., Prochiantz, A. and Doherty, P.

Inhibition of FGF-stimulated phosphatidylinositol hydrolysis and neuron outgrowth by a cell -membrane permeable phosphopeptide. Current Biology, 6:580-587, 1996.

Hammes HP, Brownlee M, Jonczyk A, Sutter A, and Preissner KT. Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization. Nature Med. 2: 529-533, 1996.

WO 02/20723 PCT/US01/28044 C Hanahan, D. and Folkman, J. (1996) Patterns and Emerging Mechanisms of the Angiogenic Switch during Tumorogenesis. Cell, 86, 353-364.

Cl Hansen, Noren, Sj6str6m,H. and Wedelin, 0. (1993) A mouse aminopeptidase-N is a marker for antigen presenting cells and apears to be coexpressed with major histocompatibility complex class II molecules. Eur. J.

CN Immunol., 23, 2358-64.

O HARLOW, and LANE, D. (1988). Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, NY).

C, Hart SL et al. Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly- Asp-containing peptide. J. Biol. Chem. 269, 12468-12474, 1994 Hemler, Weitzman, Pasqualini, Kawaguchi, Kassner, P. and Berdichevsky, F. Structure, biochemical properties, and biological functions of integrin cytoplasmic domains. In: Integrins: The Biological Problems (ed. Yoshi Takada) CRC Press, Inc., Boca Raton, FL, USA; pp.1-35, 1994.

Hendrix RW. Evolution: the long evolutionary reach of viruses. Current Biol. 9:914- 917, 1999.

HENRY, XIA, WILKE, DEISENHOFER, and GERARD, R. (1994).

Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in E. coli. J. Virol. 68; 5239-5246.

Herbst, E. Steinbrecher, G. Niedobitek, L. S. Young, L. Brooks, N. Muller- Lantzsch, and H. Stein. 1992. Distribution and phenotype of Epstein-Barr virusharboring cells in Hodgkin's disease. Blood 80:484-91.

Herbst, F. Dallenbach, M. Hummel, G. Niedobitek, S. Pileri, N. Muller-Lantzsch, and H. Stein. 1991. Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells. Proc Natl Acad Sci U S A 88:4766-70.

Hermonat and Muzycska, Proc. Natl. Acad Sci. USA, 81:6466-6470, 1984.

WO 02/20723 PCT/US01/28044 Hemdier BG, Werner A, Arstein P, Abbey NW, Demartis F, Cohen RL, Shuman MA Sand Levy, JA Characterization of a human Kaposi's sarcoma cell line that C induces angiogenic tumors in animals. AIDS 8:575-581, 1996.

Hersdorffer et al., DNA Cell Biol., 9:713-723, 1990.

in Herz and Gerard, Proc. Natl Acad. Sci. USA, 90:2812-2816, 1993.

HEYWOOD, and HOOPER, N.M. (1995). Development and application of a fluorometric assay for mammalian membrane dipeptidase. Anal. Biochem. 226; 14.

HONG, GALAUP, PEYTAVI, CHAZAL, and BOULANGER, PA.

(1999). Enhancement of adenovirus-mediated gene delivery by use of an oligopeptide with dual binding specificity. Hum. Gene Ther. 10; 2577-2586.

HONG, KARYAN, TOURNIER, CURIEL, and BOULANGER, P.A.

(1997). Adenovirus type 5 fiber knob binds to MHC class I alpha-2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO J. 16; 2294-2306.

Horwich, et al., J. ViroL, 64:642-650, 1990.

Hughes et al., Cancer Res. 49:4452-54, 1989 Hynes, R.O. Integrins: versatility, modulation and signaling in cell adhesion. Cell 69:11-25, 1992.

Ivanenkov, Felici, F. Menon, A.G. Uptake and intracellular fate of phage display vectors in mammalian cells. Biochim Biophys Acta 1448, 450-462, 1999a.

Ivanenkov, Felici, F. Menon, A.G. Targeted delivery of multivalent phage display vectors into mammalian cells. Biochim Biophys Acta 1448, 463-472, 1999b.

J. Natl. Cancer Inst. 90:273-286, 1998.

Jarrett, A. A. A. Armstrong, and E. Alexander. 1996. Epidemiology of EBV and Hodgkin's lymphoma. Ann Oncol 7:5-10.

WO 02/20723 PCT/US01/28044 CN Jarrett, R. and J. MacKenzie. 1999. Epstein-Barr virus and other candidate viruses in the pathogenesis of Hodgkin's disease. Semin Hematol 36:260-9.

Johnson et al., "Peptide Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, New York (1993).

tC Joliot, A.H. Triller, Volovitch, M. Pernelle, and Prochiantz, A. alpha-2,8- Polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide.

SNew Biol.3:1121-1131, 1991a.

Joliot, Pernelle, Deagostini-Bazin, H. and Prochiantz, A. Antennapedia homeobox peptide regulates neural morphogenesis Proc. Natl. Acad. Sci. U.S.A.

88:1864-1868, 1991b.

Jones and Shenk, Cell, 13:181-188, 1978.

Kaplan, E. L. a. P. Nonparametric estimation from incomplete observations.

Journal of the American Statistical Association 53,457-481 (1958).

Karlsson et al., EMBO 5:2377-2385, 1986.

Kasono, K. et al. Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector. Clin Cancer Res 5, 2571-2579, 1999.

Kassner, Burg, Baird, A. Larocca, D. Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells. Biochem Biophys Res Commun 264, 921-928, 1999.

Kiang et al., Chin. J. Physiol. 40:213-219, 1997 Klemke, Yebra, Bayna, E.M. and Cheresh, D.A. Receptor tyrosine kinase signaling required for integrin av85 -directed cell motility but not adhesion on vitronectin. J. Cell Biol. 127:859-866, 1994.

WO 02/20723 PCTUS1/28044 Koivunen E, Arap W, Valtanen H, Rainisalo A, Gahmberg CG, Salo T, Konttinen YT, ZSorsa T, Ruoslahti E, Pasqualini R. Tumor targeting with a selective gelatinase c1 inhibitor. Nature Biotechnol 17:768-774, 1999 Koivunen E, Gay DA and Ruoslahti E. Selection of peptides binding to the a5o1 integrin from phage display library. J. Biol. Chem. 268:20205-20210, 1993.

Koivunen E, Wang B, and Ruoslahti E. Phage display libraries displaying cyclic C peptides with different ring sizes: ligand specificities of the RGD-directed integrins. BioTechnology 13:265-270, 1995.

Koivunen, E. et al. Integrin-binding peptides derived from phage display libraries.

Methods Mol Biol 129, 3-17 (1999).

Kolanus, W. and Seed, B. Integrins and inside-out signal transduction: converging signals from PKC and PIP3. Curr. Opin. Cell Biol. 9:725-731, 1997.

Kolonin MG. Finley RL Jr. Targeting cyclin-dependent kinases in Drosophila with peptide aptamers. Proc. of the Natl. Acad. of Sci. USA..95:14266-71, 1998.

Kong HL and Crystal RG. Gene therapy strategies for tumor antiangiogenesis.

Kouzmitcheva G. A. et al. Identifying diagnostic peptides for lyme disease through epitope discovery. Clin Diagn Lab Immunol 8, 150-60 (2001).

KOZARSKY, JOOSS, DUNAHEE, STRAUSS, and WILSON, J.M.

(1996). Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene. Nat. Genet. 13; 54- 62.

KRASNYKH, DMITRIEV, MIKHEEV, MILLER, BELOUSOVA, and CURIEL, D.T. (1998). Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob. J. Virol. 72; 1844- 1852.

WO 02/20723 PCT/US01/28044 u' KRASNYKH, MIKHEEVA, DOUGLAS, and CURIEL, D.T. (1996).

SGeneration of recombinant adenovirus vectors with modified fibers for altering viral (C tropism. J. Virol. 70; 6839-6846.

Lane T. Shah J. Clinical features and management of benign prostatic hyperplasia.

Hospital Medicine. 60(10):705-9, 1999.

C Larocca D et al. Gene transfer to mammalian cells using genetically targeted

O

C1 filamentous bacteriophage. FASEB J 1999; 13:727-734.

0Larocca, Witte, Johnson, Pierce, G.F. Baird, A. Targeting bacteriophage to mammalian cell surface receptors for gene delivery. Hum Gene Ther 9, 2393- 2399,1998.

Le Gal La Salle et al., Science, 259:988-990, 1993.

Le Roux, Joliot, Bloch-Gallego, Prochiantz, A. and Volovitch, M.

Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties. Proc. Natl. Acad. Sci. U.SA. 90:9120-9124, 1993 Levrero et al., Gene, 101:195-202, 1991.

Lewis, Cheresh, D.A. and Schwartz, M.A. Protein kinase C regulates dependent cytoskeletal associations and focal adhesion kinase phosphorylation. J.

Cell Biol. 134:1323-1332, 1996.

Lin, Aplin, Shen, Chen Schaller, Romer Aukhil, I. and Juliano, R.L. Integrin-mediated activation of MAP kinase is independent of FAK: evidence for dual integrin signalling pathways in fibroblast. J. Cell Biol. 136:1385- 1395, 1997.

Longhurst, C.M. and Jennings, L.K. Integrin-mediated signal transduction. Cell Mol.

Life Sci. 54:514-526, 1998.

WO 02/20723 PCT/US01/28044 u 'Look AT, Ashmun RA, Shapiro LH and Peiper SC. Human myeloid plasma membrane glycoprotein CD13 (gpl50) is identical to aminopeptidase N. J. Clin. Invest.

83:1299-1307, 1989.

LOUIS, FENDER, BARGE, KITS, and CHROBOCZEK, J. (1994). Cellbinding domain of adenovirus serotype 2 fiber. J. Virol. 68; 4104-4106.

tC Lunardi, C. et al. Systemic sclerosis immunoglobulin G autoantibodies bind the human

O

C1 cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells S[In Process Citation]. Nat Med 6, 1183-1186 (2000).

O

Lynch, C.M. et al. Adeno-associated virus vectors for vascular gene delivery. Circ Res 80,497-505, 1997.

Lyons, S. and D. N. Liebowitz. 1998. The roles of human viruses in the pathogenesis of lymphoma. Semin Oncol 25:461-75.

MacGregor, G.R. Caskey, C.T. Construction of plasmids that express E. coli betagalactosidase in mammalian cells. Nucleic Acids Res 17, 2365, 1989.

Mahboubi et al, J. Immunol. 164:3837-3846, 2000.

Mann et al., Cell, 33:153-159, 1983.

Markowitz et al., J. Virol., 62:1120-1124, 1988.

Martin F et al. Retrovirus targeting by tropism restriction to melanoma cells. J Virol 1999;73: 6923-6929.

Martiny-Baron G, and Marme D. VEGF-mediated tumor angiogenesis: a new target for cancer therapy. Curr. Opin. Biotechnol. 6:675-680, 1995.

Mennuni, C. et al. Selection of phage-displayed peptides mimicking type 1 diabetesspecific epitopes. JAutoimmun 9, 431-436 (1996).

Merrifield, Science, 232: 341-347, 1986 WO 02/20723 PCT/US01/28044 MICHAEL, HONG, CURIEL, and ENGLER, J.A. (1995). Addition of a short peptide ligand to the adenovirus fiber protein. Gene Ther. 2; 660-668.

Mikolajczyk SD. Millar LS. Wang TJ. Rittenhouse HG. Marks LS. Song W. Wheeler TM. Slawin KM. A precursor form of prostate-specific antigen is more highly In elevated in prostatecancer compared with benign transition zone prostate tissue.

Cancer Research. 60(3):756-9, 2000.

Cl Miller CR et al. Differential susceptibility of primary and established human glioma l^- Scells to adenovirus infection: targeting via the epidermal growth factor receptor CI achieves fiber receptor independent gene transfer. Cancer Res 1998; 58: 5738- 5748.

Motti, C. et al. Recognition by human sera and immunogenicity of HBsAg mimotopes selected from an M13 phage display library. Gene 146, 191-198 (1994).

Mulligan, Science, 260:926-932, 1993.

Mustonen T and Alitalo K. Endothelial receptor tyrosine kinases involved in angiogenesis. J. Cell Biol. 129:895-898, 1995.

Muzyczka N. Adeno-associated virus (AAV) vectors: will they work? J. Clin. Invest.

94:1351, 1994 Nicolas and Rubinstein, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988.

Nicolau et al., Methods Enzymol., 149:157-176, 1987.

Old, L. J. Cancer immunology: the search for specificity--G. H. A. Clowes Memorial lecture. Cancer Res 41, 361-375 (1981).

Olofsson, B. Jeltsch, Eriksson, U. and Alitalo, K. (1999) Current Biology of VEGF- B and VEGF-C. Curr Op Biotechnol, 10, 528-535.

Olofsson, Pajusola, Kaipainen, Euler, Joukov, Saksela, Orpana, Pettersson, Alitalo, K. and Eriksson, U. (1996) Vascular Endothelial WO 02/20723 PCT/US01/28044 Growth factor B, a novel growth factor for endothelial cells. Proc Natl Acad Sci Z USA, 93, 2576-2581.

Owens, G. R. A. Williamson, M. P. Burgoon, O. Ghausi, D. R. Burton, and D. H.

Gilden. 2000. Cloning the antibody response in humans with chronic inflammatory disease: immunopanning of subacute sclerosing panencephalitis (SSPE) brain sections with antibody phage libraries prepared from SSPE brain enriches for antibody recognizing measles virus antigens in situ. J Virol 74:1533-7.

Pallesen, S. J. Hamilton-Dutoit, M. Rowe, and L. S. Young. 1991. Expression of Epstein-Barr virus latent gene products in tumour cells of Hodgkin's disease [see comments]. Lancet 337:320-2.

Paskind et al., Virology, 67:242-248, 1975.

Pasqualini R and Ruoslahti E. Organ targeting in vivo using phage display peptide libraries. Nature 380:364-366, 1996.

Pasqualini R, Koivunen E, and Ruoslahti E. A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins. J. Cell Biol. 130:1189-1196, 1995.

Pasqualini R, Koivunen E, and Ruoslahti E. av integrins as receptors for tumor targeting by circulating ligands. Nature Biotechnol 15:542-546, 1997 Pasqualini, R. and Hemler, M. E. Contrasting roles for integrin bl and b5 cytoplasmic domains in subcellular localization, cell proliferation, and cell migration. J. Cell Biol. 125:447-60, 1994.

Pasqualini, R. Vascular Targeting with Phage Display Peptide Libraries. The Quart. J.

Nucl. Med. 43:159-162, 1999.

Pasqualini, Arap Koivunen, Kain, Lahdenranta, Shapiro, L., Sakamoto, Stryn, A. and Ruoslahti, E. Aminopeptidase N is a receptor for WO 02/20723 PCT/US01/28044 tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res. S722-727, 2000.

Pelleymounter et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269: 540-543, 1994.

to) Pereboeva, L. A. V. Pereboev, and G. E. Morris. 1998. Identification of antigenic Cc sites on three hepatitis C virus proteins using phage-displayed peptide libraries. J N Med Virol 56:105-11.

0 Pereboeva, L. A. V. Pereboev, L. F. Wang, and G. E. Morris. 2000. Hepatitis C epitopes from phage-displayed cDNA libraries and improved diagnosis with a chimeric antigen. J Med Virol 60:144-51.

Potter et al., Proc. Nat. Acad Sci. USA, 81:7161-7165, 1984.

Poul, M.A. Marks, J.D. Targeted gene delivery to mammalian cells by filamentous bacteriophage. J Mol Biol 288, 203-211, 1999.

Prezzi, C. et al. Selection of antigenic and immunogenic mimics of hepatitis C virus using sera from patients. JImmunol 156, 4504-4513 (1996).

Prezzi, M. Nuzzo, A. Meola, P. Delmastro, G. Galfre, R. Cortese, A. Nicosia, and P.

Monaci. 1996. Selection of antigenic and immunogenic mimics of hepatitis C virus using sera from patients. J Immunol 156:4504-13.

PRICE, POLYZOS, ZHANG, and DANIELS, L.M. (1990).

Tumorigenicity and metastasis of human breast carcinoma cells lines in nude mice.

Cancer Res. 50; 717-721.

Puntoriero, G. et al. Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants. Embo J 17, 3521-3533 (1998).

Racher et al., Biotechnology Techniques, 9:169-174, 1995.

WO 02/20723 PCT/US01/28044 Ragot et al., Nature, 361:647-650, 1993.

Rajotte D and Ruoslahti E. Membrane dipeptidase is the receptor for a lung-targeting peptide identified by in vivo phage display. J Biol Chem 274:11593-11598, 1999 Rajotte D, Arap W, Hagedorn M, Koivunen E, Pasqualini R, and Ruoslahti E.

in Molecular heterogeneity of the vascular endothelium revealed by in vivo phage C display. J Clin Invest 102:430-437, 1998 Rak JW, St. Croix BD, and Kerbel RS. Consequences of angiogenesis for tumor progression, metastasis and cancer. Anticancer Drugs 6:3-18, 1995.

Razzaque, Y. Francillon, P. N. Jilly, and F. Varricchio. 1996. Detection of human herpesvirus 6 sequences in lymphoma tissues by immunohistochemistry and polymerase chain reactions. Cancer Lett 106:221-6.

Remington's Pharmaceutical Sciences, 15th ed., pp. 1035-1038 and 1570-1580.

Renan, Radiother. Oncol., 19:197-218, 1990.

Renata Pasqualini, W. Daniel Rajotte, and Erkki Ruoslahti. in Phage Display: A Laboratory manual (ed. Carlos F. Barbas III, D. R. Jamie K. Scott, and Gregg J.

Silverman) 22.21-22.24 (Cold Spring Harbor Laboratory Press, New York, 2001).

Rich et al., Hum. Gene Ther., 4:461-476, 1993.

Ridgeway, In: Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Rodriguez et al., eds., Stoneham: Butterworth, pp. 467-492, 1988.

Rippe et al., Mol. Cell Biol., 10:689-695, 1990.

ROELVINK, LEE, EINFELD, KOVESDI, and WICKHAM, T.J.

(1999). Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae. Science 286; 1568-1571.

ROMANCZUK, GALER, ZABNER, BARSOMIAN, WADSWORTH, and O'RIORDAN, C.R. (1999). Modification of an adenoviral vector with WO 02/20723 PCT/US01/28044 U biologically selected peptides: a novel strategy for gene delivery to cells of choice.

Z Hum. Gene Ther. 10; 2615-2626.

Rosenfeld et al., Cell, 68:143-155, 1992.

Rosenfeld et al., Science, 252:431-434, 1991.

n Rowley, M. J. et al. Prediction of the immunodominant epitope of the pyruvate dehydrogenase complex E2 in primary biliary cirrhosis using phage display. J Immunol 164,3413-3419 (2000).

SRuoslahti E. RGD and other sequence recognition sequences for integrins. Annu. Rev.

Cell Dev. Biol. 12:697-715, 1996 Sahin, U. et al. Human neoplasms elicit multiple specific immune responses in the autologous host. Proc Natl Acad Sci USA 92, 11810-11813 (1995).

Sahin, Tureci, O. Pfreundschuh, M. Serological identification of human tumor antigens. Curr Opin Immunol 9, 709-716 (1997).

Scala, G. et al. Selection of HIV-specific immunogenic epitopes by screening random peptide libraries with HIV-1-positive sera. J Immunol 162, 6155-6161 (1999).

Schlingemann RO, Rietveld FJ, de Waal RM, Ferrone S, Ruiter DJ. Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds. Am. J. Pathol.. 136:1393-1405, 1990.

Schmitz, Baumann, G. and Gram, H. Catalytic specificity of phosphotyrosine kinase Blk, Lyn, c-Src and Syk as assessed by phage display J. Mol. Biol. 260: 664-677, 1996.

Shattil, S.J. and Ginsberg, M.H. Perspectives series: cell adhesion in vascular biology.

Integrin signaling in vascular biology. J. Clin. Invest. 100:1-5, 1997.

WO 02/20723 PCT/US01/28044 Short S M, Talbott GA and Juliano RL. Integrin-mediated Signaling Events in Human SEndothelial Cells. Mol. Biol. Cell 9: 1969-1980, 1998 Silverstein, JCI 74:1625-1633, 1984 SSmith G. P. Surface presentation of protein epitopes using bacteriophage expression CN system. Curr Opin Biotechnol 2, 668-73 (1991).

O

Smith GP, and Scott JK. Libraries of peptides and proteins displayed in filamentous phage. Meth. Enzymol. 21:228-257, 1993.

Smith GP, and Scott JK. Searching for peptide ligands with an epitope library. Science 228:1315-1317,1985 Smith, D. and K. S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31- Smith, G. P. 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315-7.

Smith, G.P. Surface presentation of protein epitopes using bacteriophage expression systems. Curr. Opin. Biotechnol. 2:668-673, 1991.

Solowska J, Edelman JM, Albelda SM and Buck CA. (1991) Cytoplasmic and transmembrane domains of integrin B1 and B3 subunits are functionally interchangeable. J. Cell Biol. 114: 1079- 1088.

Staratschek-Jox, S. Kotkowski, G. Beige, T. Rudiger, J. Bullerdiek, V. Diehl, and J.

Wolf. 2000. Detection of Epstein-Barr virus in Hodgkin-Reed-Sternberg cells no evidence for the persistence of integrated viral fragments inLatent membrane protein-1 (LMP-1)-negative classical Hodgkin's disease. Am J Pathol 156:209-16.

Sternberg, N. Hoess, R.H. Display of peptides and proteins on the surface of bacteriophage lambda. Proc Natl Acad Sci U S A 92, 1609-1613, 1995.

WO 02/20723 WO 0220723PCT/US01/28044 Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co., 1984.

Stoeckle et al., Mol. Cell Biol. 8:2675-80, 1988.

Stratford-Perrcaudet and Perricaudet, In: Human Gene Transfer, 0. Cohen-Haguenauer et eds., John Libbey Eurotext, France, pp. 51-61, 199 1.

(Ni Stratford-Perricaudet et al., Hum. Gene. Ther., 1:241-256, 1990.

Tam et al., J. Am. Chem. Soc., 105:6442, 1983.

Tanaka T, Cao Y, Folkman J and Fine HA. Viral vector-targeted antiangiogenic gene (Ni therapy utilizing an angiostatin complementary DNA. Cancer Res. 58:3362-3369, 1998.

Temin, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum Press, pp. 149-188, 1986.

Theodore, Derossi, Chassaing, Uirbat, Kubes, Jordan, P., Clineiweiss, Godement, and Prochiantz, A. Tntraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse. J. Neurosci. 15:7158-7 167, 1995.

Tischer, Mitchell, Hartman, Silvia, Gospodarowicz, Fiddes, J.C.

arnd Abraham, J. (1991) the human Gene for Vascular Endothelial Growth Factor. J. Biol. Chem., 226,11947-11954.

Top et al, J. Infect. Dis., 124:155-160, 197 1.

Triantafilou et Hum. Immunol. 62:764-770, 200 1.

Tureci, Sahin, U. Pfreundschuh, M. Serological analysis of human tumor antigens: molecular definition and implications. Mol Med Today 3, 342-349 (1997).

Tur-Kaspa et Mol. Cell Biol, 6:716-718, 1986.

U.S. Patent No. 3,817,837 U.S. Patent No. 3,850,752 WO 02/20723 PCT/US01/28044 U.S. Patent No. 3,939,350 U.S. Patent No. 3,996,345 U.S. Patent No.4,275,149 SU.S. Patent No. 4,277,437 NC U.S. Patent No. 4,366,241 (I U.S. Patent No. 4,472,509 0 U.S. Patent No. 5,021,236

O

U.S. Patent No. 5,206,347 U.S. Patent No. 5,223,409 U.S. Patent No. 5,401,511 U.S. Patent No. 5,603,872 U.S. Patent No. 5,622,699 U.S. Patent No. 5,889,155 U.S. Patent No. 6,068,829 Varmus et al., Cell, 25:23-36, 1981.

Veikkola, T. and Alitalo, K. (1999) VEGFs, receptors and angiogenesis. Seminar Cancer Bio.l, 9, 211-220.

VIGNE, MAHFOUZ, L, DEDIEU, BRIE, PERRICAUDET, and YEH, P. (1999). RGD inclusion in the hexon monomer provides adenovirus type vectors with a fiber knob-independent pathway for infection. J. Virol. 73; 5156- 5161.

Vu, T.H. et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93,411-422, 1998.

Vuori K. Ruoslahti E. Association of insulin receptor substrate-1 with integrins.

Science 266:1576-1578, 1994 WO 02/20723 PCT/US01/28044 t WATKINS, MESYANZHINOV, KUROCHKINA, and HAWKINS, Z R.E. (1997). The adenobody approach to viral targeting specific and enhanced C1 adenoviral gene delivery. Gene Ther. 4; 1004-1012.

Watson CA, Camera-Benson L, Palmer-Croker R and Pober JS.Variability among human umbilical vein endothelial cell cultures. Science 268: 447-448, 1995.

c Weiss, L. J. G. Strickler, R. A. Warnke, D. T. Purtilo, and J. Sklar. 1987. Epstein-

O

CNI Barr viral DNA in tissues of Hodgkin's disease. Am J Pathol 129:86-91 SWeiss, L. Y. Y. Chen, X. F. Liu, and D. Shibata. 1991. Epstein-Barr virus and Hodgkin's disease. A correlative in situ hybridization and polymerase chain reaction study. Am J Pathol 139:1259-65.

Weitzman MD, Wilson JM and Eck SL. Adenovirus vectors in cancer gene therapy. In: Gene Therapy and Vector Systems 2: 17-25, 1997.

Wells, J.A. and Lowman, H.B. Rapid evolution of peptide and protein binding properties in vitro. Curr. Opin. Biotechnol. 3:355-362, 1992.

Wickham TJ. Haskard D. Segal D. Kovesdi I. Targeting endothelium for gene therapy via receptors up-regulated during angiogenesis and inflammation. Cancer Immunol. Immunother. 45:149-151, 1997c.

Wickham, T.J. Targeting adenovirus. Gene Ther 7, 110-114, 2000.

WICKHAM, CARRION, and KOVESDI, I. (1995). Targeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs. Gene Ther. 2; 750-756.

WICKHAM, LEE, TITUS, SCONOCCHIA, BAKACS, KOVESDI, and SEGAL, D. (1997a). Targeted adenovirus-mediated gene delivery to T-cells via CD3. J. Virol. 71; 7663-7669.

WO 02/20723 WO 0220723PCT/USOI/28044 WICKHAM, MATHIAS, CHERESH, and NEMEROW, G.R. (1993).

Z Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not attachment. Cell 73; 309-3 19.

WICKHAM, ROELVINK, BROUGH, and KOVESDI, 1. (1996b).

In Adenovirus targeted to heparan-containing receptors increases its gene delivery efficiency to multiple cell types. Nature Biotechnol. 14; 1570-1573.

WICKHAM, TJ., SEGAL, ROELVINK, CARRION, IZONO VA, LEE, and KOVESDI, L (1996a). Targeted adenovirus gene transfer to CI endothelial and smooth muscle cells by using bispecific antibodies. J. Virol. 6831-6838.

WICKHAM, TZENG, SHEARS II, ROELVINK, LI, LEE, BROUGH, LIZONOVA, and KOVESDI, I. (1997b). Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins. J. Virol. 7 1; 8221-8229.

Wong et al., Gene, 10:87-94, 1980.

Wu and Wu, Biochemnistry, 27: 887-892, 1988.

Wu and Wu, J. Biol. Chem., 262: 4429-4432, 1987.

Zetter BR. Angiogenesis and tumor metastasis. Ann Rev Med 49:407-424, 1998 Zhang et al., J. Nature 372: 425, 1994.

Zhang J and Russell S. Vectors for cancer gene therapy.Cancer Met. Rev. 3:385-401, 1996.

ZF{ANG, W. (1999). Development and application of adenoviral vectors for gene therapy of cancer. Cancer Gene Ther. 6; 113-138.

Zini, Fournie-Zaluski, Chauvel, Roques, Corvol, P. and Cortes- Llorens, C. (1996) Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin II in the control of vasopressin release. Proc Nati Acad Sci USA, 93, 11968-11973.

Claims (44)

1. A method of identifying a targeting peptide, comprising: a) obtaining a phage display library; Ci b) injecting the phage display library into the circulation of a human; c) obtaining a sample of an organ, tissue or cell type from said human; and d) identifying one or more targeting peptides from phage present in said organ, tissue or cell type.

2. The method of claim 1, wherein said human is brain dead or a terminal wean patient.

3. The method of claim 1, wherein said library contains approximately 10 14 transforming units (TU).

4. The method of claim 3, wherein said library is a primary phage library. The method of claim 3, wherein said library contains at least 10 8 different targeting peptide sequences.

6. The method of claim 1, further comprising recovering phage from said organ, tissue or cell type.

7. The method of claim 6, wherein phage are recovered by infecting bacteria.

8. The method of claim 6, wherein phage are recovered by amplifying phage inserts.

9. The method of claim 8, wherein the inserts encode targeting peptides. 110 WO 02/20723 PCT/US01/28044 The method of claim 9, further comprising ligating the amplified inserts to phage DNA to produce new phage.

11. The method of claim 1, further comprising: t a) performing a statistical analysis of the targeting peptide sequences; and O C, b) identifying statistically significant peptide motifs. N 12. The method of claim 11, further comprising a) conducting an amino acid residue sequence count; b) tracking the relative frequency of each tripeptide motif, compared to its frequency in the unselected library; and c) calculating the probability of observing each relative frequency.

13. A targeting peptide identified by the method of claim 1.

14. A targeting peptide motif identified by the method of claim 12. A method of identifying a targeting peptide, comprising: a) injecting a phage display library into the circulation of a human; b) obtaining samples of multiple organs, tissues or cell types from said human; c) recovering phage from said samples; d) amplifying said phage; WO 02/20723 PCT/US01/28044 c e) pooling the amplified phage; f) injecting the pooled phage into the circulation of a second human; h) obtaining samples of the same organs, tissues or cell types as were if obtained from the first human; and CN i) recovering phage from said samples. C 16. An isolated peptide of 100 amino acids or less in size, comprising at least 3 contiguous amino acids of a sequence selected from any of SEQ ID through SEQ ID NO:325.

17. The isolated peptide of claim 16, wherein said peptide is 50 amino acids or less in size.

18. The isolated peptide of claim 16, wherein said peptide is 25 amino acids or less in size.

19. The isolated peptide of claim 16, wherein said peptide is 10 amino acids or less in size. The isolated peptide of claim 16, wherein said peptide is 7 amino acids or less in size.

21. The isolated peptide of claim 16, wherein said peptide is 5 amino acids or less in size.

22. The isolated peptide of claim 16, wherein said peptide is attached to a molecule.

23. The isolated peptide of claim 22, wherein said molecule is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti- 112 WO 02/20723 WO 0220723PCT[USOI/28044 angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a Z peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an C1 imaging agent, an antigen, a survival factor, an anti-apoptotic agent or a hormone antagonist.

24. The isolated peptide of claim 23, wherein said pro-aptoptosis agent is selected from the group consisting of gramicidin, magainin, mnellitin, defensin, cecropin, N ~(KLAKLAK) 2 (SEQ ID NO: (KLAKKLA) 2 (SEQ ID NO:2), (KAAKKAA) 2 (SEQ ID NO:3) and (KLGKKLG) 3 (SEQ ID NO:4). The isolated peptide of claim 23, wherein said anti-angiogemc agent is selected from the group consisting of thrombospondin, angiostatin5, pigment epithelium- drived factor, angiotensin, lamninin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-lO, Gro-B, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM1O1, Marimastat, pentosan polysuiphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PN1J145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, .endostatin, paclitaxel, Docetaxel, polyamines, a proteosome inhibitor, a kinase inhibitor, a signaling inhibitor, SU5416, SU6668, accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline.

26. The isolated peptide of claim 23, wherein said cytokine is selected from the group consisting of interleukin 1 1-5, E-10, I-11, IL-12, I-18, interferon-y IF-a, IF-B, tumor necrosis factor-a (rNF-a), or GM-CSF (granulocyte macrophage colony stimulating factor).

27. The isolated peptide of claim 16, wherein said peptide is attached to a macromolecular complex. WO 02/20723 PCT/US01/28044

28. The isolated peptide of claim 27, wherein the macromolecular complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic C bead, a yeast cell, a mamalian cell or a cell. _29. The isolated peptide of claim 16, wherein said peptide is attached to a eukaryotic expression vector. cI 30. The isolated peptide of claim 16, wherein said vector is a gene therapy vector. C 31. A fusion protein, comprising at least 3 contiguous amino acids selected from any of SEQ ID NO:5 through SEQ ID NO:325.

32. A composition comprising the isolated peptide of claim 16 in a pharmaceutically acceptable carrier.

33. The composition of claim 32, wherein said peptide is attached to 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, an imaging agent, an antigen, a survival factor, an anti-apoptotic agent or a hormone antagonist..

34. The composition of claim 32, wherein said peptide is attached to a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a yeast cell, a mamalian cell, a eukaryotic expression vector or a cell A kit comprising: a) the isolated peptide of claim 16; and b) a control peptide. WO 02/20723 PCT/US01/28044

36. An antibody that selectively binds to an isolated peptide of a sequence selected Z from any of SEQ ID NO:5 through SEQ ID NO:325.

37. A gene therapy vector, wherein the vector expresses a targeting peptide sequence as part of a surface protein, the targeting peptide comprising at least l three contiguous amino acids selected from any of SEQ ID NO:5 through SEQ ID NO:325. O 38. An isolated nucleic acid of 300 nucleotides or less in size, said nucleic acid c encoding a peptide according to claim 16.

39. A vector comprising the isolated nucleic acid of claim 38. The vector of claim 39, wherein said vector is a plasmid, a cosmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a virus or a phage.

41. A method of treating a disease state comprising: a) selecting a peptide according to claim 1, wherein said peptide is targeted to cells associated with said disease state; b) attaching to said peptide one or more molecules effective to treat said disease state; and c) administering said peptide to a subject with said disease state.

42. The method of claim 41, wherein said disease state is selected from the group consisting of diabetes, inflammatory disease, arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection, viral infection, cardiovascular disease or degenerative disease. WO 02/20723 PCT/US01/28044 C 43. The isolated peptide of claim 20, wherein said sequence is selected from SEQ SID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:130. S44. The isolated peptide of claim 20, wherein said sequence is selected from SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID SNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID C. NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 or SEQ ID NO:131. The isolated peptide of claim 20, wherein said sequence is selected from any of SEQ ID NO:46 through SEQ ID

46. The isolated peptide of claim 20, wherein said sequence is selected from any of SEQ ID NO:66 through SEQ ID NO:83.

47. The isolated peptide of claim 20, wherein said sequence is selected from any of SEQ ID NO:84 through SEQ ID NO:129.

48. The isolated peptide of claim 20, wherein said sequence is selected from any of SEQ ID NO:132 through SEQ ID NO:148.

49. The isolated peptide of claim 20, wherein said sequence is selected from any of SEQ ID NO:149 through SEQ ID NO:173. A method of imaging comprising: a) selecting a peptide according to claim 1, b) attaching said peptide to an imaging agent; c) administering said peptide and agent to a human; and WO 02/20723 PCT/US01/28044 Sd) obtaining an image of the human.

51. A method of diagnosing a disease state comprising: a) selecting a peptide according to claim 1, wherein said peptide is targeted to cells associated with a disease state; b) administering said peptide to a human; and O c) determining the distribution of said peptide in said human.

52. A method, comprising the steps of: a) injecting a phage display library comprising about 108 or more targeting peptides into the circulation of a human; b) allowing the library to remain in the human for a period of time and under conditions such that phage bind to the human; and c) obtaining a sample from said human which sample comprises bound phage.

53. The method of claim 52, further comprising isolating phage bound to the sample.

54. The method of claim 52, wherein the human is a human selected from the group consisting of a brain dead human and a terminal wean human patient. The method of claim 52, wherein the library comprises about 1014 or more transforming units (TU).

56. The method of claim 55, wherein the library is a primary phage library.

57. The method of claim 52, wherein the library comprises at least 10 9 different targeting peptide sequences.

58. The method of claim 52, further comprising recovering phage from the sample. 117 WO 02/20723 PCT/US01/28044

59. The method of claim 52, wherein the phage are attached to a detectable label. The method of claim 52, wherein the sample is obtained from diseased tissue of the human after the human has died. Cct

61. A method, comprising the steps of: a) injecting a phage display library into the circulation of a human, wherein the 0library is comprised of 108 or more targeting peptides and the phage are attached to a detectable label and further wherein the human has a terminal disease and a beating heart; b) allowing the library to remain in the human for a period of time and under conditions such that phage of library bind to the human; c) obtaining a sample from the human after the heart of the human is no longer beating, wherein the sample comprises bound phage; and d) isolating phage from the sample.

62. The method of claim 52, further comprising: e) performing a statistical analysis of the targeting peptide sequences; and f) identifying statistically significant peptide motifs.

63. The method of claim 62, further comprising g) conducting an amino acid residue sequence count; h) tracking the relative frequency of each tripeptide motif, compared to its frequency in the unselected library; and i) calculating the probability of observing each relative frequency.